Sorting Nexin Research Articles

Retromer involvement in trafficking of the calcium-activated K channel, KCa3.1

The intermediate-conductance Ca 2+ -activated K + channel KCa3.1 ( KCNN4) regulates many physiological processes, including, water and electrolyte transport in polarised epithelia and vascular tone. Crucial for KCa3.1 function is the regulation of the number of channels at the plasma membrane. Even though, the mechanism of trafficking of KCa3.1 to and from the basolateral membrane (BLM) of epithelial cells has been advanced recently, there is opposing evidence suggesting that KCa3.1 is recycled back to the leading edge of migrating cells; while others have reported that KCa3.1 does not enter recycling endosomes, but is targeted for degradation. The highly conserved multi-protein complex Retromer has been demonstrated to regulate the membrane retrieval and recycling of many membrane-bound proteins, including ion channels. Using mass-spectrometry, it was reported that when VPS35, a core protein of the Retromer complex, was knocked-down, the membrane expression of KCa3.1 was reduced 1.4 fold in HeLa cells; suggesting a link between Retromer and trafficking of KCa3.1. In contrast, our data indicate KCa3.1 was not affected by knockdown of VPS35 in our FRT (Fischer rat thyroid)-KCa3.1 stable cell line. Here, we have examined other key components (sorting nexins SNX1, SNX4, SNX27; cargo binding proteins) of the Retromer complex to determine if those proteins aid in trafficking of KCa3.1 directly to the BLM. We conducted immunoblot and Ussing chamber experiments to examine the effects of knockdown of SNX 1, SNX2 or SNX27 on the BLM population of KCa3.1 and functional expression of the channel (K + current), respectively. Using our FRT-KCa3.1 cell line, we demonstrate knockdown of SNX1, SNX4 or SNX27 decreases KCa3.1 current. Further, we demonstrate knockdown of SNX4 or SNX27 decreases BLM expression of KCa3.1, consistent with the decrease in current observed.These data, along with our previous data, strongly indicate that the role of Retromer in the trafficking of KCa3.1 to the BLM of epithelial cells is quite dynamic. Reducing SNX4 or SNX27 have notable effects on trafficking of KCa3.1. While knockdown of SNX1 might affect the trafficking of KCa3.1 by altering other proteins of the Retromer complex. This work was supported by the National Institutes of Health grants HL083060 and HL092157 to DCD, Lottery Health New Zealand (R-LHR-2019-101706) to FJM, Aim Fund grant to KLH, and the Department of Physiology, University of Otago. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The association of lipid transfer protein VPS13A with endosomes is mediated by sorting nexin SNX5.

Human VPS13 proteins are implicated in severe neurological diseases. These proteins play an important role in lipid transport at membrane contact sites between different organelles. Identification of adaptors that regulate the subcellular localization of these proteins at specific membrane contact sites is essential to understand their function and role in disease. We have identified the sorting nexin SNX5 as an interactor of VPS13A that mediates its association with endosomal subdomains. As for the yeast sorting nexin and Vps13 endosomal adaptor Ypt35, this association involves the VPS13 adaptor-binding (VAB) domain in VPS13A and a PxP motif in SNX5. Notably, this interaction is impaired by mutation of a conserved asparagine residue in the VAB domain, which is also required for Vps13-adaptor binding in yeast and is pathogenic in VPS13D. VPS13A fragments containing the VAB domain co-localize with SNX5, whereas the more C-terminal part of VPS13A directs its localization to the mitochondria. Overall, our results suggest that a fraction of VPS13A localizes to junctions between the endoplasmic reticulum, mitochondria, and SNX5-containing endosomes.

Sorting nexin 10 sustains PDGF receptor signaling in glioblastoma stem cells via endosomal protein sorting.

Glioblastoma is the most malignant primary brain tumor, the prognosis of which remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared with NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor β (PDGFRβ) proliferative and stem cell signaling pathways through posttranscriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.

Fumarate induces vesicular release of mtDNA to drive innate immunity

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING)–TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.

Dermal macrophages set pain sensitivity by modulating the amount of tissue NGF through an SNX25–Nrf2 pathway

Cross-talk between peripheral neurons and immune cells is important in pain sensation. We identified Snx25 as a pain-modulating gene in a transgenic mouse line with reduced pain sensitivity. Conditional deletion of Snx25 in monocytes and macrophages, but not in peripheral sensory neurons, in mice (Snx25cKO mice) reduced pain responses in both normal and neuropathic conditions. Bone marrow transplantation using Snx25cKO and wild-type mice indicated that macrophages modulated pain sensitivity. Expression of sorting nexin (SNX)25 in dermal macrophages enhanced expression of the neurotrophic factor NGF through the inhibition of ubiquitin-mediated degradation of Nrf2, a transcription factor that activates transcription of Ngf. As such, dermal macrophages set the threshold for pain sensitivity through the production and secretion of NGF into the dermis, and they may cooperate with dorsal root ganglion macrophages in pain perception.

