Long-read and short-read RNA-seq reveal the transcriptional regulation characteristics of PICK1 in Baoshan pig testis.

PICK1 plays a crucial role in mammalian spermatogenesis. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine PICK1 expression patterns in adult Baoshan pig (BS) testes. We identified the most important transcript ENSSSCT00000000120 of PICK1, obtaining its full-length coding sequence (CDS) spanning 1254 bp. Gene structure analysis located PICK1 on pig chromosome 5 with 14 exons. Protein structure analysis reflected that PICK1 consisted of 417 amino acids containing two conserved domains, PDZ and BAR_PICK1. Phylogenetic analysis underscored the evolutionary conservation and homology of PICK1 across different mammalian species. Evaluation of protein interaction network, KEGG, and GO pathways implied that interacted with 50 proteins, predominantly involved in glutamatergic synapses, amphetamine addiction, neuroactive ligand-receptor interactions, dopaminergic synapses, and synaptic vesicle recycling, and PICK1 exhibited significant correlation with DLG4 and TBC1D20. Functional annotation identified that PICK1 was involved in 9 GOs, including seven cellular components and two molecular functions. ceRNA network analysis suggested BS PICK1 was regulated by seven miRNA targets. Moreover, qPCR expression analysis across 15 tissues highlighted that PICK1 was highly expressed in the bulbourethral gland and testis. Subcellular localization analysis in ST (Swine Tesits) cells demonstrated that PICK1 significantly localized within the cytoplasm. Overall, our findings shed new light on PICK1's role in BS reproduction, providing a foundation for further functional studies of PICK1.

Utilizing AfDesign for Developing a Small Molecule Inhibitor of PICK 1-PDZ.

INTRODUCTION: The PICK1 PDZ domain has been identified as a potential drug target for neurological disorders. After many years of effort, a few inhibitors, such as TAT-C5 and mPD5, have been discovered experimentally to bind to the PDZ domain with a relatively high binding affinity. With the rapid growth of computational research, there is an urgent need for more efficient computational methods to design viable ligands that target proteins. METHOD: Recently, a newly developed program called AfDesign (part of ColabDesign) at https:// github.com/sokrypton/ColabDesign), an open-source software built on AlphaFold, has been suggested to be capable of generating ligands that bind to targeted proteins, thus potentially facilitating the ligand development process. To evaluate the performance of this program, we explored its ability to target the PICK1 PDZ domain, given our current understanding of it. We found that the designated length of the ligand and the number of recycles play vital roles in generating ligands with optimal properties. RESULTS: Utilizing AfDesign with a sequence length of 5 for the ligand produced the highest comparable ligands to that of prior identified ligands. Moreover, these designed ligands displayed significantly lower binding energy compared to manually created sequences. CONCLUSION: This work demonstrated that AfDesign can potentially be a powerful tool to facilitate the exploration of the ligand space for the purpose of targeting PDZ domains.

ICA1 affects APP processing through the PICK1-PKCα signaling pathway.

AIMS: Islet cell autoantigen 1 (ICA1) is involved in autoimmune diseases and may affect synaptic plasticity as a neurotransmitter. Databases related to Alzheimer's disease (AD) have shown decreased ICA1 expression in patients with AD. However, the role of ICA1 in AD remains unclear. Here, we report that ICA1 expression is decreased in the brains of patients with AD and an AD mouse model. RESULTS: The ICA1 increased the expression of amyloid precursor protein (APP), disintegrin and metalloprotease 10 (ADAM10), and disintegrin and metalloprotease 17 (ADAM17), but did not affect protein half-life or mRNA levels. Transcriptome sequencing analysis showed that ICA1 regulates the G protein-coupled receptor signaling pathway. The overexpression of ICA1 increased PKCα protein levels and phosphorylation. CONCLUSION: Our results demonstrated that ICA1 shifts APP processing to non-amyloid pathways by regulating the PICK1-PKCα signaling pathway. Thus, this study suggests that ICA1 is a novel target for the treatment of AD.

