Family Of Scaffolding Proteins Research Articles

IQGAP3 Is an Important Mediator of Skin Inflammatory Diseases.

IQGAP3 (IQ Motif Containing GTPase Activating Protein 3) is member of the IQGAP family of scaffold proteins, which are essential for assembling multiprotein complexes that coordinate various intracellular signaling pathways. Previous research has shown that IQGAP3 is overexpressed in psoriatic skin lesions. Given its involvement in processes like cell proliferation and chemokine signaling, we sought to explore its molecular role in driving the psoriatic phenotype of keratinocytes. By conducting transcriptome profiling of HaCaT keratinocytes, we identified numerous psoriasis-associated pathways that were affected when IQGAP3 was knocked down. These included alterations in NFkB signaling, EGFR signaling, activation of p38/MAPK and ERK1/ERK2, lipid metabolism, cytokine production, and the response to inflammatory cytokine stimulation. Real-time analysis further revealed changes in cell growth dynamics, including proliferation and wound healing. The balance between cell proliferation and apoptosis was altered, as were skin barrier functions and the production of IL-6 and IFNγ. Despite these significant findings, the diversity of the alterations observed in the knockdown cells led us to conclude that IQGAP3 may not be the best target for the therapeutic inhibition to normalize the phenotype of keratinocytes in psoriasis.

Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere

Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localise to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52-53, localised to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four and half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in maintenance and/or regulation of sarcomeric organisation and function.

CCDC88B interacts with RASAL3 and ARHGEF2 and regulates dendritic cell function in neuroinflammation and colitis

CCDC88B is a risk factor for several chronic inflammatory diseases in humans and its inactivation causes a migratory defect in DCs in mice. CCDC88B belongs to a family of cytoskeleton-associated scaffold proteins that feature protein:protein interaction domains. Here, we identified the Rho/Rac Guanine Nucleotide Exchange Factor 2 (ARHGEF2) and the RAS Protein Activator Like 3 (RASAL3) as CCDC88B physical and functional interactors. Mice defective in Arhgef2 or Rasal3 show dampened neuroinflammation, and display altered cellular response and susceptibility to colitis; ARHGEF2 maps to a human Chromosome 1 locus associated with susceptibility to IBD. Arhgef2 and Rasal3 mutant DCs show altered migration and motility in vitro, causing either reduced (Arhgef2) or enhanced (Rasal3) migratory properties. The CCDC88B/RASAL3/ARHGEF2 complex appears to regulate DCs migration by modulating activation of RHOA, with ARHGEF2 and RASAL3 acting in opposite regulatory fashions, providing a molecular mechanism for the involvement of these proteins in DCs immune functions.

IQ Motif Containing GTPase-Activating Protein 3 Is Associated with Cancer Stemness and Survival in Pancreatic Ductal Adenocarcinoma

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of malignancy, with poor prognosis and rising incidence. IQ motif containing GTPase-activating protein 3 (IQGAP3) is a member of the IQGAPs family of scaffolding proteins that govern multiple cellular activities like cytoskeletal remodeling and cellular signal transduction. This study aimed to analyze the expression and biological function of IQGAP3 in PDAC. Methods: We analyzed IQGAP3 expression in 81 PDAC samples by immunohistochemistry. RNA interference was used to inhibit IQGAP3 expression in PDAC cell lines. Results: Immunohistochemical analysis of IQGAP3 showed that 54.3% of PDACs were positive for cytoplasmic expression of IQGAP3, with no expression found in non-neoplastic tissue. Furthermore, IQGAP3 expression was an independent poor prognostic factor in our immunostaining-based studies and analyses of public databases. Our cohort and the Cancer Genome Atlas database indicated that IQGAP3 is co-localized with kinesin family member C1 (KIFC1), which we previously reported as a cancer stem cell-associated protein. IQGAP3 small interfering RNA treatment decreased PDAC cell proliferation and spheroid colony formation via ERK and AKT pathways. Discussion/Conclusion: These results suggest that IQGAP3, a transmembrane protein, is involved in survival and stemness and may be a promising new therapeutic target for PDAC.

