Structural basis for coupling of the WASH subunit FAM21 with the endosomal SNX27-Retromer complex.

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains.

HPV-18 E6 enhances the interaction between EMILIN2 and SNX27 to promote WNT signaling.

Oncogenic HPV E6 proteins have a PDZ-binding motif (PBM) which plays important roles in both the viral life cycle and tumor development. The PBM confers interaction with a large number of different PDZ domain-containing substrates, one of which is Sorting Nexin 27. This protein is part of the retromer complex and plays an important role in endocytic sorting pathways. It has been shown that at least two SNX27 interacting partners, GLUT1 and TANC2, are aberrantly trafficked due to the E6 PBM-dependent interaction with SNX27. To investigate further which other components of the endocytic trafficking pathway might be affected by the SNX27-HPV E6 interaction, we analyzed the SNX27 proteome interaction profile in a previously described HeLa cell line expressing GFP-SNX27, both in the presence and absence of the HPV-18 E6 oncoprotein. In this study, we identify a novel interacting partner of SNX27, secreted glycoprotein EMILIN2, whose release is blocked by HPV18 E6 in a PBM-dependent manner. Mechanistically, E6 can block EMILIN2 interaction with the WNT1 ligand, thereby enhancing WNT1 signaling and promoting cell proliferation. IMPORTANCE: This study demonstrates that HPV E6 blocks EMILIN2 inhibition of WNT1 signaling, thereby enhancing cell proliferation in HPV-positive tumor cells. This involves a novel mechanism whereby the E6 PBM actually contributes toward enhancing the interaction between SNX27 and EMILIN2, suggesting that the mode of recognition of SNX27 by E6 and EMILIN2 is different. This is the first example of the E6 PBM altering a PDZ domain-containing protein to enhance potential substrate recognition.

SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry.

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

The retromer complex regulates C. elegans development and mammalian ciliogenesis.

The mammalian retromer consists of subunits VPS26 (either VPS26A or VPS26B), VPS29 and VPS35, and a loosely associated sorting nexin (SNX) heterodimer or a variety of other SNX proteins. Despite involvement in yeast and mammalian cell trafficking, the role of retromer in development is poorly understood, and its impact on primary ciliogenesis remains unknown. Using CRISPR/Cas9 editing, we demonstrate that vps-26-knockout worms have reduced brood sizes, impaired vulval development and decreased body length, all of which have been linked to ciliogenesis defects. Although preliminary studies did not identify worm ciliary defects, and impaired development limited additional ciliogenesis studies, we turned to mammalian cells to investigate the role of retromer in ciliogenesis. VPS35 localized to the primary cilium of mammalian cells, and depletion of VPS26, VPS35, VPS29, SNX1, SNX2, SNX5 or SNX27 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the centriolar protein, CP110 (also known as CCP110), and was required for its removal from the mother centriole. Herein, we characterize new roles for retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, suggesting a novel role for retromer in CP110 removal from the mother centriole.

Partial loss of Sorting Nexin 27 resembles age- and Down syndrome-associated T cell dysfunctions.

BACKGROUND: Sorting Nexin 27 (SNX27)-retromer complex facilitates cargo recycling from endosomes to the plasma membrane. SNX27 downregulation in neurons, as the result of Trisomy 21 (T21), has been linked with cognitive deficits due to impairment of AMPA and NMDA receptor recycling. Studies in human T cell lines likewise demonstrated that SNX27 regulates the correct delivery of cargoes to the immune synapse limiting the activation of pro-inflammatory pathways. Nevertheless, the physiological consequences of partial SNX27 loss in T cell homeostasis are still unclear. RESULTS: In this study, we have explored the consequences of T cell specific partial SNX27 downregulation in mice. T cells with partial SNX27 deficiency show a marked deficit in the CD4+ T cell pool, a hallmark of aging in mice and humans, and a well-characterized comorbidity of individuals with Down syndrome (DS). When analyzed ex vivo, CD4+ T cells with partial SNX27 deletion demonstrate enhanced proliferation but diminished IL-2 production. In contrast, the CD8+ population show enhanced expression of pro-inflammatory cytokines and lytic enzymes. CONCLUSIONS: This mouse model supports the relevance of SNX27 in the organization of the immune synapse, previously described in cell lines, as well as in the control of T cell homeostasis. Individuals with DS experiment an acceleration of the aging process, which particularly affects the immune and central nervous systems. Thus, we hypothesize that reduced SNX27 expression in DS could contribute to the dysregulation of these systems and further research in SNX27 will shed light on the molecular factors underlying the phenotypes observed in people with DS and its contribution to aging.

SNX27-FERM-SNX1 complex structure rationalizes divergent trafficking pathways by SNX17 and SNX27.