Non-coding RNA yREX3 from human extracellular vesicles exerts macrophage-mediated cardioprotection via a novel gene-methylating mechanism.

BACKGROUND AND AIMS: Extracellular vesicles (EVs) secreted by cardiosphere-derived cells exert immunomodulatory effects through the transmission of small non-coding RNAs. METHODS: The mechanism and role of yREX3, a small Y RNA abundant in EVs in myocardial injury, was investigated. RESULTS: yREX3 attenuates cardiac ischaemic injury by selective DNA methylation. Synthetic yREX3 encapsulated in lipid nanoparticles triggers broad transcriptomic changes in macrophages, localizes to the nucleus, and mediates epigenetic silencing of protein interacting with C kinase-1 (Pick1) through methylation of upstream CpG sites. Moreover, yREX3 interacts with polypyrimidine tract binding protein 3 (PTBP3) to methylate the Pick1 gene locus in a DNA methyltransferase-dependent manner. Suppression of Pick1 in macrophages potentiates Smad3 signalling and enhances efferocytosis, minimizing heart necrosis in rats with myocardial infarction. Adoptive transfer of Pick1-deficient macrophages recapitulates the cardioprotective effects of yREX3 in vivo. CONCLUSIONS: These findings highlight the role of a small Y RNA mined from EVs with a novel gene-methylating mechanism.

Unconventional PDZ Recognition Revealed in α7 nAChR-PICK1 Complexes.

PDZ domains are modular domains that conventionally bind to C terminal or internal motifs of target proteins to control cellular functions through the regulation of protein complex assemblies. Almost all reported structures of PDZ-target protein complexes rely on fragments or peptides as target proteins. No intact target protein complexed with PDZ was structurally characterized. In this study, we used NMR spectroscopy and other biochemistry and biophysics tools to uncover insights into structural coupling between the PDZ domain of protein interacting with C-kinase 1 (PICK1) and α7 nicotinic acetylcholine receptors (α7 nAChR). Notably, the intracellular domains of both α7 nAChR and PICK1 PDZ exhibit a high degree of plasticity in their coupling. Specifically, the MA helix of α7 nAChR interacts with residues lining the canonical binding site of the PICK1 PDZ, while flexible loops also engage in protein-protein interactions. Both hydrophobic and electrostatic interactions mediate the coupling. Overall, the resulting structure of the α7 nAChR-PICK1 complex reveals an unconventional PDZ binding mode, significantly expanding the repertoire of functionally important PDZ interactions.

Multi-omics study identifies that PICK1 deficiency causes male infertility by inhibiting vesicle trafficking in Sertoli cells.

BACKGROUND: Infertility affects approximately 10-15% of reproductive-age men worldwide, and genetic causes play a role in one-third of cases. As a Bin-Amphiphysin-Rvs (BAR) domain protein, protein interacting with C-kinase 1 (PICK1) deficiency could lead to impairment of acrosome maturation. However, its effects on auxiliary germ cells such as Sertoli cells are unknown. PURPOSE: The present work was aimed to use multi-omics analysis to research the effects of PICK1 deficiency on Sertoli cells and to identify effective biomarkers to distinguish fertile males from infertile males caused by PICK1 deficiency. METHODS: Whole-exome sequencing (WES) was performed on 20 infertility patients with oligozoospermia to identify pathogenic PICK1 mutations. Multi-omics analysis of a PICK1 knockout (KO) mouse model was utilized to identify pathogenic mechanism. Animal and cell function experiments of Sertoli cell-specific PICK1 KO mouse were performed to verify the functional impairment of Sertoli cells. RESULTS: Two loss-of-function deletion mutations c.358delA and c.364delA in PICK1 resulting in transcription loss of BAR functional domain were identified in infertility patients with a specific decrease in serum inhibin B, indicating functional impairment of Sertoli cells. Multi-omics analysis of PICK1 KO mouse illustrated that targeted genes of differentially expressed microRNAs and mRNAs are significantly enriched in the negative regulatory role in the vesicle trafficking pathway, while metabolomics analysis showed that the metabolism of amino acids, lipids, cofactors, vitamins, and endocrine factors changed. The phenotype of PICK1 KO mouse showed a reduction in testis volume, a decreased number of mature spermatozoa and impaired secretory function of Sertoli cells. In vitro experiments confirmed that the expression of growth factors secreted by Sertoli cells in PICK1 conditional KO mouse such as Bone morphogenetic protein 4 (BMP4) and Fibroblast growth factor 2 (FGF2) were decreased. CONCLUSIONS: Our study attributed male infertility caused by PICK1 deficiency to impaired vesicle-related secretory function of Sertoli cells and identified a variety of significant candidate biomarkers for male infertility induced by PICK1 deficiency.