Gab3 Plays an Essential Role in CD8 + T-Cell Expansion through Regulating IL-2-Mediated Activation of PI3K/AKT/mTOR and Erk/FoxO Pathways

Grb2-associated binding protein 3 (Gab3) is a member of the Gab family of scaffolding proteins. Its role in T cells is largely unknown. In the current study, we investigated its in vivo function in CD8 + T cells using the lymphocytic choriomeningitis virus (LCMV) Armstrong infection model. We created BM chimera mice with a specific deficiency of Gab3 in CD8 + T cells. This was achieved by transplanting a mixture of BM cells obtained from CD8-deficient mice and Gab3-deficient (Gab3 -/-) mice (1) into lethally irradiated CD45.1 mice. The control chimera mice were lethally irradiated CD45.1 mice transplanted with a mixture of BM cells from CD8-deficient mice and WT mice. Twelve weeks after the BM transplantation, the Gab3 -/- and control BM chimera mice were infected with Armstrong. On day eight following infection, Gab3 -/- CD8 + T cells exhibited reduced expansion compared to WT CD8 + T cells. Importantly, Gab3 -/- LCMV antigen specific CD8 + T cells, GP33 + and NP396 + CD8 + T cells, were significantly reduced compared to their WT counterparts. Furthermore, CD8 + T cells in Gab3 -/- BM chimera mice showed significant increase of apoptosis compared to that in WT BM chimera mice following LCMV infection. To explore the mechanism underlying the observed phenotype, we used a primary CD8 + T-cell culture system, in which we stimulated CD8 + T cells with anti-CD3 and anti-CD28 followed by expanding the cells in IL-2. We observed that Gab3 deficiency severely impaired IL-2-induced CD8 + T cell expansion. Gab3 -/- CD8 + T cells in this culture system displayed increased apoptosis and reduced proliferation compared to wild-type (WT) controls, which may account for the reduced CD8 + T cell expansion. We then performed Seahorse analysis of the cultured CD8 + T cells after IL-2 stimulation. We found that IL-2-mediated extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were largely impaired in Gab3 -/- CD8 + T cells compared to WT CD8 + T cells. This data indicates a crucial role of Gab3 in both glycolysis and mitochondrial respiration. We further investigated how Gab3 regulates the IL-2 signaling pathway by conducting transcriptomic and phosphoproteomic analysis. RNA-seq analysis revealed that genes associated with mTOR signaling, glucose metabolism, cell cycle, and Erk/MAPK targets were highly enriched in WT CD8 + T cells, whereas genes related to apoptosis, autophagy, and FoxO3 targets were highly enriched in Gab3 -/- CD8 + T cells. In contrast, the gene set enrichment analysis (GSEA) showed that IL-2-induced Stat5 target gene expression was comparable between WT and Gab3 -/- CD8 + T cells, indicating an important role of Gab3 specifically in the activation of the mTOR and Erk/MAPK pathways downstream of IL-2 signaling. Consistent with that observed in the transcriptomic analysis, phosphoproteomics analysis revealed a significant reduction in IL-2-induced phosphorylation of key components in ERK/FOXO signaling pathway, including RafA, MAPK1, FoxO3, AMPK, and mTOR downstream molecule Eif4B in Gab3 -/- CD8 + T cells compared to WT CD8 + cells. Furthermore, phosphorylation of Stat5 at different sites was comparable between Gab3 -/- and WT CD8 + T cells, further supporting an important role of Gab3 specifically in IL-2 mediated mTOR and Erk/MAPK activation. Finally, we validated the results obtained from the transcriptomic and phosphoproteomic analyses using western blot analysis. Our findings revealed that in Gab3 -/- CD8 + T cells, the IL-2-induced phosphorylation of signaling molecules in the mTOR pathway (S6K, S6, 4EBP1, AKT) and the Erk pathway (Erk) was impaired. Notably, the phosphorylation of Stat5 remained intact despite the absence of Gab3. In summary, our studies have demonstrated that Gab3 plays an essential role in CD8 + T-cell expansion by promoting cell proliferation and prohibiting cell apoptosis through regulating IL-2-mediated activation of PI3K/AKT/mTOR and Erk-FoxO3 pathways. (1) We gratefully acknowledge that Gab3-deficient mice were originally generated by Dr. Larry Rohrschneider at Fred Hutchinson Cancer Research Center ( Mol Cell Biol 2003 Apr;23(7):2415-24. doi: 10.1128/MCB.23.7.2415-2424.2003.). The mice were subsequently backcrossed to C57BL/6.

Is Insulin Receptor Substrate4 (IRS4) a Platform Involved in the Activation of Several Oncogenes?

