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• Expanding GABAAR pharmacology via receptor-associated proteins Schulte, Clemens; Maric, Hans Michael in Current Opinion in Pharmacology (2021). 57 98–106.
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  Drugs directly targeting γ-aminobutyric acid type A receptors (GABAARs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence establishes intracellularly GABAAR-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABAAR-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.

  @article{schulte2021expanding, abstract = {Drugs directly targeting γ-aminobutyric acid type A receptors (GABAARs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence establishes intracellularly GABAAR-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABAAR-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.}, author = {Schulte, Clemens and Maric, Hans Michael}, journal = {Current Opinion in Pharmacology}, keywords = {maric}, pages = {98–106}, title = {Expanding GABAAR pharmacology via receptor-associated proteins}, volume = 57, year = 2021 }

  %0 Journal Article %1 schulte2021expanding %A Schulte, Clemens %A Maric, Hans Michael %D 2021 %J Current Opinion in Pharmacology %P 98--106 %R https://doi.org/10.1016/j.coph.2021.01.004 %T Expanding GABAAR pharmacology via receptor-associated proteins %U https://www.sciencedirect.com/science/article/pii/S1471489221000059 %V 57 %X Drugs directly targeting γ-aminobutyric acid type A receptors (GABAARs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence establishes intracellularly GABAAR-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABAAR-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.
• Targeting the APP-Mint2 Protein–Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-β Formation Bartling, Christian R. O.; Jensen, Thomas M. T.; Henry, Shawna M.; Colliander, Anna L.; Sereikaite, Vita; Wenzler, Marcella; Jain, Palash; Maric, Hans M.; Harpsøe, Kasper; Pedersen, Søren W.; Clemmensen, Louise S.; Haugaard-Kedström, Linda M.; Gloriam, David E.; Ho, Angela; Strømgaard, Kristian in J. Am. Chem. Soc. (2021). 143(2) 891–901.
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  There is an urgent need for novel therapeutic approaches to treat Alzheimer’s disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-β (Aβ) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aβ. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aβ generation. Herein, we deciphered the APP-Mint2 protein–protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aβ42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aβ levels.

  @article{bartling2021targeting, abstract = {There is an urgent need for novel therapeutic approaches to treat Alzheimer’s disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-β (Aβ) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aβ. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aβ generation. Herein, we deciphered the APP-Mint2 protein–protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aβ42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aβ levels.}, author = {Bartling, Christian R. O. and Jensen, Thomas M. T. and Henry, Shawna M. and Colliander, Anna L. and Sereikaite, Vita and Wenzler, Marcella and Jain, Palash and Maric, Hans M. and Harpsøe, Kasper and Pedersen, Søren W. and Clemmensen, Louise S. and Haugaard-Kedström, Linda M. and Gloriam, David E. and Ho, Angela and Strømgaard, Kristian}, booktitle = {Journal of the American Chemical Society}, journal = {J. Am. Chem. Soc.}, keywords = {maric}, month = {jan}, number = 2, pages = {891–901}, publisher = {American Chemical Society}, title = {Targeting the APP-Mint2 Protein–Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-β Formation}, volume = 143, year = 2021 }

  %0 Journal Article %1 bartling2021targeting %A Bartling, Christian R. O. %A Jensen, Thomas M. T. %A Henry, Shawna M. %A Colliander, Anna L. %A Sereikaite, Vita %A Wenzler, Marcella %A Jain, Palash %A Maric, Hans M. %A Harpsøe, Kasper %A Pedersen, Søren W. %A Clemmensen, Louise S. %A Haugaard-Kedström, Linda M. %A Gloriam, David E. %A Ho, Angela %A Strømgaard, Kristian %B Journal of the American Chemical Society %D 2021 %I American Chemical Society %J J. Am. Chem. Soc. %N 2 %P 891--901 %R 10.1021/jacs.0c10696 %T Targeting the APP-Mint2 Protein–Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-β Formation %U https://doi.org/10.1021/jacs.0c10696 %V 143 %X There is an urgent need for novel therapeutic approaches to treat Alzheimer’s disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-β (Aβ) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aβ. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aβ generation. Herein, we deciphered the APP-Mint2 protein–protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aβ42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aβ levels.
• Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases Moreno-Yruela, Carlos; Bæk, Michael; Vrsanova, Adela-Eugenie; Schulte, Clemens; Maric, Hans M.; Olsen, Christian A. in Nature Communications (2021). 12(1) 62-.
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  Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.

  @article{morenoyruela2021hydroxamic, abstract = {Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.}, author = {Moreno-Yruela, Carlos and Bæk, Michael and Vrsanova, Adela-Eugenie and Schulte, Clemens and Maric, Hans M. and Olsen, Christian A.}, journal = {Nature Communications}, keywords = {maric}, number = 1, pages = {62–}, title = {Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases}, volume = 12, year = 2021 }

  %0 Journal Article %1 morenoyruela2021hydroxamic %A Moreno-Yruela, Carlos %A Bæk, Michael %A Vrsanova, Adela-Eugenie %A Schulte, Clemens %A Maric, Hans M. %A Olsen, Christian A. %D 2021 %J Nature Communications %N 1 %P 62-- %R 10.1038/s41467-020-20250-9 %T Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases %U https://doi.org/10.1038/s41467-020-20250-9 %V 12 %X Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.
• High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale Schulte, Clemens; Khayenko, Vladimir; Nordblom, Noah Frieder; Tippel, Franziska; Peck, Violetta; Gupta, Amit Jean; Maric, Hans Michael in iScience (2021). 24(1) 101898-.
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  Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.