Retromer oligomerization drives SNX-BAR coat assembly and membrane constriction.

Proteins exit from endosomes through tubular carriers coated by retromer, a complex that impacts cellular signaling, lysosomal biogenesis and numerous diseases. The coat must overcome membrane tension to form tubules. We explored the dynamics and driving force of this process by reconstituting coat formation with yeast retromer and the BAR-domain sorting nexins Vps5 and Vps17 on oriented synthetic lipid tubules. This coat oligomerizes bidirectionally, forming a static tubular structure that does not exchange subunits. High concentrations of sorting nexins alone constrict membrane tubes to an invariant radius of 19 nm. At lower concentrations, oligomers of retromer must bind and interconnect the sorting nexins to drive constriction. Constricting less curved membranes into tubes, which requires more energy, coincides with an increased surface density of retromer on the sorting nexin layer. Retromer-mediated crosslinking of sorting nexins at variable densities may thus tune the energy that the coat can generate to deform the membrane. In line with this, genetic ablation of retromer oligomerization impairs endosomal protein exit in yeast and human cells.

Canonical and Non-Canonical Roles of SNX1 and SNX2 in Endosomal Membrane Dynamics.

Sorting nexins (SNXs) are a family of membrane-binding proteins known to play a critical role in regulating endocytic pathway sorting and endosomal membrane trafficking. Among them, SNX1 and SNX2 are members of the SNX-BAR subfamily and possess a membrane-curvature domain and a phosphoinositide-binding domain, which enables their stabilization at the phosphatidylinositol-3-phosphate (PI3P)-positive surface of endosomes. While their binding to PI3P-positive platforms facilitates interaction with endosomal partners and stabilization at the endosomal membrane, their SNX-BAR region is pivotal for generating membrane tubulation from endosomal compartments. In this context, their primary identified biological roles-and their partnership-are tightly associated with the retromer and endosomal SNX-BAR sorting complex for promoting exit 1 complex trafficking, facilitating the transport of cargoes from early endosomes to the secretory pathway. However, recent literature indicates that these proteins also possess biological functions in other aspects of endosomal features and sorting processes. Notably, SNX2 has been found to regulate endosome-endoplasmic reticulum (ER) contact sites through its interaction with VAP proteins at the ER membrane. Furthermore, data from our laboratory show that SNX1 and SNX2 are involved in the tubulation of early endosomes toward ER sites associated with autophagy initiation during starvation. These findings shed light on a novel role of SNXs in inter-organelle tethering and communication. In this concise review, we will explore the non-retromer functions of SNX1 and SNX2, specifically focusing on their involvement in endosomal membrane dynamics during stress sensing and autophagy-associated processes.

A SNX1-SNX2-VAPB partnership regulates endosomal membrane rewiring in response to nutritional stress.

Nutrient deprivation ("starvation") is a major catabolic stress faced by mammalian cells in both pathological and physiological situations. Starvation induces autophagosome biogenesis in the immediate vicinity of ER and leads to lysosome spatial repositioning, but little is known about the consequences of nutritional stress on endosomes. Here, we report that starvation induces tethering of endosomal tubules to ER subregions, fostering autophagosome assembly. We show that this endosomal membrane generation is regulated by sorting nexin 1 (SNX1) protein and is important for the autophagic response. These newly formed SNX1 endosomal tubules establish connections with ER subdomains engaged in early autophagic machinery mobilization. Such endosome-ER transient tethers are regulated by a local dialog between SNX2, an endosomal partner of SNX1, and VAPB, an ER protein associated with autophagy initiation stage regulation. We propose that in a very early response to starvation, SNX1 and SNX2 cooperation induces and regulates endosomal membrane tubulation towards VAPB-positive ER subdomains involved in autophagosome biogenesis, highlighting the contribution of early endosomes in the cellular response to nutritional stress.

Endosomal Sorting Protein SNX27 and Its Emerging Roles in Human Cancers.