Post-feeding transcriptomics reveals essential genes expressed in the midgut of the desert locust.

The digestive tract constitutes an important interface between an animal's internal and external environment. In insects, available gut transcriptome studies are mostly exploratory or look at changes upon infection or upon exposure to xenobiotics, mainly performed in species belonging to holometabolan orders, such as Diptera, Lepidoptera or Coleoptera. By contrast, studies focusing on gene expression changes after food uptake and during digestion are underrepresented. We have therefore compared the gene expression profiles in the midgut of the desert locust, Schistocerca gregaria, between three different time points after feeding, i.e., 24 h (no active digestion), 10 min (the initial stage of feeding), and 2 h (active food digestion). The observed gene expression profiles were consistent with the polyphagous herbivorous lifestyle of this hemimetabolan (orthopteran) species. Our study reveals the upregulation of 576 genes 2 h post-feeding. These are mostly predicted to be associated with digestive physiology, such as genes encoding putative digestive enzymes or nutrient transporters, as well as genes putatively involved in immunity or in xenobiotic metabolism. The 10 min time point represented an intermediate condition, suggesting that the S. gregaria midgut can react rapidly at the transcriptional level to the presence of food. Additionally, our study demonstrated the critical importance of two transcripts that exhibited a significant upregulation 2 h post-feeding: the vacuolar-type H(+)-ATPase and the sterol transporter Niemann-Pick 1b protein, which upon RNAi-induced knockdown resulted in a marked increase in mortality. Their vital role and accessibility via the midgut lumen may make the encoded proteins promising insecticidal target candidates, considering that the desert locust is infamous for its huge migrating swarms that can devastate the agricultural production in large areas of Northern Africa, the Middle East, and South Asia. In conclusion, the transcriptome datasets presented here will provide a useful and promising resource for studying the midgut physiology of S. gregaria, a socio-economically important pest species.

Circulating ovarian hormones interact with protein interacting with C kinase (PICK1) within the medial prefrontal cortex to influence cocaine seeking in female mice.

Protein interacting with C kinase 1 (PICK1) is an AMPA receptor binding protein that works in conjunction with glutamate receptor interacting protein (GRIP) to balance the number of GluA2-containing AMPARs in the synapse. In male mice, disrupting PICK1 in the medial prefrontal cortex (mPFC) leads to a decrease in cue-induced cocaine seeking and disrupting GRIP in the mPFC has the opposing effect, consistent with other evidence that removal of GluA2-containing AMPARs potentiates reinstatement. However, PICK1 does not seem to play the same role in female mice, as knockdown of either PICK1 or GRIP in the mPFC leads to similar increases in cue-induced cocaine seeking. These previous findings indicate that the role of PICK1 in the prefrontal cortex is sex specific. The goal of the current study was to examine whether ovarian hormones contribute to the effect of prefrontal PICK1 knockdown on reinstatement of cocaine seeking. While we replicated the increased cue-induced cocaine seeking in prefrontal PICK1 knockdown sham mice, we did not see any difference between the GFP control mice and PICK1 knockdowns following ovariectomy. However, this effect was driven primarily by an increase in cocaine seeking in ovariectomized GFP control mice while there was no effect ovariectomy in PICK1 knockdown mice. Taken together, these findings suggest that circulating ovarian hormones interact with the effects of PICK1 on cue-induced reinstatement.

