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Key Points The postsynaptic density (PSD) is a large, dynamic protein assembly that orchestrates the densities and activities of both AMPA-type and NMDA-type glutamate receptors in excitatory synapses. The membrane-associated guanylate kinase (MAGUK) family of scaffold proteins, including Discs large homologues (DLGs) and membrane-associated guanylate kinase inverted (MAGI) proteins, are key organizers of PSDs. They act as an interface between the upstream membrane-spanning glutamate receptors and cell adhesion proteins and the downstream synapse-associated protein 90/postsynaptic density protein 95 (PSD95)-associated protein (SAPAP)–SRC homology 3 (SH3) and multiple ankyrin repeat domains protein (SHANK) complexes and the cytoskeleton. The guanylate kinase-like (GK) domain of MAGUKs binds to target proteins in a phosphorylation-dependent manner. This phosphorylation-dependent target recognition by MAGUKs suggests that the assembly of PSD is activity-dependent. Each MAGUK contains a highly conserved domain organization with PSD95–DLG1–Zonula occludens 1 (PDZ)–SH3–GK domains arranged in tandem. The PDZ–SH3–GK tandem forms a supramodule allowing the MAGUK scaffolds to bind to target proteins with high specificity as well as to cluster transmembrane ion channels and receptors. Biochemical and structural studies of MAGUKs have offered insights into why mutations affecting genes encoding MAGUKs and their target proteins may alter synaptic protein organization and lead to defects of synaptic development and signalling. Membrane-associated guanylate kinases (MAGUKs) are synaptic scaffold proteins involved in organizing protein complexes that are required for synaptic development and plasticity. Placing their focus on recent biochemical and structural data, Zhang and colleagues review the role of MAGUKs in synaptic protein complex formation and regulation. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffold proteins that are highly enriched in synapses and are responsible for organizing the numerous protein complexes required for synaptic development and plasticity. Mutations in genes encoding MAGUKs and their interacting proteins can cause a broad spectrum of human psychiatric disorders. Here, we review MAGUK-mediated synaptic protein complex formation and regulation by focusing on findings from recent biochemical and structural investigations. These mechanistic-based studies show that the formation of MAGUK-organized complexes is often directly regulated by protein phosphorylation, suggesting a close connection between neuronal activity and the assembly of dynamic protein complexes in synapses.

Human guanylate kinase (GMPK) as the sole enzyme for GDP biosynthesis plays pivotal roles in antiviral prodrug activation and tumorigenesis. Despite its biological significance, the catalytic mechanism remains poorly understood. Here, we resolve crystal structures of GMPK in free and GMP-bound form, revealing the interdomain motions of GMPBD and LID relative to the CORE domain. Biochemical assays demonstrate potassium’s dual functionality in substrate recognition and phosphoryl transfer catalysis. Structural analyses uncover intradomain conformational motion within the LID domain and essential interactions for ADP/ATP binding. Notably, the cooperative ATPγS binding potentiated by prior GMP binding are structurally elucidated. Three key complexes, pre-reaction state (GMP/ATPγS), transition state (AlF 4 - mimic), and post-reaction state (GDP/ADP), collectively delineate the reversible catalytic pathway. This comprehensive structural characterization of GMPK’s dynamic landscape establishes a foundation for developing conformation-specific inhibitors through structure-guided drug design. Human guanylate kinase (GMPK) is the sole enzyme for GDP biosynthesis contributing to antiviral prodrug activation and tumorigenesis. Here, authors profile the ligand-specific conformations of human GMPK along with the reversible catalytic pathway.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a membrane-associated guanylate kinase (MAGUK) protein that is associated with neurodevelopmental disorders. CASK is thought to have both pre- and postsynaptic functions, but the mechanism and consequences of its functions in the brain have yet to be elucidated, because homozygous CASK-knockout (CASK-KO) mice die before brain maturation. Taking advantage of the X-chromosome inactivation (XCI) mechanism, here we examined the synaptic functions of CASK-KO neurons in acute brain slices of heterozygous CASK-KO female mice. We also analyzed CASK-knockdown (KD) neurons in acute brain slices generated by in utero electroporation. Both CASK-KO and CASK-KD neurons showed a disruption of the excitatory and inhibitory (E/I) balance. We further found that the expression level of the N-methyl-d-aspartate receptor subunit GluN2B was decreased in CASK-KD neurons and that overexpressing GluN2B rescued the disrupted E/I balance in CASK-KD neurons. These results suggest that the down-regulation of GluN2B may be involved in the mechanism of the disruption of synaptic E/I balance in CASK-deficient neurons.