SNX27 belongs to the sorting nexin (SNX) family of proteins that play a critical role in protein sorting and trafficking in the endocytosis pathway. This protein family is characterized by the presence of a Phox (PX) domain; however, SNX27 is unique in containing an additional PDZ domain. Recently, SNX27 has gained popularity as an important sorting protein that is associated with the retromer complex and mediates the recycling of internalized proteins from endosomes to the plasma membrane in a PDZ domain-dependent manner. Over 100 cell surface proteins have been identified as binding partners of the SNX27-retromer complex. However, the roles and underlying mechanisms governed by SNX27 in tumorigenesis remains to be poorly understood. Many of its known binding partners include several G-protein coupled receptors, such as β2-andrenergic receptor and parathyroid hormone receptor, are associated with multiple pathways implicated in oncogenic signaling and tumorigenesis. Additionally, SNX27 mediates the recycling of GLUT1 and the activation of mTORC1, both of which can regulate intracellular energy balance and promote cell survival and proliferation under conditions of nutrient deprivation. In this review, we summarize the structure and fundamental roles of SNX proteins, with a focus on SNX27, and provide the current evidence indicating towards the role of SNX27 in human cancers. We also discuss the gap in the field and future direction of SNX27 research. Insights into the emerging roles and mechanism of SNX27 in cancers will provide better development strategies to prevent and treat tumorigenesis.

Overexpression of lncRNA HOXA-AS2 promotes the progression of oral squamous cell carcinoma by mediating SNX5 expression

BackgroundOral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers. Long non-coding RNA HOXA-AS2 (lncRNA HOXA-AS2) have been extensively studied in various cancers. However, the expression and function of HOXA-AS2 in OSCC still remain unknown. The aim of this study is to investigate the roles of HOXA-AS2 in OSCC.MethodsOSCC tissues and adjacent normal tissues were obtained from OSCC patients. RT-qPCR and Western blot assays were used to detect the expression of target genes in OSCC tissues or cells. Cells proliferation, migration and invasion were detected by CCK-8 and transwell assays, respectively. The target gene of HOXA-AS2 was confirmed by dual-luciferase reporter gene assay.ResultsWe found that HOXA-AS2 expression was remarkably upregulated in OSCC tissues and cell lines. The downregulation of HOXA-AS2 inhibited cells proliferation, migration and invasion. Our bioinformatics analysis found that HOXA-AS2 can target miR-520c-3p, which was confirmed by dual-luciferase reporter gene assay. The expression of HOXA-AS2 was found to be negatively associated with miR-520c-3p in OSCC tissues. Moreover, sorting nexin 5 (SNX5), a downstream target of miR-520c-3p, was inhibited by miR-520c-3p overexpression. SNX5 was also increased in OSCC tissues and cell lines. Additionally, we found that the higher expression of SNX5 was strongly associated with the tumor grade of OSCC patients in Oncomine database. Most importantly, the knockdown of HOXA-AS2 induced cells apoptosis by promoting autophagy by regulating SNX5.ConclusionHOXA-AS2 served an oncogene and promoted OSCC progression via the miR-520c-3p/SNX5 axis. Thus, HOXA-AS2 may be a new biomarker for diagnosis and treatment of OSCC.

Cancer-associated fibroblast-derived exosome miR-181b-3p promotes the occurrence and development of colorectal cancer by regulating SNX2 expression

Tumor microenvironment (TME) (e.g., stromal cells) has been closely related to the pathological process of colorectal cancer (CRC). In TME, tumor-associated fibroblasts (CAFs) are the main stromal cells. The studies have showed that CAFs promoted tumor growth and metastasis in CRC and led to poor prognosis. Mounting evidence indicates that CAFs-mediated exosomes regulate the pathological process of neighboring tumor cells through the transmission of miRNAs. In our study, we aimed to explore the function of CAFs-derived exosome miR-181b-3p in CRC. First, the expression of miR-181b-3p in CRC was found to be up-regulated and its expression was dramatically up-regulated in CRC cells after co-incubation of CAFs-mediated exosomes with CRC cells. Then, it was found that the CAFs-derived exosomes were markedly enhanced the proliferation and migration of the CRC cells, and substantially reduced apoptosis. To elucidate the influence of CAFs-derived exosome miR-181b-3p on CRC, we overexpressed and knocked down the miR-181b-3p expression in CAFs, respectively. It was found that miR-181b-3p significantly increased the proliferation and migration of CRC cells. Furthermore, we conducted in vivo experiments. Finally, we demonstrated that CAF-derived exosome miR-181b-3p regulated sorting nexin 2 (SNX2) expression in CRC cells by bioinformatics prediction combined with luciferase reporter assay. Further cellular and animal experiments jointly elucidated that miR-181b-3p promoted the pathological process of CRC by SNX2 expression. In brief, our results demonstrated that CAFs-derived exosome miR-181b-3p promoted the pathogenesis of CRC by regulating SNX2 expression, which provides a novel idea for CRC treatment.