ICA69 regulates activity-dependent synaptic strengthening and learning and memory.

Long-term potentiation (LTP) is one of the major cellular mechanisms for learning and memory. Activity-dependent increases in surface AMPA receptors (AMPARs) are important for enhanced synaptic efficacy during LTP. Here, we report a novel function of a secretory trafficking protein, ICA69, in AMPAR trafficking, synaptic plasticity, and animal cognition. ICA69 is first identified as a diabetes-associated protein well characterized for its function in the biogenesis of secretory vesicles and trafficking of insulin from ER, Golgi to post-Golgi in pancreatic beta cells. In the brain, ICA69 is found in the AMPAR protein complex through its interaction with PICK1, which binds directly to GluA2 or GluA3 AMPAR subunits. Here, we showed that ICA69 regulates PICK1's distribution in neurons and stability in the mouse hippocampus, which in turn can impact AMPAR function in the brain. Biochemical analysis of postsynaptic density (PSD) proteins from hippocampi of mice lacking ICA69 (Ica1 knockout) and their wild-type littermates revealed comparable AMPAR protein levels. Electrophysiological recording and morphological analysis of CA1 pyramidal neurons from Ica1 knockout also showed normal AMPAR-mediated currents and dendrite architecture, indicating that ICA69 does not regulate synaptic AMPAR function and neuron morphology at the basal state. However, genetic deletion of ICA69 in mice selectively impairs NMDA receptor (NMDAR)-dependent LTP but not LTD at Schaffer collateral to CA1 synapses, which correlates with behavioral deficits in tests of spatial and associative learning and memory. Together, we identified a critical and selective role of ICA69 in LTP, linking ICA69-mediated synaptic strengthening to hippocampus-dependent learning and memory.

PICK1-Deficient Mice Maintain Their Glucose Tolerance During Diet-Induced Obesity.

Context: Metabolic disorders such as obesity represent a major health challenge. Obesity alone has reached epidemic proportions, with at least 2.8 million people worldwide dying annually from diseases caused by overweight or obesity. The brain-metabolic axis is central to maintain homeostasis under metabolic stress via an intricate signaling network of hormones. Protein interacting with C kinase 1 (PICK1) is important for the biogenesis of various secretory vesicles, and we have previously shown that PICK1-deficient mice have impaired secretion of insulin and growth hormone. Objective: The aim was to investigate how global PICK1-deficient mice respond to high-fat diet (HFD) and assess its role in insulin secretion in diet-induced obesity. Methods: We characterized the metabolic phenotype through assessment of body weight, composition, glucose tolerance, islet morphology insulin secretion in vivo, and glucose-stimulated insulin secretion ex vivo. Results: PICK1-deficient mice displayed similar weight gain and body composition as wild-type (WT) mice following HFD. While HFD impaired glucose tolerance of WT mice, PICK1-deficient mice were resistant to further deterioration of their glucose tolerance compared with already glucose-impaired chow-fed PICK1-deficient mice. Surprisingly, mice with ß-cell-specific knockdown of PICK1 showed impaired glucose tolerance both on chow and HFD similar to WT mice. Conclusion: Our findings support the importance of PICK1 in overall hormone regulation. However, importantly, this effect is independent of the PICK1 expression in the ß-cell, whereby global PICK1-deficient mice resist further deterioration of their glucose tolerance following diet-induced obesity.

Investigating the Mechanical Properties and Flexibility of N-BAR Domains in PICK1 by Molecular Dynamics Simulations.