  @article{schulte2021highthroughput, abstract = {Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.}, author = {Schulte, Clemens and Khayenko, Vladimir and Nordblom, Noah Frieder and Tippel, Franziska and Peck, Violetta and Gupta, Amit Jean and Maric, Hans Michael}, journal = {iScience}, keywords = {maric}, number = 1, pages = {101898–}, title = {High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale}, volume = 24, year = 2021 }

  %0 Journal Article %1 schulte2021highthroughput %A Schulte, Clemens %A Khayenko, Vladimir %A Nordblom, Noah Frieder %A Tippel, Franziska %A Peck, Violetta %A Gupta, Amit Jean %A Maric, Hans Michael %D 2021 %J iScience %N 1 %P 101898-- %R https://doi.org/10.1016/j.isci.2020.101898 %T High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale %U https://www.sciencedirect.com/science/article/pii/S2589004220310956 %V 24 %X Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.

• Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytes Gründl, Marco; Walz, Susanne; Hauf, Laura; Schwab, Melissa; Werner, Kerstin Marcela; Spahr, Susanne; Schulte, Clemens; Maric, Hans Michael; Ade, Carsten P.; Gaubatz, Stefan in PLOS Genetics (2020). 16(5) e1008818-.
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  Author summary YAP, the major downstream transducer of the Hippo pathway, is a potent inducer of proliferation. Here we show that the Myb-MuvB complex (MMB) mediates cardiomyocyte proliferation by YAP. We find that YAP and MMB regulate an overlapping set of pro-proliferative genes which involves binding of MMB to the promoters of these genes. We also identified a direct interaction between the B-MYB subunit of MMB and YAP. Based on the binding studies, we created a tool called MY-COMP that interferes with the association YAP to B-MYB and strongly inhibits proliferation of cardiomyocytes. Together, our data suggests that the YAP-MMB interaction is essential for division of cardiomyocytes, underscoring the functional relevance of the crosstalk between these two pathways for proper heart development.

  @article{grundl2020interaction, abstract = {Author summary YAP, the major downstream transducer of the Hippo pathway, is a potent inducer of proliferation. Here we show that the Myb-MuvB complex (MMB) mediates cardiomyocyte proliferation by YAP. We find that YAP and MMB regulate an overlapping set of pro-proliferative genes which involves binding of MMB to the promoters of these genes. We also identified a direct interaction between the B-MYB subunit of MMB and YAP. Based on the binding studies, we created a tool called MY-COMP that interferes with the association YAP to B-MYB and strongly inhibits proliferation of cardiomyocytes. Together, our data suggests that the YAP-MMB interaction is essential for division of cardiomyocytes, underscoring the functional relevance of the crosstalk between these two pathways for proper heart development.}, author = {Gründl, Marco and Walz, Susanne and Hauf, Laura and Schwab, Melissa and Werner, Kerstin Marcela and Spahr, Susanne and Schulte, Clemens and Maric, Hans Michael and Ade, Carsten P. and Gaubatz, Stefan}, journal = {PLOS Genetics}, keywords = {maric}, month = {may}, number = 5, pages = {e1008818–}, publisher = {Public Library of Science}, title = {Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytes}, volume = 16, year = 2020 }

  %0 Journal Article %1 grundl2020interaction %A Gründl, Marco %A Walz, Susanne %A Hauf, Laura %A Schwab, Melissa %A Werner, Kerstin Marcela %A Spahr, Susanne %A Schulte, Clemens %A Maric, Hans Michael %A Ade, Carsten P. %A Gaubatz, Stefan %D 2020 %I Public Library of Science %J PLOS Genetics %N 5 %P e1008818-- %T Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytes %U https://doi.org/10.1371/journal.pgen.1008818 %V 16 %X Author summary YAP, the major downstream transducer of the Hippo pathway, is a potent inducer of proliferation. Here we show that the Myb-MuvB complex (MMB) mediates cardiomyocyte proliferation by YAP. We find that YAP and MMB regulate an overlapping set of pro-proliferative genes which involves binding of MMB to the promoters of these genes. We also identified a direct interaction between the B-MYB subunit of MMB and YAP. Based on the binding studies, we created a tool called MY-COMP that interferes with the association YAP to B-MYB and strongly inhibits proliferation of cardiomyocytes. Together, our data suggests that the YAP-MMB interaction is essential for division of cardiomyocytes, underscoring the functional relevance of the crosstalk between these two pathways for proper heart development.
• A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition Langlhofer, Georg; Schaefer, Natascha; Maric, Hans M.; Keramidas, Angelo; Zhang, Yan; Baumann, Peter; Blum, Robert; Breitinger, Ulrike; Strømgaard, Kristian; Schlosser, Andreas; Kessels, Michael M.; Koch, Dennis; Qualmann, Britta; Breitinger, Hans-Georg; Lynch, Joseph W.; Villmann, Carmen in J. Neurosci. (2020). 40(25) 4954-.
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  Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.

  @article{langlhofer2020novel, abstract = {Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.}, author = {Langlhofer, Georg and Schaefer, Natascha and Maric, Hans M. and Keramidas, Angelo and Zhang, Yan and Baumann, Peter and Blum, Robert and Breitinger, Ulrike and Strømgaard, Kristian and Schlosser, Andreas and Kessels, Michael M. and Koch, Dennis and Qualmann, Britta and Breitinger, Hans-Georg and Lynch, Joseph W. and Villmann, Carmen}, journal = {J. Neurosci.}, keywords = {maric}, month = {jun}, number = 25, pages = {4954–}, title = {A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition}, volume = 40, year = 2020 }

  %0 Journal Article %1 langlhofer2020novel %A Langlhofer, Georg %A Schaefer, Natascha %A Maric, Hans M. %A Keramidas, Angelo %A Zhang, Yan %A Baumann, Peter %A Blum, Robert %A Breitinger, Ulrike %A Strømgaard, Kristian %A Schlosser, Andreas %A Kessels, Michael M. %A Koch, Dennis %A Qualmann, Britta %A Breitinger, Hans-Georg %A Lynch, Joseph W. %A Villmann, Carmen %D 2020 %J J. Neurosci. %N 25 %P 4954-- %R 10.1523/JNEUROSCI.2490-19.2020 %T A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition %U http://www.jneurosci.org/content/40/25/4954.abstract %V 40 %X Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.
• Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells Prieto-Garcia, Cristian; Hartmann, Oliver; Reissland, Michaela; Braun, Fabian; Fischer, Thomas; Walz, Susanne; Schülein-Völk, Christina; Eilers, Ursula; Ade, Carsten P; Calzado, Marco A; Orian, Amir; Maric, Hans M; Münch, Christian; Rosenfeldt, Mathias; Eilers, Martin; Diefenbacher, Markus E in EMBO Molecular Medicine (2020). 12(4) e11101-.
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  Abstract The transcription factor ?Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ?Np63 and maintains elevated ?NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ?Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.