Synaptic plasticity is the ability of synapses to modulate the strength of neuronal connections; however, the molecular factors that regulate this feature are incompletely understood. Here, we demonstrated that mice lacking brain-specific angiogenesis inhibitor 1 (BAI1) have severe deficits in hippocampus-dependent spatial learning and memory that are accompanied by enhanced long-term potentiation (LTP), impaired long-term depression (LTD), and a thinning of the postsynaptic density (PSD) at hippocampal synapses. We showed that compared with WT animals, mice lacking Bai1 exhibit reduced protein levels of the canonical PSD component PSD-95 in the brain, which stems from protein destabilization. We determined that BAI1 prevents PSD-95 polyubiquitination and degradation through an interaction with murine double minute 2 (MDM2), the E3 ubiquitin ligase that regulates PSD-95 stability. Restoration of PSD-95 expression in hippocampal neurons in BAI1-deficient mice by viral gene therapy was sufficient to compensate for Bai1 loss and rescued deficits in synaptic plasticity. Together, our results reveal that interaction of BAI1 with MDM2 in the brain modulates PSD-95 levels and thereby regulates synaptic plasticity. Moreover, these results suggest that targeting this pathway has therapeutic potential for a variety of neurological disorders.

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

During critical periods, all cortical neural circuits are refined to optimize their functional properties. The prevailing notion is that the balance between excitation and inhibition determines the onset and closure of critical periods. In contrast, we show that maturation of silent glutamatergic synapses onto principal neurons was sufficient to govern the duration of the critical period for ocular dominance plasticity in the visual cortex of mice. Specifically, postsynaptic density protein-95 (PSD-95) was absolutely required for experience-dependent maturation of silent synapses, and its absence before the onset of critical periods resulted in lifelong juvenile ocular dominance plasticity. Loss of PSD-95 in the visual cortex after the closure of the critical period reinstated silent synapses, resulting in reopening of juvenile-like ocular dominance plasticity. Additionally, silent synapse-based ocular dominance plasticity was largely independent of the inhibitory tone, whose developmental maturation was independent of PSD-95. Moreover, glutamatergic synaptic transmission onto parvalbumin-positive interneurons was unaltered in PSD-95 KO mice. These findings reveal not only that PSD-95–dependent silent synapse maturation in visual cortical principal neurons terminates the critical period for ocular dominance plasticity but also indicate that, in general, once silent synapses are consolidated in any neural circuit, initial experience-dependent functional optimization and critical periods end.

Oncogenic protein E6 from Human Papilloma Virus 16 (HPV-16) mediates the degradation of Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1), throughout the interaction of its protein binding motif (PBM) with the Discs-large homologous regions 1 (PDZ1) domain of MAG1-1. Generic variation in the E6 gene that translates to changes in the protein’s amino acidic sequence modifies the interaction of E6 with the cellular protein MAGI-1. MAGI-1 is a scaffolding protein found at tight junctions of epithelial cells, where it interacts with a variety of proteins regulating signaling pathways. MAGI-1 is a multidomain protein containing two WW (rsp-domain-9), one guanylate kinase-like, and six PDZ domains. PDZ domains played an important role in the function of MAGI-1 and served as targets for several viral proteins including the HPV-16 E6. The aim of this work was to evaluate, with an in silico approach, employing molecular dynamics simulation and protein–protein docking, the interaction of the intragenic variants E-G350 (L83V), E-C188/G350 (E29Q/L83V), E-A176/G350 (D25N/L83V), E6-AAa (Q14H/H78Y/83V) y E6-AAc (Q14H/I27RH78Y/L83V) and E6-reference of HPV-16 with MAGI-1. We found that variants E-G350, E-C188/G350, E-A176/G350, AAa and AAc increase their affinity to our two models of MAGI-1 compared to E6-reference.