INTRODUCTION: The proteins of the Bin/Amphiphysin/Rvs167 (BAR) domain superfamily are believed to induce membrane curvature. PICK1 is a distinctive protein that consists of both a BAR and a PDZ domain, and it has been associated with numerous diseases. It is known to facilitate membrane curvature during receptor-mediated endocytosis. In addition to understanding how the BAR domain facilitates membrane curvature, it's particularly interesting to unravel the hidden links between the structural and mechanical properties of the PICK1 BAR domain. METHODS: This paper employs steered molecular dynamics (SMD) to investigate the mechanical properties associated with structural changes in the PICK1 BAR domains. RESULTS: Our findings suggest that not only do helix kinks assist in generating curvature of BAR domains, but they may also provide the additional flexibility required to initiate the binding between BAR domains and the membrane. CONCLUSION: We have observed a complex interaction network within the BAR monomer and at the binding interface of the two BAR monomers. This network is crucial for maintaining the mechanical properties of the BAR dimer. Owing to this interaction network, the PICK1 BAR dimer exhibits different responses to external forces applied in opposite directions.

A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis.

Group I metabotropic glutamate receptors (mGluRs) play important roles in many neuronal processes and are believed to be involved in synaptic plasticity underlying the encoding of experience, including classic paradigms of learning and memory. These receptors have also been implicated in various neurodevelopmental disorders, such as Fragile X syndrome and autism. Internalization and recycling of these receptors in the neuron are important mechanisms to regulate the activity of the receptor and control the precise spatiotemporal localization of these receptors. Applying a "molecular replacement" approach in hippocampal neurons derived from mice, we demonstrate a critical role for protein interacting with C kinase 1 (PICK1) in regulating the agonist-induced internalization of mGluR1. We show that PICK1 specifically regulates the internalization of mGluR1, but it does not play any role in the internalization of the other member of group I mGluR family, mGluR5. Various regions of PICK1 viz., the N-terminal acidic motif, PDZ domain, and BAR domain play important roles in the agonist-mediated internalization of mGluR1. Finally, we demonstrate that PICK1-mediated internalization of mGluR1 is critical for the resensitization of the receptor. Upon knockdown of endogenous PICK1, mGluR1s stayed on the cell membrane as inactive receptors, incapable of triggering the MAP kinase signaling. They also could not induce AMPAR endocytosis, a cellular correlate for mGluR-dependent synaptic plasticity. Thus, this study unravels a novel role for PICK1 in the agonist-mediated internalization of mGluR1 and mGluR1-mediated AMPAR endocytosis that might contribute to the function of mGluR1 in neuropsychiatric disorders.

Truncating PICK1 Variant Identified in Azoospermia Affected Mitochondrial Dysfunction in Knockout Mice.

OBJECTIVE: The protein interacting with C kinase 1 (PICK1) plays a critical role in vesicle trafficking, and its deficiency in sperm cells results in abnormal vesicle trafficking from Golgi to acrosome, which eventually disrupts acrosome formation and leads to male infertility. METHODS: An azoospermia sample was filtered, and the laboratory detection and clinical phenotype indicated typical azoospermia in the patient. We sequenced all of the exons in the PICK1 gene and found that there was a novel homozygous variant in the PICK1 gene, c.364delA (p.Lys122SerfsX8), and this protein structure truncating variant seriously affected the biological function. Then we constructed a PICK1 knockout mouse model using clustered regularly interspaced short palindromic repeat cutting technology (CRISPRc). RESULTS: The sperm from PICK1 knockout mice showed acrosome and nucleus abnormalities, as well as dysfunctional mitochondrial sheath formation. Both the total sperm and motility sperm counts were decreased in the PICK1 knockout mice compared to wild-type mice. Moreover, the mitochondrial dysfunction was verified in the mice. These defects in the male PICK1 knockout mice may have eventually led to complete infertility. CONCLUSION: The c.364delA novel variant in the PICK1 gene associated with clinical infertility, and pathogenic variants in the PICK1 may cause azoospermia or asthenospermia by impairing mitochondrial function in both mice and humans.

Hypoxia-induced degradation of PICK1 by RBCK1 promotes the proliferation of nasopharyngeal carcinoma cells.