  @article{prietogarcia2020maintaining, abstract = {Abstract The transcription factor ?Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ?Np63 and maintains elevated ?NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ?Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.}, author = {Prieto-Garcia, Cristian and Hartmann, Oliver and Reissland, Michaela and Braun, Fabian and Fischer, Thomas and Walz, Susanne and Schülein-Völk, Christina and Eilers, Ursula and Ade, Carsten P and Calzado, Marco A and Orian, Amir and Maric, Hans M and Münch, Christian and Rosenfeldt, Mathias and Eilers, Martin and Diefenbacher, Markus E}, booktitle = {EMBO Molecular Medicine}, journal = {EMBO Molecular Medicine}, keywords = {maric}, month = {apr}, number = 4, pages = {e11101–}, publisher = {John Wiley & Sons, Ltd}, title = {Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells}, volume = 12, year = 2020 }

  %0 Journal Article %1 prietogarcia2020maintaining %A Prieto-Garcia, Cristian %A Hartmann, Oliver %A Reissland, Michaela %A Braun, Fabian %A Fischer, Thomas %A Walz, Susanne %A Schülein-Völk, Christina %A Eilers, Ursula %A Ade, Carsten P %A Calzado, Marco A %A Orian, Amir %A Maric, Hans M %A Münch, Christian %A Rosenfeldt, Mathias %A Eilers, Martin %A Diefenbacher, Markus E %B EMBO Molecular Medicine %D 2020 %I John Wiley & Sons, Ltd %J EMBO Molecular Medicine %N 4 %P e11101-- %R 10.15252/emmm.201911101 %T Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells %U https://doi.org/10.15252/emmm.201911101 %V 12 %X Abstract The transcription factor ?Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ?Np63 and maintains elevated ?NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ?Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.

• MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation Sdelci, Sara; Rendeiro, André F.; Rathert, Philipp; You, Wanhui; Lin, Jung-Ming G.; Ringler, Anna; Hofstätter, Gerald; Moll, Herwig P.; Gürtl, Bettina; Farlik, Matthias; Schick, Sandra; Klepsch, Freya; Oldach, Matthew; Buphamalai, Pisanu; Schischlik, Fiorella; Májek, Peter; Parapatics, Katja; Schmidl, Christian; Schuster, Michael; Penz, Thomas; Buckley, Dennis L.; Hudecz, Otto; Imre, Richard; Wang, Shuang-Yan; Maric, Hans Michael; Kralovics, Robert; Bennett, Keiryn L.; Müller, Andre C.; Mechtler, Karl; Menche, Jörg; Bradner, James E.; Winter, Georg E.; Klavins, Kristaps; Casanova, Emilio; Bock, Christoph; Zuber, Johannes; Kubicek, Stefan in Nature Genetics (2019). 51(6) 990–998.
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  The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

  @article{sdelci2019mthfd1, abstract = {The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.}, author = {Sdelci, Sara and Rendeiro, André F. and Rathert, Philipp and You, Wanhui and Lin, Jung-Ming G. and Ringler, Anna and Hofstätter, Gerald and Moll, Herwig P. and Gürtl, Bettina and Farlik, Matthias and Schick, Sandra and Klepsch, Freya and Oldach, Matthew and Buphamalai, Pisanu and Schischlik, Fiorella and Májek, Peter and Parapatics, Katja and Schmidl, Christian and Schuster, Michael and Penz, Thomas and Buckley, Dennis L. and Hudecz, Otto and Imre, Richard and Wang, Shuang-Yan and Maric, Hans Michael and Kralovics, Robert and Bennett, Keiryn L. and Müller, Andre C. and Mechtler, Karl and Menche, Jörg and Bradner, James E. and Winter, Georg E. and Klavins, Kristaps and Casanova, Emilio and Bock, Christoph and Zuber, Johannes and Kubicek, Stefan}, journal = {Nature Genetics}, keywords = {maric}, number = 6, pages = {990–998}, title = {MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation}, volume = 51, year = 2019 }

  %0 Journal Article %1 sdelci2019mthfd1 %A Sdelci, Sara %A Rendeiro, André F. %A Rathert, Philipp %A You, Wanhui %A Lin, Jung-Ming G. %A Ringler, Anna %A Hofstätter, Gerald %A Moll, Herwig P. %A Gürtl, Bettina %A Farlik, Matthias %A Schick, Sandra %A Klepsch, Freya %A Oldach, Matthew %A Buphamalai, Pisanu %A Schischlik, Fiorella %A Májek, Peter %A Parapatics, Katja %A Schmidl, Christian %A Schuster, Michael %A Penz, Thomas %A Buckley, Dennis L. %A Hudecz, Otto %A Imre, Richard %A Wang, Shuang-Yan %A Maric, Hans Michael %A Kralovics, Robert %A Bennett, Keiryn L. %A Müller, Andre C. %A Mechtler, Karl %A Menche, Jörg %A Bradner, James E. %A Winter, Georg E. %A Klavins, Kristaps %A Casanova, Emilio %A Bock, Christoph %A Zuber, Johannes %A Kubicek, Stefan %D 2019 %J Nature Genetics %N 6 %P 990--998 %R 10.1038/s41588-019-0413-z %T MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation %U https://doi.org/10.1038/s41588-019-0413-z %V 51 %X The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.
• MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation Baluapuri, Apoorva; Hofstetter, Julia; Dudvarski Stankovic, Nevenka; Endres, Theresa; Bhandare, Pranjali; Vos, Seychelle Monique; Adhikari, Bikash; Schwarz, Jessica Denise; Narain, Ashwin; Vogt, Markus; Wang, Shuang-Yan; Düster, Robert; Jung, Lisa Anna; Vanselow, Jens Thorsten; Wiegering, Armin; Geyer, Matthias; Maric, Hans Michael; Gallant, Peter; Walz, Susanne; Schlosser, Andreas; Cramer, Patrick; Eilers, Martin; Wolf, Elmar in Molecular Cell (2019). 74(4) 674–687.e11.
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  Summary The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