The IRS (insulin receptor substrate) family of scaffold proteins includes insulin receptor substrate-4 (IRS4), which is expressed only in a few cell lines, including human kidney, brain, liver, and thymus and some cell lines. Its N-terminus carries a phosphotyrosine-binding (PTB) domain and a pleckstrin homology domain (PH), which distinguishes it as a member of this family. In this paper, we collected data about the molecular mechanisms that explain the relevance of IRS4 in the development of cancer and identify IRS4 differences that distinguish it from IRS1 and IRS2. Search engines and different databases, such as PubMed, UniProt, ENSEMBL and SCANSITE 4.0, were used. We used the name of the protein that it encodes "(IRS-4 or IRS4)", or the combination of these terms with the word "(cancer)" or "(human)", for searches. Terms related to specific tumor pathologies ("breast", "ovary", "colon", "lung", "lymphoma", etc.) were also used. Despite the lack of knowledge on IRS4, it has been reported that some cancers and benign tumors are characterized by high levels of IRS-4 expression. Specifically, the role of IRS-4 in different types of digestive tract neoplasms, gynecological tumors, lung cancers, melanomas, hematological tumors, and other less common types of cancers has been shown. IRS4 differs from IRS1 and IRS2 in that can activate several oncogenes that regulate the PI3K/Akt cascade, such as BRK and FER, which are characterized by tyrosine kinase-like activity without regulation via extracellular ligands. In addition, IRS4 can activate the CRKL oncogene, which is an adapter protein that regulates the MAP kinase cascade. Knowledge of the role played by IRS4 in cancers at the molecular level, specifically as a platform for oncogenes, may enable the identification and validation of new therapeutic targets.

The Shank/ProSAP N-Terminal (SPN) Domain of Shank3 Regulates Targeting to Postsynaptic Sites and Postsynaptic Signaling

Members of the Shank family of postsynaptic scaffold proteins (Shank1–3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of Ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, αCaMKII, and cytoskeletal proteins. Here, we analyze the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive αCaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on αCaMKII activity at postsynaptic sites in mice in vivo. The relevance of the SPN-Ank intramolecular interaction was further analyzed in primary cultured neurons; here, we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of clusters in spine-like protrusions. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signaling and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.

The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes.

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.

Neurodevelopmental Consequences of Dietary Zinc Deficiency: A Status Report.

Zinc is a tightly regulated trace mineral element playing critical roles in growth, immunity, neurodevelopment, and synaptic and hormonal signaling. Although severe dietary zinc deficiency is relatively uncommon in the United States, dietary zinc deficiency is a substantial public health concern in low- and middle-income countries. Zinc status may be a key determinant of neurodevelopmental processes. Indeed, limited cohort studies have shown that serum zinc is lower in people diagnosed with autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), and depression. These observations have sparked multiple studies investigating the mechanisms underlying zinc status and neurodevelopmental outcomes. Animal models of perinatal and adult dietary zinc restriction yield distinct behavioral phenotypes reminiscent of features of ASD, ADHD, and depression, including increased anxiety and immobility, repetitive behaviors, and altered social behaviors. At the cellular and molecular level, zinc has demonstrated roles in neurogenesis, regulation of cellular redox status, transcription factor trafficking, synaptogenesis, and the regulation of synaptic architecture via the Shank family of scaffolding proteins. Although mechanistic questions remain, the current evidence suggests that zinc status is important for adequate neuronal development and may be a yet overlooked factor in the pathogenesis of several psychiatric conditions. This review aims to summarize current knowledge of the role of zinc in the neurophysiology of the perinatal period, the many cellular targets of zinc in the developing brain, and the potential consequences of alterations in zinc homeostasis in early life.

Spoonbill positively regulates JNK signalling mediated apoptosis in Drosophila melanogaster