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a neurodevelopmental disorder caused by the deficiency of the X-chromosomal gene CASK. However, the molecular mechanisms by which CASK deficiency causes cerebellar hypoplasia in this syndrome remain elusive. In this study, we used CASK knockout (KO) mice as models for MICPCH syndrome and investigated the effect of CASK mutants. Female CASK heterozygote KO mice replicate the progressive cerebellar hypoplasia observed in MICPCH syndrome. CASK KO cultured cerebellar granule (CG) cells show progressive cell death that can be rescued by co-infection with lentivirus expressing wild-type CASK. Rescue experiments with CASK deletion mutants identify that the CaMK, PDZ, and SH3, but not L27 and guanylate kinase domains of CASK are required for the survival of CG cells. We identify missense mutations in the CaMK domain of CASK derived from human patients that fail to rescue the cell death of cultured CASK KO CG cells. Machine learning-based structural analysis using AlphaFold 2.2 predicts that these mutations disrupt the structure of the binding interface with Liprin-α2. These results suggest that the interaction with Liprin-α2 via the CaMK domain of CASK may be involved in the pathophysiology of cerebellar hypoplasia in MICPCH syndrome.

Traumatic fear memories are highly durable but also dynamic, undergoing repeated reactivation and rehearsal over time. Although overly persistent fear memories underlie anxiety disorders, such as posttraumatic stress disorder, the key neural and molecular mechanisms underlying fear memory durability remain unclear. Postsynaptic density 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory. Using a loss-of-function mutant mouse lacking the guanylate kinase domain of PSD-95 (PSD-95 GK ), we analyzed the contribution of PSD-95 to fear memory formation and retrieval, and sought to identify the neural basis of PSD-95-mediated memory maintenance using ex vivo immediate-early gene mapping, in vivo neuronal recordings and viral-mediated knockdown (KD) approaches. We show that PSD-95 is dispensable for the formation and expression of recent fear memories, but essential for the formation of precise and flexible fear memories and for the maintenance of memories at remote time points. The failure of PSD-95 GK mice to retrieve remote cued fear memory was associated with hypoactivation of the infralimbic (IL) cortex (but not the anterior cingulate cortex (ACC) or prelimbic cortex), reduced IL single-unit firing and bursting, and attenuated IL gamma and theta oscillations. Adeno-associated virus-mediated PSD-95 KD in the IL, but not the ACC, was sufficient to impair recent fear extinction and remote fear memory, and remodel IL dendritic spines. Collectively, these data identify PSD-95 in the IL as a critical mechanism supporting the durability of fear memories over time. These preclinical findings have implications for developing novel approaches to treating trauma-based anxiety disorders that target the weakening of overly persistent fear memories.

A membrane skeletal molecular complex, protein 4.1G–membrane palmitoylated protein 6 (MPP6)–Lin7–cell adhesion molecule 4 (CADM4), is incorporated in Schwann cells, especially in Schmidt–Lanterman incisures (SLIs), in the mouse peripheral nervous system (PNS). MPP6, Lin7, and CADM4 are transported to SLIs by 4.1G. In this study, we created MPP6-deficient mice and evaluated myelin structure and MPP6 protein complexes. In SLIs in MPP6-deficient nerves, Lin7 was rarely detected by immunohistochemistry and western blotting, but the localization and amount of CADM4 and 4.1G were not altered. Motor activity was not significantly impaired in a tail-suspension test, but the sciatic nerves of MPP6-deficient mice had thicker myelin in internodes by electron microscopy compared to that of wild-type mice. These results indicate that the MPP6–Lin7 complex regulates myelin formation.