  @article{baluapuri2019recruits, abstract = {Summary The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.}, author = {Baluapuri, Apoorva and Hofstetter, Julia and Dudvarski Stankovic, Nevenka and Endres, Theresa and Bhandare, Pranjali and Vos, Seychelle Monique and Adhikari, Bikash and Schwarz, Jessica Denise and Narain, Ashwin and Vogt, Markus and Wang, Shuang-Yan and Düster, Robert and Jung, Lisa Anna and Vanselow, Jens Thorsten and Wiegering, Armin and Geyer, Matthias and Maric, Hans Michael and Gallant, Peter and Walz, Susanne and Schlosser, Andreas and Cramer, Patrick and Eilers, Martin and Wolf, Elmar}, journal = {Molecular Cell}, keywords = {maric}, number = 4, pages = {674–687.e11}, title = {MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation}, volume = 74, year = 2019 }

  %0 Journal Article %1 baluapuri2019recruits %A Baluapuri, Apoorva %A Hofstetter, Julia %A Dudvarski Stankovic, Nevenka %A Endres, Theresa %A Bhandare, Pranjali %A Vos, Seychelle Monique %A Adhikari, Bikash %A Schwarz, Jessica Denise %A Narain, Ashwin %A Vogt, Markus %A Wang, Shuang-Yan %A Düster, Robert %A Jung, Lisa Anna %A Vanselow, Jens Thorsten %A Wiegering, Armin %A Geyer, Matthias %A Maric, Hans Michael %A Gallant, Peter %A Walz, Susanne %A Schlosser, Andreas %A Cramer, Patrick %A Eilers, Martin %A Wolf, Elmar %D 2019 %J Molecular Cell %N 4 %P 674--687.e11 %R https://doi.org/10.1016/j.molcel.2019.02.031 %T MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation %U http://www.sciencedirect.com/science/article/pii/S109727651930142X %V 74 %X Summary The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.
• Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins Kasaragod, Vikram Babu; Hausrat, Torben Johann; Schaefer, Natascha; Kuhn, Maximilian; Christensen, Nikolaj Riis; Tessmer, Ingrid; Maric, Hans Michael; Madsen, Kenneth Lindegaard; Sotriffer, Christoph; Villmann, Carmen; Kneussel, Matthias; Schindelin, Hermann in Neuron (2019). 101(4) 673–689.e11.
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  Summary The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Å resolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABAARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.

  @article{kasaragod2019elucidating, abstract = {Summary The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Å resolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABAARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.}, author = {Kasaragod, Vikram Babu and Hausrat, Torben Johann and Schaefer, Natascha and Kuhn, Maximilian and Christensen, Nikolaj Riis and Tessmer, Ingrid and Maric, Hans Michael and Madsen, Kenneth Lindegaard and Sotriffer, Christoph and Villmann, Carmen and Kneussel, Matthias and Schindelin, Hermann}, journal = {Neuron}, keywords = {maric}, number = 4, pages = {673–689.e11}, title = {Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins}, volume = 101, year = 2019 }

  %0 Journal Article %1 kasaragod2019elucidating %A Kasaragod, Vikram Babu %A Hausrat, Torben Johann %A Schaefer, Natascha %A Kuhn, Maximilian %A Christensen, Nikolaj Riis %A Tessmer, Ingrid %A Maric, Hans Michael %A Madsen, Kenneth Lindegaard %A Sotriffer, Christoph %A Villmann, Carmen %A Kneussel, Matthias %A Schindelin, Hermann %D 2019 %J Neuron %N 4 %P 673--689.e11 %R https://doi.org/10.1016/j.neuron.2019.01.001 %T Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins %U http://www.sciencedirect.com/science/article/pii/S0896627319300029 %V 101 %X Summary The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Å resolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABAARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.
• Targeting GABAAR-Associated Proteins: New Modulators, Labels and Concepts Khayenko, Vladimir; Maric, Hans Michael in Frontiers in Molecular Neuroscience (2019). 12 162-.
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  γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review we introduce a new strategy to modulate GABAeric signaling: Targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with nanometer resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in-vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.

  @article{khayenko2019targeting, abstract = {γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review we introduce a new strategy to modulate GABAeric signaling: Targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with nanometer resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in-vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.}, author = {Khayenko, Vladimir and Maric, Hans Michael}, journal = {Frontiers in Molecular Neuroscience}, keywords = {maric}, pages = {162–}, title = {Targeting GABAAR-Associated Proteins: New Modulators, Labels and Concepts}, volume = 12, year = 2019 }

  %0 Journal Article %1 khayenko2019targeting %A Khayenko, Vladimir %A Maric, Hans Michael %D 2019 %J Frontiers in Molecular Neuroscience %P 162-- %T Targeting GABAAR-Associated Proteins: New Modulators, Labels and Concepts %U https://www.frontiersin.org/article/10.3389/fnmol.2019.00162 %V 12 %X γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review we introduce a new strategy to modulate GABAeric signaling: Targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with nanometer resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in-vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.

• Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms Bleem, Alissa; Christiansen, Gunna; Madsen, Daniel J.; Maric, Hans; Strømgaard, Kristian; Bryers, James D.; Daggett, Valerie; Meyer, Rikke L.; Otzen, Daniel E. in Journal of Molecular Biology (2018). 430(20) 3751–3763.
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  Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.