AIMS: Hypoxia is an important feature of nasopharyngeal carcinoma (NPC). "Protein interacting with PRKCA 1" (PICK1) is commonly downregulated in human malignancies and is functionally related to poor prognosis. However, there is a limited understanding of the upstream mechanisms regulating PICK1 currently. MAIN METHODS: PICK1 and HIF-1α expression levels were analyzed by Immunohistochemistry (IHC), western blotting, and quantitative real-time PCR assay. Protein stability and ubiquitin assays were used to investigate PICK1 protein degradation. Immunofluorescence and co-immunoprecipitation assays were used to demonstrate the interaction between RBCK1 and PICK1. Gene knockdown by siRNA transfection was used to investigate the role of HIF-1α and RBCK1 in hypoxia-induced PICK1 degradation. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) assays and subcutaneous xenograft nude models were used to explore the roles of RBCK1 and PICK1 in NPC cell proliferation. KEY FINDINGS: PICK1 expression in NPC tissue was negatively relative to that of HIF-1α. HIF-1α downregulated PICK1 expression by facilitating its ubiquitination by the E3 ligases RANBP2-type and C3HC4-type zinc finger containing 1 (RBCK1), thereby enhancing proteasome-mediated PICK1 degradation. RBCK1 knockdown inhibited NPC cell proliferation, which was ameliorated by double knockdown of RBCK1/PICK1. SIGNIFICANCE: These data provide evidence for an NPC cell adaptation mechanism to hypoxia, where HIF-1α regulates RBCK1, which targets PICK1 for degradation to promote cell proliferation.

Exosomal miR-3131 derived from endothelial cells with KRAS mutation promotes EndMT by targeting PICK1 in brain arteriovenous malformations.

AIMS: To explore the underlying mechanism by which low-frequency KRAS mutations result in extensive EndMT occurrence. METHODS: Exosomes derived from primarily cultured brain arteriovenous malformation (bAVMs) and human umbilical vein endothelial cells (HUVECs) transfected with KRASG12D , KRASWT , or KRASNC lentiviruses were isolated, and their effects on HUVECs were identified by western blotting and immunofluorescence staining. The expression levels of exosomal microRNAs (miRNAs) were evaluated by miRNA microarray, followed by functional experiments on miR-3131 and detection of its downstream target, and miR-3131 inhibitor in reversing the EndMT process induced by KRASG12D -transfected HUVECs and bAVM endothelial cells (ECs) were explored. RESULTS: Exosomes derived from KRASG12D bAVM ECs and KRASG12D -transfected HUVECs promoted EndMT in HUVECs. MiR-3131 levels were highest in the exosomes of KRASG12D -transfected HUVECs, and HUVECs transfected with the miR-3131 mimic acquired mesenchymal phenotypes. RNA-seq and dual-luciferase reporter assays revealed that PICK1 is the direct downstream target of miR-3131. Exosomal miR-3131 was highly expressed in KRASG12D bAVMexos compared with non-KRAS-mutant bAVMexos or HUVECexos . Finally, a miR-3131 inhibitor reversed EndMT in HUVECs treated with exosomes or the supernatant of KRASG12D -transfected HUVECs and KRASG12D bAVM ECs. CONCLUSION: Exosomal miR-3131 promotes EndMT in KRAS-mutant bAVMs, and miR-3131 might be a potential biomarker and therapeutic target in KRASG12D -mutant bAVMs.

The M1 muscarinic acetylcholine receptor regulates the surface expression of the AMPA receptor subunit GluA2 via PICK1.