A-kinase anchoring protein (AKAP) comprises a family of scaffold proteins, which decides the subcellular localisation of a combination of signalling molecules. Spoonbill (Spoon) is a putative A-kinase anchoring protein in Drosophila. We have earlier reported that Spoon suppresses ribonuclear foci formed by trinucleotide repeat expanded transcripts associated with Spinocerebellar Ataxia 8 neurodegeneration in Drosophila. However, the role of Spoonbill in cellular signalling was unexplored. In this report, we have unravelled a novel function of Spoon protein in the regulation of the apoptotic pathway. The Drosophila TNFα homolog, Eiger, induces apoptosis via activation of the JNK pathway. We have shown here that Spoonbill is a positive regulator of the Eiger-induced JNK signalling. Further genetic interaction studies show that the spoon interacts with components of the JNK pathway, TGF-β activated kinase 1 (Tak1 - JNKKK), hemipterous (hep - JNKK) and basket (bsk - JNK). Interestingly, Spoonbill alone can also induce ectopic activation of the JNK pathway in a context-specific manner. To understand the molecular mechanism underlying Spoonbill-mediated modulation of the JNK pathway, the interaction between Spoon and Drosophila JNK was assessed. basket encodes the only known JNK in Drosophila. This serine/threonine-protein kinase phosphorylates Jra/Kay, which transcriptionally regulate downstream targets like Matrix metalloproteinase 1 (Mmp1), puckered (puc), and proapoptotic genes hid, reaper and grim. Interestingly, we found that Spoonbill colocalises and co-immunoprecipitates with the Basket protein in the developing photoreceptor neurons. Hence, we propose that Spoon plays a vital role in JNK-induced apoptosis. Furthermore, stress-induced JNK activation underlying Parkinson's Disease was also examined. In the Parkinson’s Drosophila model of neurodegeneration, depletion of Spoonbill leads to a partial reduction of JNK pathway activation, along with improvement in adult motor activity. These observations suggest that the putative scaffold protein Spoonbill is a functional and physical interacting partner of the Drosophila JNK protein, Basket. Spoon protein is localised on the outer mitochondrial membrane (OMM), which may perhaps provide a suitable subcellular niche for activation of Drosophila Basket protein by its kinases which induce apoptosis.

The Antithetic Roles of IQGAP2 and IQGAP3 in Cancers

The scaffold protein family of IQ motif-containing GTPase-activating proteins (IQGAP1, 2, and 3) share a high degree of homology and comprise six functional domains. IQGAPs bind and regulate the cytoskeleton, interact with MAP kinases and calmodulin, and have GTPase-related activity, as well as a RasGAP domain. Thus, IQGAPs regulate multiple cellular processes and pathways, affecting cell division, growth, cell-cell interactions, migration, and invasion. In the past decade, significant evidence on the function of IQGAPs in signal transduction during carcinogenesis has emerged. Compared with IQGAP1, IQGAP2 and IQGAP3 were less analyzed. In this review, we summarize the different signaling pathways affected by IQGAP2 and IQGAP3, and the antithetic roles of IQGAP2 and IQGAP3 in different types of cancer. IQGAP2 expression is reduced and plays a tumor suppressor role in most solid cancer types, while IQGAP3 is overexpressed and acts as an oncogene. In lymphoma, for example, IQGAPs have partially opposite functions. There is considerable evidence that IQGAPs regulate a multitude of pathways to modulate cancer processes and chemoresistance, but some questions, such as how they trigger this signaling, through which domains, and why they play opposite roles on the same pathways, are still unanswered.

Tetraspanin CD82 Regulates Hematopoietic Stem and Progenitor Cell Quiescence & Regeneration