  @article{bleem2018protein, abstract = {Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.}, author = {Bleem, Alissa and Christiansen, Gunna and Madsen, Daniel J. and Maric, Hans and Strømgaard, Kristian and Bryers, James D. and Daggett, Valerie and Meyer, Rikke L. and Otzen, Daniel E.}, booktitle = {Functional Amyloids in Health and Disease}, journal = {Journal of Molecular Biology}, keywords = {maric}, number = 20, pages = {3751–3763}, title = {Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms}, volume = 430, year = 2018 }

  %0 Journal Article %1 bleem2018protein %A Bleem, Alissa %A Christiansen, Gunna %A Madsen, Daniel J. %A Maric, Hans %A Strømgaard, Kristian %A Bryers, James D. %A Daggett, Valerie %A Meyer, Rikke L. %A Otzen, Daniel E. %B Functional Amyloids in Health and Disease %D 2018 %J Journal of Molecular Biology %N 20 %P 3751--3763 %R https://doi.org/10.1016/j.jmb.2018.06.043 %T Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms %U http://www.sciencedirect.com/science/article/pii/S0022283618307046 %V 430 %X Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.
• Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin Hines, Rochelle M.; Maric, Hans Michael; Hines, Dustin J.; Modgil, Amit; Panzanelli, Patrizia; Nakamura, Yasuko; Nathanson, Anna J.; Cross, Alan; Deeb, Tarek; Brandon, Nicholas J.; Davies, Paul; Fritschy, Jean-Marc; Schindelin, Hermann; Moss, Stephen J. in Nature Communications (2018). 9(1) 3130-.
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  Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

  @article{hines2018developmental, abstract = {Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.}, author = {Hines, Rochelle M. and Maric, Hans Michael and Hines, Dustin J. and Modgil, Amit and Panzanelli, Patrizia and Nakamura, Yasuko and Nathanson, Anna J. and Cross, Alan and Deeb, Tarek and Brandon, Nicholas J. and Davies, Paul and Fritschy, Jean-Marc and Schindelin, Hermann and Moss, Stephen J.}, journal = {Nature Communications}, keywords = {maric}, number = 1, pages = {3130–}, title = {Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin}, volume = 9, year = 2018 }

  %0 Journal Article %1 hines2018developmental %A Hines, Rochelle M. %A Maric, Hans Michael %A Hines, Dustin J. %A Modgil, Amit %A Panzanelli, Patrizia %A Nakamura, Yasuko %A Nathanson, Anna J. %A Cross, Alan %A Deeb, Tarek %A Brandon, Nicholas J. %A Davies, Paul %A Fritschy, Jean-Marc %A Schindelin, Hermann %A Moss, Stephen J. %D 2018 %J Nature Communications %N 1 %P 3130-- %R 10.1038/s41467-018-05481-1 %T Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin %U https://doi.org/10.1038/s41467-018-05481-1 %V 9 %X Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

• Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle Büchel, Gabriele; Carstensen, Anne; Mak, Ka-Yan; Roeschert, Isabelle; Leen, Eoin; Sumara, Olga; Hofstetter, Julia; Herold, Steffi; Kalb, Jacqueline; Baluapuri, Apoorva; Poon, Evon; Kwok, Colin; Chesler, Louis; Maric, Hans Michael; Rickman, David S.; Wolf, Elmar; Bayliss, Richard; Walz, Susanne; Eilers, Martin in Cell Reports (2017). 21(12) 3483–3497.
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  Summary MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.

  @article{buchel2017association, abstract = {Summary MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.}, author = {Büchel, Gabriele and Carstensen, Anne and Mak, Ka-Yan and Roeschert, Isabelle and Leen, Eoin and Sumara, Olga and Hofstetter, Julia and Herold, Steffi and Kalb, Jacqueline and Baluapuri, Apoorva and Poon, Evon and Kwok, Colin and Chesler, Louis and Maric, Hans Michael and Rickman, David S. and Wolf, Elmar and Bayliss, Richard and Walz, Susanne and Eilers, Martin}, journal = {Cell Reports}, keywords = {maric}, number = 12, pages = {3483–3497}, title = {Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle}, volume = 21, year = 2017 }

  %0 Journal Article %1 buchel2017association %A Büchel, Gabriele %A Carstensen, Anne %A Mak, Ka-Yan %A Roeschert, Isabelle %A Leen, Eoin %A Sumara, Olga %A Hofstetter, Julia %A Herold, Steffi %A Kalb, Jacqueline %A Baluapuri, Apoorva %A Poon, Evon %A Kwok, Colin %A Chesler, Louis %A Maric, Hans Michael %A Rickman, David S. %A Wolf, Elmar %A Bayliss, Richard %A Walz, Susanne %A Eilers, Martin %D 2017 %J Cell Reports %N 12 %P 3483--3497 %R https://doi.org/10.1016/j.celrep.2017.11.090 %T Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle %U http://www.sciencedirect.com/science/article/pii/S2211124717317576 %V 21 %X Summary MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.

• Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling Cheng, Jin; Sahani, Sadhna; Hausrat, Torben Johann; Yang, Jenq-Wei; Ji, Haichao; Schmarowski, Nikolai; Endle, Heiko; Liu, Xinfeng; Li, Yunbo; Böttche, Rahel; Radyushkin, Konstantin; Maric, Hans M; Hoerder-Suabedissen, Anna; Molnár, Zoltán; Prouvot, Pierre-Hugues; Trimbuch, Thorsten; Ninnemann, Olaf; Huai, Jisen; Fan, Wei; Visentin, Barbara; Sabbadini, Roger; Strømgaard, Kristian; Stroh, Albrecht; Luhmann, Heiko J; Kneussel, Matthias; Nitsch, Robert; Vogt, Johannes in Neuron (2016). 92(1) 126–142.
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  Summary Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2−/− thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2−/− animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.