Muscarinic acetylcholine receptors (mAChRs) have been shown to play significant roles in the regulation of normal cognitive processes in the hippocampus, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are also involved in these processes. This study aims to explore the mAChR-mediated regulation of AMPARs GluA2 trafficking and to reveal the key proteins and the signaling cascade involved in this process. Primary hippocampal neurons, as cell models, were treated with agonist 77-LH-28-1 and antagonist VU0255035, Fsc231, and APV. C57BL/6J male mice were stereotactically injected with 77-LH-28-1 and Fsc231 to obtain hippocampal slices. The trafficking of GluA2 was detected by surface biotinylation and immunostaining. Activation of M1 mAChRs promoted endocytosis and decreased the postsynaptic localization of the AMPA receptor subunit GluA2 and that phosphorylation of GluA2 at Ser880 was increased by M1 mAChR activity. Fsc231 blocked the endocytosis and postsynaptic localization of GluA2 induced by 77-LH-28-1 without affecting the phosphorylation of Ser880. PICK1 was required for M1 mAChR-mediated GluA2 endocytosis and downstream of phosphorylation of GluA2-Ser880, and the PICK1-GluA2 interaction was essential for M1 mAChR-mediated postsynaptic expression of GluA2. Taken together, our results show a functional correlation of M1 mAChRs with GluA2 and the role of PICK1 in their interplay. The schematic diagram for the modulation of GluA2 trafficking by M1 mAChRs. Activation of M1 mAChRs induces PKC activation, and the interaction of PICK1-GluA2 determines the endocytosis and postsynaptic localization of GluA2.

AMPA GluA2 subunit competitive inhibitors for PICK1 PDZ domain: Pharmacophore-based virtual screening, molecular docking, molecular dynamics simulation, and ADME studies.

PICK1 (Protein interacting with C kinase-1) plays a key role in the regulation of intracellular trafficking of AMPA GluA2 subunit that is linked with synaptic plasticity. PICK1 is a scaffolding protein and binds numerous proteins through its PDZ domain. Research showed that synaptic plasticity is altered upon disrupting the GluA2-PDZ interactions. Inhibiting PDZ and GluA2 binding lead to beneficial effects in the cure of neurological diseases thus, targeting PDZ domain is proposed as a novel therapeutic target in such diseases. For this, various classes of synthetic peptides were tested. Though small organic molecules have been utilized to prevent these interactions, the number of such molecules is inadequate. Hence, in this study, ten molecular libraries containing large number of molecules were screened against the PDZ domain using pharmacophore-based virtual screening to find the best hits for the PDZ domain. Molecular docking and molecular dynamics simulation studies revealed that Hit_II is a potent inhibitor for the PDZ domain and confirm the allosteric nature of Hit_III. Additionally, ADME analysis suggests the drug-likeness of both Hit_II and Hit_III. This study suggests that tested hits may have potency against the PDZ domain and can be considered effective to treat neurological disorders.Communicated by Ramaswamy H. Sarma.

Modulation of Hippocampal Network Oscillation by PICK1-Dependent Cell Surface Expression of mGlu3 Receptors.

Metabotropic glutamate receptor Type 3 (mGlu3) controls the sleep/wake architecture, which plays a role in the glutamatergic pathophysiology of schizophrenia. Interestingly, mGlu3 receptor expression is decreased in the brain of schizophrenic patients. However, little is known about the molecular mechanisms regulating mGlu3 receptors at the cell membrane. Subcellular receptor localization is strongly dependent on protein-protein interactions. Here we show that mGlu3 interacts with PICK1 and that this scaffolding protein is important for mGlu3 surface expression and function in hippocampal primary cultures. Disruption of their interaction via an mGlu3 C-terminal mimicking peptide or an inhibitor of the PDZ domain of PICK1 altered the functional expression of mGlu3 receptors in neurons. We next investigated the impact of disrupting the mGlu3-PICK1 interaction on hippocampal theta oscillations in vitro and in vivo in WT male mice. We found a decreased frequency of theta oscillations in organotypic hippocampal slices, similar to what was previously observed in mGlu3 KO mice. In addition, hippocampal theta power was reduced during rapid eye movement sleep, non-rapid eye movement (NREM) sleep, and wake states after intraventricular administration of the mGlu3 C-terminal mimicking peptide. Targeting the mGlu3-PICK1 complex could thus be relevant to the pathophysiology of schizophrenia.SIGNIFICANCE STATEMENT Dysregulation of the glutamatergic system might play a role in the pathophysiology of schizophrenia. Metabotropic glutamate receptors Type 3 (mGlu3) have been proposed as potential targets for schizophrenia. Understanding the molecular mechanisms regulating mGlu3 receptor at the cell membrane is critical toward comprehending how their dysfunction contributes to the pathogenesis of schizophrenia. Here we describe that the binding of the signaling and scaffolding protein PICK1 to mGlu3 receptors is important for their localization and physiological functions. The identification of new proteins that associate specifically to mGlu3 receptors will advance our understanding of the regulatory mechanisms associated with their targeting and function and ultimately might provide new therapeutic strategies to counter these psychiatric conditions.