The significant cellular demand of the hematopoietic system is maintained by a rare pool of tissue-specific, hematopoietic stem and progenitor cells (HSPCs) that are primarily found in a quiescent state. Upon hematopoietic stress, HSPCs undergo rapid cell cycle activation, but ultimately must return to quiescence to prevent hematopoietic exhaustion. While the diverse mechanisms used by HSPCs to regulate quiescence are still being defined, it is clear that a combination of cell-intrinsic and bone marrow niche-mediated signaling pathways are involved. Tetraspanins are a unique family of membrane scaffold proteins with the capacity to modulate intrinsic and extrinsic signaling, through the formation of tetraspanin-enriched microdomains. Collectively, tetraspanins, and the microdomains they assemble, act as molecular facilitators that spatiotemporally compartmentalize membrane-associated signaling and adhesion proteins, providing a means to regulate the cell-cell communication and signal transduction associated with a breadth of cellular processes. More specifically, studies from our laboratory identified a critical role for the tetraspanin CD82 in the regulation of HSPC maintenance where we find a reduction in long term-HSPCs (LT-HSPCs) in CD82 knockout (CD82KO) mice, resulting from increased HSPC activation and a reduction of quiescent G0 cells. To further delineate how the CD82 scaffold modulates HSPC quiescence, we transplanted CD82 overexpressing (CD82OE) leukemia cell model into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Despite the fact that CD82OE cells display similar proliferation rates to control cells in culture, once localized to the bone marrow, CD82OE cells demonstrate reduced proliferation and increased quiescence, as indicated by diminished BrdU incorporation and IVIS signal. Additional mechanistic insight is provided by analysis of cells expressing a mutant form of CD82, where three N-linked glycosylation sites are inhibited (Ngly-CD82). Ngly-CD82 cells do not exhibit the same level of quiescence as CD82OE cells when injected into NSG mice, demonstrating an important role for CD82 glycosylation in the regulation of HSPC quiescence. Mechanistically, the cytokine TGF-β plays an essential role in supporting HSPC quiescence and activation, as one of the most potent inhibitors of HSPC growth. To determine if CD82 plays a role in TGF-β signaling, we stimulated WT and CD82KO HSPCs with TGF-β and measured nuclear translocation of SMAD2/3, a downstream indicator of TGF-β activation. Upon TGF-β activation, CD82KO HSPCs display decreased canonical TGF-β signaling, although our data indicate that the non-canonical TGF-β signaling pathway is not impacted. Additionally, we analyzed TGF-β signaling in primary human CD34+ HSPCs, finding that CD34+ human HSPCs with low CD82 expression also have decreased SMAD2/3 nuclear translocation suggestive of a reduced response to TGF-β stimulation. Analysis of the cell line models with differential CD82 expression suggests that cells with higher CD82 expression have increased SMAD2/3 activation following TGFβ stimulation when compared to lower expressing CD82 cells or Ngly-CD82 cells, but only when cells are engaged with fibronectin. Together, these data indicate that the CD82 scaffold has the capacity to modulate TGFb signaling through niche-mediated interactions and implicate integrins in the modulation of signal transduction.

Elevation of SHANK3 Levels by Antisense Oligonucleotides Directed Against the 3'-UTR of the Human SHANK3 mRNA.

SHANK3 is a member of the SHANK family of scaffolding proteins that localize to the postsynaptic density of excitatory synapses. Mutations within the SHANK3 gene or SHANK3 haploinsufficiency is thought to be one of the major causes for Phelan-McDermid Syndrome (PMDS) that is characterized by a broad spectrum of autism-related behavioral alterations. Several approaches have already been proposed to elevate SHANK3 protein levels in PMDS patients like transcriptional activation or inhibition of SHANK3 degradation. We undertook a systematic screening approach and tested whether defined antisense oligonucleotides (ASOs) directed against the 3' untranslated region (3'-UTR) of the human SHANK3 mRNA are suitable to elevate SHANK3 protein levels. Using human induced pluripotent stem cells (hiPSCs) and hiPSCs-derived motoneurons from controls and PMDS patients we eventually identified two 18 nucleotide ASOs (ASO 4-5.2-4 and 4-5.2-6) that were able to increase SHANK3 protein levels in vitro by about 1.3- to 1.6-fold. These findings were confirmed by co-transfection of the identified ASOs with a GFP-SHANK3-3'-UTR construct in HEK293T cells using GFP protein expression as read-out. Based on these results we propose a novel approach to elevate SHANK3 protein concentrations by 3'-UTR specific ASOs. Further research is needed to test the suitability of SHANK3-specific ASOs as pharmacological compounds also in vivo.

Genetic Analysis Implicates Dysregulation of SHANK2 in Renal Cell Carcinoma Progression.

SH3 and multiple ankyrin repeat domains (SHANK) is a family of scaffold proteins that were first identified to be involved in balancing synaptic transmission via regulation of intracellular signalling crosstalk and have been linked to various cancers. However, the role of the SHANK genes in renal cell carcinoma (RCC) remains to be elucidated. In this study, we aimed to evaluate whether genetic variants in SHANK family genes affect the risk of RCC and survival of patients. A genetic association study was conducted using logistic regression and Cox regression analyses, followed by the correction for a false discovery rate (FDR), in 630 patients with RCC and controls. A pooled analysis was further performed to summarise the clinical relevance of SHANK gene expression in RCC. After adjustment for known risk factors and the FDR, the SHANK2 rs10792565 T allele was found to be associated with an increased risk of RCC (adjusted odds ratio = 1.79, 95% confidence interval = 1.32–2.44, p = 1.96 × 10−4, q = 0.030), whereas no significant association was found with RCC survival. A pooled analysis of 19 independent studies, comprising 1509 RCC and 414 adjacent normal tissues, showed that the expression of SHANK2 was significantly lower in RCC than in normal tissues (p < 0.001). Furthermore, low expression of SHANK2 was correlated with an advanced stage and poor prognosis for patients with clear cell and papillary RCC. This study suggests that SHANK2 rs10792565 is associated with an increased risk of RCC and that SHANK2 may play a role in RCC progression.