  @article{cheng2016precise, abstract = {Summary Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2−/− thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2−/− animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.}, author = {Cheng, Jin and Sahani, Sadhna and Hausrat, Torben Johann and Yang, Jenq-Wei and Ji, Haichao and Schmarowski, Nikolai and Endle, Heiko and Liu, Xinfeng and Li, Yunbo and Böttche, Rahel and Radyushkin, Konstantin and Maric, Hans M and Hoerder-Suabedissen, Anna and Molnár, Zoltán and Prouvot, Pierre-Hugues and Trimbuch, Thorsten and Ninnemann, Olaf and Huai, Jisen and Fan, Wei and Visentin, Barbara and Sabbadini, Roger and Strømgaard, Kristian and Stroh, Albrecht and Luhmann, Heiko J and Kneussel, Matthias and Nitsch, Robert and Vogt, Johannes}, journal = {Neuron}, keywords = {maric}, number = 1, pages = {126–142}, title = {Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling}, volume = 92, year = 2016 }

  %0 Journal Article %1 cheng2016precise %A Cheng, Jin %A Sahani, Sadhna %A Hausrat, Torben Johann %A Yang, Jenq-Wei %A Ji, Haichao %A Schmarowski, Nikolai %A Endle, Heiko %A Liu, Xinfeng %A Li, Yunbo %A Böttche, Rahel %A Radyushkin, Konstantin %A Maric, Hans M %A Hoerder-Suabedissen, Anna %A Molnár, Zoltán %A Prouvot, Pierre-Hugues %A Trimbuch, Thorsten %A Ninnemann, Olaf %A Huai, Jisen %A Fan, Wei %A Visentin, Barbara %A Sabbadini, Roger %A Strømgaard, Kristian %A Stroh, Albrecht %A Luhmann, Heiko J %A Kneussel, Matthias %A Nitsch, Robert %A Vogt, Johannes %D 2016 %J Neuron %N 1 %P 126--142 %R https://doi.org/10.1016/j.neuron.2016.08.035 %T Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling %U http://www.sciencedirect.com/science/article/pii/S0896627316305281 %V 92 %X Summary Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2−/− thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2−/− animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.
• Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska; Dalby, Nils Ole; Doose, Sören; Sauer, Markus; Kneussel, Matthias; Strømgaard, Kristian in Nature Chemical Biology (2016). 13 153-.
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  γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

  @article{maric2016gephyrinbinding, abstract = {γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.}, author = {Maric, Hans Michael and Hausrat, Torben Johann and Neubert, Franziska and Dalby, Nils Ole and Doose, Sören and Sauer, Markus and Kneussel, Matthias and Strømgaard, Kristian}, journal = {Nature Chemical Biology}, keywords = {maric}, month = {nov}, pages = {153–}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission}, volume = 13, year = 2016 }

  %0 Journal Article %1 maric2016gephyrinbinding %A Maric, Hans Michael %A Hausrat, Torben Johann %A Neubert, Franziska %A Dalby, Nils Ole %A Doose, Sören %A Sauer, Markus %A Kneussel, Matthias %A Strømgaard, Kristian %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nature Chemical Biology %P 153-- %T Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission %U https://doi.org/10.1038/nchembio.2246 %V 13 %X γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

• Design and Synthesis of High-Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors Maric, Hans Michael; Kasaragod, Vikram Babu; Haugaard-Kedström, Linda; Hausrat, Torben Johann; Kneussel, Matthias; Schindelin, Hermann; Strømgaard, Kristian in Angewandte Chemie International Edition (2015). 54(2) 490–494.
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  Abstract Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8?nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin?receptor interplay.

  @article{maric2015design, abstract = {Abstract Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8?nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin?receptor interplay.}, author = {Maric, Hans Michael and Kasaragod, Vikram Babu and Haugaard-Kedström, Linda and Hausrat, Torben Johann and Kneussel, Matthias and Schindelin, Hermann and Strømgaard, Kristian}, booktitle = {Angewandte Chemie International Edition}, journal = {Angewandte Chemie International Edition}, keywords = {maric}, month = {jan}, number = 2, pages = {490–494}, publisher = {John Wiley & Sons, Ltd}, title = {Design and Synthesis of High-Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors}, volume = 54, year = 2015 }

  %0 Journal Article %1 maric2015design %A Maric, Hans Michael %A Kasaragod, Vikram Babu %A Haugaard-Kedström, Linda %A Hausrat, Torben Johann %A Kneussel, Matthias %A Schindelin, Hermann %A Strømgaard, Kristian %B Angewandte Chemie International Edition %D 2015 %I John Wiley & Sons, Ltd %J Angewandte Chemie International Edition %N 2 %P 490--494 %R 10.1002/anie.201409043 %T Design and Synthesis of High-Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors %U https://doi.org/10.1002/anie.201409043 %V 54 %X Abstract Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8?nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin?receptor interplay.

• Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin Maric, Hans Michael; Kasaragod, Vikram Babu; Hausrat, Torben Johann; Kneussel, Matthias; Tretter, Verena; Strømgaard, Kristian; Schindelin, Hermann in Nature Communications (2014). 5 5767-.
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  γ-Aminobutyric acid type A and glycine receptors (GABAARs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABAARs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin–GABAAR α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR β subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABAAR α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin–receptor interactions identify two residues as primary determinants for gephyrin’s subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABAARs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin–GABAAR interaction.

  @article{maric2014molecular, abstract = {γ-Aminobutyric acid type A and glycine receptors (GABAARs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABAARs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin–GABAAR α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR β subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABAAR α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin–receptor interactions identify two residues as primary determinants for gephyrin’s subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABAARs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin–GABAAR interaction.}, author = {Maric, Hans Michael and Kasaragod, Vikram Babu and Hausrat, Torben Johann and Kneussel, Matthias and Tretter, Verena and Strømgaard, Kristian and Schindelin, Hermann}, journal = {Nature Communications}, keywords = {maric}, month = {dec}, pages = {5767–}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin}, volume = 5, year = 2014 }