Three Binding Conformations of BIO124 in the Pocket of the PICK1 PDZ Domain.

The PDZ family has drawn attention as possible drug targets because of the domains' wide ranges of function and highly conserved binding pockets. The PICK1 PDZ domain has been proposed as a possible drug target because the interactions between the PICK1 PDZ domain and the GluA2 subunit of the AMPA receptor have been shown to progress neurodegenerative diseases. BIO124 has been identified as a sub µM inhibitor of the PICK1-GluA2 interaction. Here, we use all-atom molecular dynamics simulations to reveal the atomic-level interaction pattern between the PICK1 PDZ domain and BIO124. Our simulations reveal three unique binding conformations of BIO124 in the PICK1 PDZ binding pocket, referred to here as state 0, state 1, and state 2. Each conformation is defined by a unique hydrogen bonding network and a unique pattern of hydrophobic interactions between BIO124 and the PICK1 PDZ domain. Interestingly, each conformation of BIO124 results in different dynamic changes to the PICK1 PDZ domain. Unlike states 1 and 2, state 0 induces dynamic coupling between BIO124 and the αA helix. Notably, this dynamic coupling with the αA helix is similar to what has been observed in other PDZ-ligand complexes. Our analysis indicates that the interactions formed between BIO124 and I35 may be the key to inducing dynamic coupling with the αA helix. Lastly, we suspect that the conformational shifts observed in our simulations may affect the stability and thus the overall effectiveness of BIO124. We propose that a physically larger inhibitor may be necessary to ensure sufficient interactions that permit stable binding between a drug and the PICK1 PDZ domain.

The adaptor protein PICK1 targets the sorting receptor SorLA.

SorLA is a member of the Vps10p-domain (Vps10p-D) receptor family of type-I transmembrane proteins conveying neuronal endosomal sorting. The extracellular/luminal moiety of SorLA has a unique mosaic domain composition and interacts with a large number of different and partially unrelated ligands, including the amyloid precursor protein as well as amyloid-ß. Several studies support a strong association of SorLA with sporadic and familial forms of Alzheimer's disease (AD). Although SorLA seems to be an important factor in AD, the large number of different ligands suggests a role as a neuronal multifunctional receptor with additional intracellular sorting capacities. Therefore, understanding the determinants of SorLA's subcellular targeting might be pertinent for understanding neuronal endosomal sorting mechanisms in general. A number of cytosolic adaptor proteins have already been demonstrated to determine intracellular trafficking of SorLA. Most of these adaptors and several ligands of the extracellular/luminal moiety are shared with the Vps10p-D receptor Sortilin. Although SorLA and Sortilin show both a predominant intracellular and endosomal localization, they are targeted to different endosomal compartments. Thus, independent adaptor proteins may convey their differential endosomal targeting. Here, we hypothesized that Sortilin and SorLA interact with the cytosolic adaptors PSD95 and PICK1 which have been shown to bind the Vps10p-D receptor SorCS3. We observed only an interaction for SorLA and PICK1 in mammalian-two-hybrid, pull-down and cellular recruitment experiments. We demonstrate by mutational analysis that the C-terminal minimal PDZ domain binding motif VIA of SorLA mediates the interaction. Moreover, we show co-localization of SorLA and PICK1 at vesicular structures in primary neurons. Although the physiological role of the interaction between PICK1 and SorLA remains unsolved, our study suggests that PICK1 partakes in regulating SorLA's intracellular itinerary.