Moesin Serves as Scaffold Protein for PD-L1 in Human Uterine Cervical Squamous Carcinoma Cells.

Immune checkpoint blockade (ICB) therapy targeting the programmed death ligand-1 (PD-L1)/PD-1 axis has emerged as a promising treatment for uterine cervical cancer; however, only a small subset of patients with uterine cervical squamous cell carcinoma (SCC) derives clinical benefit from ICB therapies. Thus, there is an urgent unmet medical need for novel therapeutic strategies to block the PD-L1/PD-1 axis in patients with uterine cervical SCC. Here, we investigated the involvement of ezrin/radixin/moesin (ERM) family scaffold proteins, which crosslink several plasma membrane proteins with the actin cytoskeleton, on the plasma membrane localization of PD-L1 in BOKU and HCS-2 cells derived from human uterine cervical SCC. Immunofluorescence analysis showed that PD-L1 colocalized with all three ERM proteins in the plasma membrane. Gene knockdown of moesin, but not ezrin and radixin, substantially reduced the plasma membrane expression of PD-L1, with limited effect on mRNA expression. An immunoprecipitation assay demonstrated the molecular interaction between PD-L1 and moesin. Moreover, phosphorylated, i.e., activated, moesin was highly colocalized with PD-L1 in the plasma membrane. In conclusion, moesin may be a scaffold protein responsible for the plasma membrane expression of PD-L1 in human uterine cervical SCC.

Arrestin-Coding Genes Regulate Endocytosis, Sporulation, Pathogenicity, and Stress Resistance in Arthrobotrys oligospora.

Arrestins are a family of scaffold proteins that play a crucial role in regulating numerous cellular processes, such as GPCR signaling. The Arthrobotrys oligospora arrestin family contains 12 members, which have highly conserved N-terminal and C-terminal domains. In the presence of ammonia, A. oligospora can change its lifestyle from saprotrophic to carnivorous. During this transition, the expression pattern of arrestin-coding (AoArc) genes was markedly upregulated. Therefore, we disrupted seven AoArc genes from A. oligospora to identify their functions. Although individual arrestin mutant strains display similar pathogenesis, phenotypes, and stress resistance, the fundamental data on the roles of AoArc genes in A. oligospora are obtained in this study. Membrane endocytosis in AoArc mutants was significantly reduced. Meanwhile, the capacity of trap device formation against nematodes and ammonia was impaired due to AoArc deletions. We also found that AoArc genes could regulate conidial phenotypes, cell nuclear distribution, pH response, and stress resistance. Results of qRT-PCR assays revealed that sporulation-regulated genes were affected after the deletion of AoArc genes. In particular, among the 12 arrestins, AoArc2 mediates pH signaling in the fungus A. oligospora. Notably, combined with the classical paradigm of arrestin–GPCR signal transduction, we suggest that arrestin-regulated trap formation in A. oligospora may be directly linked to the receptor endocytosis pathway.

Zinc deficiency and supplementation in autism spectrum disorder and Phelan-McDermid syndrome.

Approximately 1 in 36 children are diagnosed with autism spectrum disorder (ASD). The disorder is four times more common in males than in females. Zinc deficiency and mutations in SHANK2 and SHANK3 (members of a family of excitatory postsynaptic scaffolding proteins) are all risk factors that may contribute to the pathophysiology of the disease. The presence of shankopathies (loss of one copy of the SHANK3 gene) can lead to the development of Phelan-McDermid syndrome (PMDS)-a rare genetic disorder characterized by developmental delay, intellectual disability, poor motor tone, and ASD-like symptoms. We reviewed the relationship between zinc, ASD, and PMDS as well as the effect of zinc supplementation in improving symptoms of ASD and PMDS based on 22 studies published within 6years (2015-2020). Zinc deficiency (assessed by either dietary intake, blood, hair, or tooth matrix) was shown to be highly prevalent in ASD and PMDS patients as well as in preclinical models of ASD and PMDS. Zinc supplements improved the behavioral deficits in animal models of ASD and PMDS. Clinical trials are still needed to validate the beneficial therapeutic effects of zinc supplements in ASD and PMDS patients.