  %0 Journal Article %1 maric2014molecular %A Maric, Hans Michael %A Kasaragod, Vikram Babu %A Hausrat, Torben Johann %A Kneussel, Matthias %A Tretter, Verena %A Strømgaard, Kristian %A Schindelin, Hermann %D 2014 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nature Communications %P 5767-- %T Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin %U https://doi.org/10.1038/ncomms6767 %V 5 %X γ-Aminobutyric acid type A and glycine receptors (GABAARs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABAARs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin–GABAAR α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR β subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABAAR α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin–receptor interactions identify two residues as primary determinants for gephyrin’s subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABAARs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin–GABAAR interaction.
• Modulation of Gephyrin-Glycine Receptor Affinity by Multivalency Maric, Hans Michael; Kasaragod, Vikram Babu; Schindelin, Hermann in ACS Chem. Biol. (2014). 9(11) 2554–2562.
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  Gephyrin is a major determinant for the accumulation and anchoring of glycine receptors (GlyRs) and the majority of γ-aminobutyric acid type A receptors (GABAARs) at postsynaptic sites. Here we explored the interaction of gephyrin with a dimeric form of a GlyR β-subunit receptor-derived peptide. A 2 Å crystal structure of the C-terminal domain of gephyrin (GephE) in complex with a 15-residue peptide derived from the GlyR β-subunit defined the core binding site, which we targeted with the dimeric peptide. Biophysical analyses via differential scanning calorimetry (DSC), thermofluor, and isothermal titration calorimetry (ITC) demonstrated that this dimeric ligand is capable of binding simultaneously to two receptor binding sites and that this multivalency results in a 25-fold enhanced affinity. Our study therefore suggests that the oligomeric state of gephyrin and the number of gephyrin-binding subunits in the pentameric GABAARs and GlyRs together control postsynaptic receptor clustering.

  @article{maric2014modulation, abstract = {Gephyrin is a major determinant for the accumulation and anchoring of glycine receptors (GlyRs) and the majority of γ-aminobutyric acid type A receptors (GABAARs) at postsynaptic sites. Here we explored the interaction of gephyrin with a dimeric form of a GlyR β-subunit receptor-derived peptide. A 2 Å crystal structure of the C-terminal domain of gephyrin (GephE) in complex with a 15-residue peptide derived from the GlyR β-subunit defined the core binding site, which we targeted with the dimeric peptide. Biophysical analyses via differential scanning calorimetry (DSC), thermofluor, and isothermal titration calorimetry (ITC) demonstrated that this dimeric ligand is capable of binding simultaneously to two receptor binding sites and that this multivalency results in a 25-fold enhanced affinity. Our study therefore suggests that the oligomeric state of gephyrin and the number of gephyrin-binding subunits in the pentameric GABAARs and GlyRs together control postsynaptic receptor clustering.}, author = {Maric, Hans Michael and Kasaragod, Vikram Babu and Schindelin, Hermann}, booktitle = {ACS Chemical Biology}, journal = {ACS Chem. Biol.}, keywords = {maric}, month = {nov}, number = 11, pages = {2554–2562}, publisher = {American Chemical Society}, title = {Modulation of Gephyrin-Glycine Receptor Affinity by Multivalency}, volume = 9, year = 2014 }

  %0 Journal Article %1 maric2014modulation %A Maric, Hans Michael %A Kasaragod, Vikram Babu %A Schindelin, Hermann %B ACS Chemical Biology %D 2014 %I American Chemical Society %J ACS Chem. Biol. %N 11 %P 2554--2562 %R 10.1021/cb500303a %T Modulation of Gephyrin-Glycine Receptor Affinity by Multivalency %U https://doi.org/10.1021/cb500303a %V 9 %X Gephyrin is a major determinant for the accumulation and anchoring of glycine receptors (GlyRs) and the majority of γ-aminobutyric acid type A receptors (GABAARs) at postsynaptic sites. Here we explored the interaction of gephyrin with a dimeric form of a GlyR β-subunit receptor-derived peptide. A 2 Å crystal structure of the C-terminal domain of gephyrin (GephE) in complex with a 15-residue peptide derived from the GlyR β-subunit defined the core binding site, which we targeted with the dimeric peptide. Biophysical analyses via differential scanning calorimetry (DSC), thermofluor, and isothermal titration calorimetry (ITC) demonstrated that this dimeric ligand is capable of binding simultaneously to two receptor binding sites and that this multivalency results in a 25-fold enhanced affinity. Our study therefore suggests that the oligomeric state of gephyrin and the number of gephyrin-binding subunits in the pentameric GABAARs and GlyRs together control postsynaptic receptor clustering.

• Gephyrin, the enigmatic organizer at GABAergic synapses Tretter, Verena; Mukherjee, Jayanta; Maric, Hans; Schindelin, Hermann; Sieghart, Werner; Moss, Stephen in Frontiers in Cellular Neuroscience (2012). 6
  • [ Abstract ]
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  GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin. Gephyrin-interacting domains have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but is also a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signalling proteins into close spatial proximity.

  @article{tretter2012gephyrin, abstract = {GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin. Gephyrin-interacting domains have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but is also a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signalling proteins into close spatial proximity.}, author = {Tretter, Verena and Mukherjee, Jayanta and Maric, Hans and Schindelin, Hermann and Sieghart, Werner and Moss, Stephen}, journal = {Frontiers in Cellular Neuroscience}, keywords = {maric}, series = 23, title = {Gephyrin, the enigmatic organizer at GABAergic synapses}, volume = 6, year = 2012 }

  %0 Journal Article %1 tretter2012gephyrin %A Tretter, Verena %A Mukherjee, Jayanta %A Maric, Hans %A Schindelin, Hermann %A Sieghart, Werner %A Moss, Stephen %B 23 %D 2012 %J Frontiers in Cellular Neuroscience %T Gephyrin, the enigmatic organizer at GABAergic synapses %U https://www.frontiersin.org/article/10.3389/fncel.2012.00023 %V 6 %X GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin. Gephyrin-interacting domains have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but is also a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signalling proteins into close spatial proximity.

• Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site Maric, Hans-Michael; Mukherjee, Jayanta; Tretter, Verena; Moss, Stephen J; Schindelin, Hermann in The Journal of biological chemistry (2011). 286(49) 42105–42114.
  • [ Abstract ]
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  Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.