Effects of Genetic Variation of the Sorting Nexin 29 (SNX29) Gene on Growth Traits of Xiangdong Black Goat.

Previous studies have found that the copy number variation (CNV) and insertion/deletion (indels) located in the sorting nexin 29 (SNX29) gene, which is an important candidate gene related to meat production and quality, are associated with growth traits of African goats and Shaanbei white cashmere goats. However, the genetic effects of SNX29 genetic variation on growth traits of Xiangdong black (XDB) goat (a representative meat goat breed in China) are still unclear. The purpose of this study was to detect the mRNA expression level of SNX29 and to explore the genetic effects of CNV and indel within SNX29 on growth traits and gene expression in XDB goat. The SNX29 mRNA expression profile showed that the SNX29 was highly expressed in adipose tissues, indicating that the SNX29 gene could play a key role in subcutaneous adipose deposition of XDB goat. 17 bp indel (g.10559298-10559314), 21 bp indel (g.10918982-10919002) and CNV were detected in 516 individuals of XDB goat by PCR or qPCR. The association analysis of SNX29 CNV with growth traits in XDB goats showed that SNX29 CNV was significantly correlated with chest circumference and abdominal circumference (p < 0.01), and the normal type of SNX29 CNV goat individuals were more advantageous. For the mRNA expression of SNX29 gene, individuals with SNX29 copy number normal type had a higher trend than that of SNX29 gene with copy number gain type in longissimus dorsi muscle (p = 0.07), whereas individuals with SNX29 copy number gain type had a higher trend in abdominal adipose (p = 0.09). Overall, these results suggested that the SNX29 gene could play an important role in growth and development of XDB goats and could be used for marker-assisted selection (MAS) in XDB goats.

Functional Interpretation of Alzheimer’s Disease Genetic Risk and Systematic Gene Prioritization

AbstractBackgroundGenome‐wide association studies (GWAS) of Alzheimer’s disease (AD) risk identified over 100 genome‐wide significant susceptibility loci in the past 15 years, but remaining challenges include resolving the mechanism of action of how these associations functionally contribute to AD risk and prioritizing the candidate risk genes in the complex risk loci. To address these, we have recently developed a systematic gene prioritization pipeline that nominated highly likely candidate risk genes within the majority of 42 new loci identified by our recent AD GWAS meta‐analysis. In this subsequent work, we investigated the complete landscape of AD risk‐associated molecular phenotypes and further extended our analyses to known AD risk loci and subthreshold significant loci.MethodWe investigated downstream effects of AD‐associated variants on molecular phenotypes (e.g. gene expression, splicing, methylation, protein expression, histone modifications) through molecular quantitative trait loci (QTL) catalogues measured in disease‐relevant tissues and cells. We queried the lead variants in these QTL catalogues and performed genetic colocalization analyses (coloc) & transcriptome/methylome/proteome‐wide association studies (TWAS/EWAS/PWAS) using the EADB stage I meta‐analysis GWAS on 85,934 cases and 401,577 controls. Evidence for candidate genes in new, known, and subthreshold loci were assessed by the gene prioritization pipeline that was updated with new analyses. Moreover, we investigated AD‐associated cryptic splicing events using Nanopore sequencing.ResultOur results implicated >140 protein‐coding genes with AD risk in 42 new loci, >275 in 34 known loci, and over >65 in 31 subthreshold loci. Within MINDY2 known locus, evidence from multiple QTL‐GWAS integration domains confirmed ADAM10 as a prioritized gene. Moreover, we obtained first QTL‐based evidences for candidate genes within previously‐unresolved new risk loci such as RASGEF1C. In a subthreshold locus, fine‐mapped expression TWAS associations were observed for SNX31 (a member of sorting nexins together with SNX1 in novel loci) consistently across multiple brain expression reference panels, implicating genetic upregulation of SNX31 with increased AD risk. Furthermore, we could confirm AD‐associated novel splicing events within prioritized genes such as DOC2A. ConclusionOur comprehensive study effectively and systematically prioritized AD risk genes and aided functional interpretation of AD GWAS by contributing to better understanding of genetically‐associated molecular mechanisms underlying AD risk.

Two siblings, distinct characteristics

In this issue of Structure, Healy etal. discovered the PDLIM family of proteins as novel interactors of the sorting nexin SNX17, determined the NMR structure of PDLIM7 with the bound SNX17 C terminus, and suggest that PDLIM proteins could represent novel regulators of endosomal trafficking together with SNX17.