Dishevelled coordinates phosphoinositide kinases PI4KIIIα and PIP5KIγ for efficient PtdInsP2 synthesis.

Phosphatidylinositol(4,5)-bisphosphate (PtdInsP2) is an important modulator of many cellular processes, and its abundance in the plasma membrane is closely regulated. We examined the hypothesis that members of the Dishevelled scaffolding protein family can bind the lipid kinases phosphatidylinositol 4-kinase (PI4K) and phosphatidylinositol 4-phosphate 5-kinase (PIP5K), facilitating synthesis of PtdInsP2 directly from phosphatidylinositol. We used several assays for PtdInsP2 to examine the cooperative function of phosphoinositide kinases and the Dishevelled protein Dvl3 in the context of two receptor signaling cascades. Simultaneous overexpression of PI4KIIIα (also known as PI4KA) and PIP5KIγ (also known as PIP5K1C) had a synergistic effect on PtdInsP2 synthesis that was recapitulated by overexpression of Dvl3. Increasing the activity of Dvl3 by overexpression increased resting plasma membrane PtdInsP2. Knockdown of Dvl3 reduced resting plasma membrane PtdInsP2 and slowed PtdInsP2 resynthesis following receptor activation. We confirm that Dvl3 promotes coupling of PI4KIIIα and PIP5KIγ and show that this interaction is essential for efficient resynthesis of PtdInsP2 following receptor activation.

MPP1 Determines the Mobility of Flotillins and Controls the Confinement of Raft-Associated Molecules.

MPP1 (membrane palmitoylated protein 1) belongs to the MAGUK (membrane-associated guanylate kinase homologs) scaffolding protein family. These proteins organize molecules into complexes, thereby maintaining the structural heterogeneity of the plasma membrane (PM). Our previous results indicated that direct, high-affinity interactions between MPP1 and flotillins (raft marker proteins) display dominant PM-modulating capacity in erythroid cells. In this study, with high-resolution structured illuminated imaging, we investigated how these complexes are organized within erythroid cells on the nanometer scale. Furthermore, using other spectroscopic techniques, namely fluorescence recovery after photobleaching (FRAP) and spot-variation fluorescence correlation spectroscopy (svFCS), we revealed that MPP1 acts as a key raft-capturing molecule, regulating temporal immobilization of flotillin-based nanoclusters, and controls local concentration and confinement of sphingomyelin and Thy-1 in raft nanodomains. Our data enabled us to uncover molecular principles governing the key involvement of MPP1-flotillin complexes in the dynamic nanoscale organization of PM of erythroid cells.

Ezrin and Radixin Differentially Modulate Cell Surface Expression of Programmed Death Ligand-1 in Human Pancreatic Ductal Adenocarcinoma KP-2 Cells

Immune checkpoint blockade (ICB) therapies, such as immune checkpoint inhibitors against programmed death ligand-1 (PD-L1), have not been successful in treating patients with pancreatic ductal adenocarcinoma (PDAC). Despite the critical role of PD-L1 in various types of cancers, the regulatory mechanism of PD-L1 expression on the cell surface of PDAC is poorly understood. Therefore, uncovering potential modulators of cell surface localisation of PD-L1 may provide a new strategy to improve ICB therapy in patients with PDAC. Here, we examined the role of ezrin/radixin/moesin (ERM) family scaffold proteins that crosslink transmembrane proteins with the actin cytoskeleton in the surface localisation of PD-L1 in KP-2 cells, a human PDAC cell line. Our results demonstrated the abundant protein expression of PD-L1, ezrin, and radixin, but not moesin, as well as their colocalisation in the plasma membrane. Interestingly, immunoprecipitation analysis detected the molecular interaction of PD-L1 with ezrin and radixin. Moreover, gene silencing of ezrin moderately decreased the mRNA and cell surface expression of PD-L1, while that of radixin greatly decreased the surface expression of PD-L1 without altering the mRNA levels. Thus, radixin and ezrin differentially modulate the cell surface localisation of PD-L1 in KP-2 cells, highlighting a potential therapeutic target to improve the current ICB therapy in PDAC.