  @article{maric2011gephyrinmediated, abstract = {Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.}, author = {Maric, Hans-Michael and Mukherjee, Jayanta and Tretter, Verena and Moss, Stephen J and Schindelin, Hermann}, journal = {The Journal of biological chemistry}, keywords = {maric}, month = {dec}, number = 49, pages = {42105–42114}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site}, volume = 286, year = 2011 }

  %0 Journal Article %1 maric2011gephyrinmediated %A Maric, Hans-Michael %A Mukherjee, Jayanta %A Tretter, Verena %A Moss, Stephen J %A Schindelin, Hermann %D 2011 %I American Society for Biochemistry and Molecular Biology %J The Journal of biological chemistry %N 49 %P 42105--42114 %R 10.1074/jbc.M111.303412 %T Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site %U https://www.ncbi.nlm.nih.gov/pubmed/22006921 %V 286 %X Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.
• The Residence Time of GABA_ARs at Inhibitory Synapses Is Determined by Direct Binding of the Receptor α1 Subunit to Gephyrin Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine; Maric, Hans-Michael; Ramsden, Sarah; Tretter, Verena; Harvey, Kirsten; Davies, Paul A.; Triller, Antoine; Schindelin, Hermann; Moss, Stephen J. in J. Neurosci. (2011). 31(41) 14677-.
  • [ Abstract ]
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  The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABAARs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360–375 within the intracellular domain of this receptor subunit. Mutating residues 360–375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360–375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition.

  @article{mukherjee2011residence, abstract = {The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABAARs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360–375 within the intracellular domain of this receptor subunit. Mutating residues 360–375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360–375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition.}, author = {Mukherjee, Jayanta and Kretschmannova, Karla and Gouzer, Geraldine and Maric, Hans-Michael and Ramsden, Sarah and Tretter, Verena and Harvey, Kirsten and Davies, Paul A. and Triller, Antoine and Schindelin, Hermann and Moss, Stephen J.}, journal = {J. Neurosci.}, keywords = {maric}, month = {oct}, number = 41, pages = {14677–}, title = {The Residence Time of GABA_ARs at Inhibitory Synapses Is Determined by Direct Binding of the Receptor α1 Subunit to Gephyrin}, volume = 31, year = 2011 }

  %0 Journal Article %1 mukherjee2011residence %A Mukherjee, Jayanta %A Kretschmannova, Karla %A Gouzer, Geraldine %A Maric, Hans-Michael %A Ramsden, Sarah %A Tretter, Verena %A Harvey, Kirsten %A Davies, Paul A. %A Triller, Antoine %A Schindelin, Hermann %A Moss, Stephen J. %D 2011 %J J. Neurosci. %N 41 %P 14677-- %R 10.1523/JNEUROSCI.2001-11.2011 %T The Residence Time of GABA_ARs at Inhibitory Synapses Is Determined by Direct Binding of the Receptor α1 Subunit to Gephyrin %U http://www.jneurosci.org/content/31/41/14677.abstract %V 31 %X The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABAARs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360–375 within the intracellular domain of this receptor subunit. Mutating residues 360–375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360–375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition.
• Molecular Basis of the γ-Aminobutyric Acid A Receptor α3 Subunit Interaction with the Clustering Protein Gephyrin Tretter, Verena; Kerschner, Bernd; Milenkovic, Ivan; Ramsden, Sarah L.; Ramerstorfer, Joachim; Saiepour, Leila; Maric, Hans-Michael; Moss, Stephen J.; Schindelin, Hermann; Harvey, Robert J.; Sieghart, Werner; Harvey, Kirsten in Journal of Biological Chemistry (2011). 286(43) 37702–37711.
  • [ Abstract ]
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  The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABAA receptor (GABAAR) subtypes. We report a direct interaction between the GABAAR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3–M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABAAR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABAARs in recombinant systems. Interestingly, motifs responsible for interactions with GABAAR α2, GABAAR α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABAAR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABAAR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.

  @article{Tretter28102011, abstract = {The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABAA receptor (GABAAR) subtypes. We report a direct interaction between the GABAAR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3–M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABAAR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABAARs in recombinant systems. Interestingly, motifs responsible for interactions with GABAAR α2, GABAAR α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABAAR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABAAR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.}, author = {Tretter, Verena and Kerschner, Bernd and Milenkovic, Ivan and Ramsden, Sarah L. and Ramerstorfer, Joachim and Saiepour, Leila and Maric, Hans-Michael and Moss, Stephen J. and Schindelin, Hermann and Harvey, Robert J. and Sieghart, Werner and Harvey, Kirsten}, journal = {Journal of Biological Chemistry}, keywords = {maric}, number = 43, pages = {37702-37711}, title = {Molecular Basis of the γ-Aminobutyric Acid A Receptor α3 Subunit Interaction with the Clustering Protein Gephyrin}, volume = 286, year = 2011 }

  %0 Journal Article %1 Tretter28102011 %A Tretter, Verena %A Kerschner, Bernd %A Milenkovic, Ivan %A Ramsden, Sarah L. %A Ramerstorfer, Joachim %A Saiepour, Leila %A Maric, Hans-Michael %A Moss, Stephen J. %A Schindelin, Hermann %A Harvey, Robert J. %A Sieghart, Werner %A Harvey, Kirsten %D 2011 %J Journal of Biological Chemistry %N 43 %P 37702-37711 %R 10.1074/jbc.M111.291336 %T Molecular Basis of the γ-Aminobutyric Acid A Receptor α3 Subunit Interaction with the Clustering Protein Gephyrin %U http://www.jbc.org/content/286/43/37702.abstract %V 286 %X The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABAA receptor (GABAAR) subtypes. We report a direct interaction between the GABAAR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3–M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABAAR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABAARs in recombinant systems. Interestingly, motifs responsible for interactions with GABAAR α2, GABAAR α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABAAR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABAAR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.