Long noncoding RNA H19 synergizes with STAT1 to regulate SNX10 in rheumatoid arthritis

Erosive destruction of joint structures is an important event in the rheumatoid arthritis (RA) development where fibroblast-like synoviocytes (FLS) represent the main effectors. The implication of long noncoding RNAs (lncRNAs) in RA has not been clearly established. Here, we sought to assess the function of lncRNA H19 in RA by assessing its contribution to the phenotype of FLS. H19 was overexpressed in RA-FLS, and H19 promoted RA-FLS proliferation, invasion as well as angiogenesis and reduced RA-FLS apoptosis. Moreover, H19 loss significantly alleviated joint redness and swelling and reduced inflammatory response, synovial hyperplasia and cartilage damage in arthritic mice induced by collagen. Mechanistically, H19 significantly increased the transcription of sorting nexin (SNX) 10 in RA-FLS by promoting STAT1 translocation into the nucleus. Overexpression of SNX10 or STAT1 mitigated the repressing effects of H19 loss on RA in mice. Our findings highlight that H19 upregulation may result in the development of FLS-mediated RA via the STAT1/SNX10 axis. H19 might serve as a possible therapeutic target for RA treatment.

The Mechanism of SNHG8/Microrna-421-3p/Sorting Nexin 8 Axis on Dopaminergic Neurons in Substantia Nigra in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease affecting the aging population. Particularly, long non-coding RNAs (lncRNAs) have been demonstrated to play vital roles in PD, while the role of lncRNA SNHG8 in PD remains to be further explored. C57BL/6 mice were induced by rotenone to establish a PD model in vivo, and then the dopaminergic (DA) neuronal damage and locomotor dysfunction in rotenone-treated mice were evaluated. Murine DA cell line MN9D was treated with rotenone to establish a cellular PD model in vitro. Then, the viability, apoptosis, mitochondrial dysfunction, endoplasmic reticulum stress, and autophagy in rotenone-treated MN9D cells were assessed. Expression levels of SNHG8, microRNA-421-3p (miR-421-3p), and sorting nexin 8 (SNX8) in the substantia nigra (SN) of PD mice and rotenone-treated MN9D cells were detected. The interaction between SNHG8 and miR-421-3p, and the targeting relationship between SNX8 and miR-421-3p were confirmed. SNHG8 and SNX8 expression levels were decreased while miR-421-3p expression level was increased in the SN of PD mice and rotenone-treated MN9D cells. Upregulated SNHG8 ameliorated dopaminergic neuron damage and locomotor dysfunction in PD mice. Meanwhile, upregulated SNHG8 enhanced viability, diminished apoptosis, and alleviated mitochondrial dysfunction, endoplasmic reticulum stress, and autophagy in rotenone-treated MN9D cells. Mechanistically, SNHG8 bound to miR-421-3p, and miR-421-3p targeted SNX8. Overexpressed SNHG8 downregulates miR-421-3p to alleviate rotenone-induced dopaminergic neuron injury in PD via upregulating SNX8.

Transmembrane Membrane Readers form a Novel Class of Proteins That Include Peripheral Phosphoinositide Recognition Domains and Viral Spikes.

Membrane proteins are broadly classified as transmembrane (TM) or peripheral, with functions that pertain to only a single bilayer at a given time. Here, we explicate a class of proteins that contain both transmembrane and peripheral domains, which we dub transmembrane membrane readers (TMMRs). Their transmembrane and peripheral elements anchor them to one bilayer and reversibly attach them to another section of bilayer, respectively, positioning them to tether and fuse membranes while recognizing signals such as phosphoinositides (PIs) and modifying lipid chemistries in proximity to their transmembrane domains. Here, we analyze full-length models from AlphaFold2 and Rosetta, as well as structures from nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, using the Membrane Optimal Docking Area (MODA) program to map their membrane-binding surfaces. Eukaryotic TMMRs include phospholipid-binding C1, C2, CRAL-TRIO, FYVE, GRAM, GTPase, MATH, PDZ, PH, PX, SMP, StART and WD domains within proteins including protrudin, sorting nexins and synaptotagmins. The spike proteins of SARS-CoV-2 as well as other viruses are also TMMRs, seeing as they are anchored into the viral membrane while mediating fusion with host cell membranes. As such, TMMRs have key roles in cell biology and membrane trafficking, and include drug targets for diseases such as COVID-19.

Regulatory role of phosphoproteins in the development of bovine small intestine during early life.

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.

Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.