The molecular events that determine the recycling versus degradation fates of internalized membrane proteins remain poorly understood. Two of the three members of the SNX-FERM family, SNX17 and SNX31, utilize their FERM domain to mediate endocytic trafficking of cargo proteins harboring the NPxY/NxxY motif. In contrast, SNX27 does not recycle NPxY/NxxY-containing cargo but instead recycles cargo containing PDZ-binding motifs via its PDZ domain. The underlying mechanism governing this divergence in FERM domain binding is poorly understood. Here, we report that the FERM domain of SNX27 is functionally distinct from SNX17 and interacts with a novel DLF motif localized within the N terminus of SNX1/2 instead of the NPxY/NxxY motif in cargo proteins. The SNX27-FERM-SNX1 complex structure reveals that the DLF motif of SNX1 binds to a hydrophobic cave surrounded by positively charged residues on the surface of SNX27. The interaction between SNX27 and SNX1/2 is critical for efficient SNX27 recruitment to endosomes and endocytic recycling of multiple cargoes. Finally, we show that the interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish. Altogether, our study solves a long-standing puzzle in the field and suggests that SNX27 and SNX17 mediate endocytic recycling through fundamentally distinct mechanisms.

HPV-18E6 Inhibits Interactions between TANC2 and SNX27 in a PBM-Dependent Manner and Promotes Increased Cell Proliferation.

Cancer-causing HPV E6 oncoproteins contain a PDZ-binding motif at the extreme carboxy terminus, which plays an important role in the viral life cycle and in the development of malignancy. Through this motif, HPV E6 targets a large number of cellular substrates, many of which are involved in processes related to the regulation of cell polarity. Recent studies also demonstrated E6's PDZ binding motif (PBM)-dependent association with SNX27, with a potential role in the perturbation of endocytic transport. Here, we have performed a proteomic analysis to identify SNX27-interacting partners whose binding to SNX27 is specifically perturbed in an E6-dependent manner. Extracts of HeLa cells that express GFP-tagged SNX27, transfected with control siRNA or siRNA targeting E6AP, were subject to GFP immunoprecipitation followed by mass spectroscopy, which identified TANC2 as an interacting partner of SNX27. Furthermore, we demonstrate that HPV E6 inhibits association between SNX27 and TANC2 in a PBM-dependent manner, resulting in an increase in TANC2 protein levels. In the absence of E6, SNX27 directs TANC2 toward lysosomal degradation. TANC2, in the presence of HPV-18E6, enhances cell proliferation in a PBM-dependent manner, indicating that HPV E6 targets the SNX27-mediated transport of TANC2 to promote cellular proliferation. IMPORTANCE While a great deal is known about the role of the E6 PDZ binding motif (PBM) in modulating the cellular proteins involved in regulating cell polarity, much less is known about the consequences of E6's interactions with SNX27 and the endocytic sorting machinery. We reasoned that a potential consequence of such interactions could be to affect the fate of multiple SNX27 endosomal partners, such as transmembrane proteins or soluble accessory proteins. Using a proteomic approach in HPV-18-positive cervical tumor-derived cells, we demonstrate that TANC2 is an interacting partner of SNX27, whose interaction is blocked by E6 in a PBM-dependent manner. This study therefore begins to shed new light on how E6 can regulate the endocytic transport of multiple SNX27-binding proteins, thereby expanding our understanding of the functions of the E6 PBM.

Circ_SNX27 regulates hepatocellular carcinoma development via miR-637/FGFR1 axis.

BACKGROUND: Circular RNAs (circRNAs) serve as critical regulatory factors in cancer development. Nonetheless, the potential regulatory mechanism of circRNA sorting nexin 27 (circ_SNX27) in hepatocellular carcinoma (HCC) is still unknown. METHODS: The circ_SNX27, microRNA-637 (miR-637), and fibroblast growth factor receptor 1 (FGFR1) levels were quantified by quantitative real-time polymerase chain reaction and western blot analysis. Next, function experiments were conducted using in vitro assays and in vivo senograft study. The relationship between miR-637 with circ_SNX27 or FGFR1 was uncovered by dual-luciferase reporter and RNA pull-down assays. RESULTS: The circ_SNX27 and FGFR1 levels were up-regulated, but miR-637 content was reduced in HCC. Circ_SNX27 down-regulation inhibited HCC cell proliferation, motility, and invasion and promoted apoptosis in vitro, as well as weakened tumor growth in vivo. Circ_SNX27 served as a sponge of miR-637 to promote FGFR1 expression. MiR-637 reduction abolished the restrained effect of circ_SNX27 absence on HCC cell development. Moreover, miR-637 curbed HCC cell malignant phenotype by regulating FGFR1. CONCLUSION: Circ_SNX27 contributed to HCC development via miR-637/FGFR1 axis, offering a new idea for the treatment of HCC.

SNX27 regulates DRA activity and mediates its direct recycling by PDZ-interaction in early endosomes at the apical pole of Caco2 cells.

DRA (downregulated in adenoma, SLC26A3) and NHE3 (Na+/H+ exchanger 3, SLC9A3) together mediate intestinal electroneutral NaCl absorption. Both transporters contain PDZ (postsynaptic density 95, disc large, zonula occludens 1) binding motifs and interact with PDZ adaptor proteins regulating their activity and recycling. SNX27 (sorting nexin 27) contains a PDZ domain and is involved in the recycling of cargo proteins including NHE3. The interaction of SNX27 with DRA and its potential role for the activity and recycling of DRA have been evaluated in this study. SNX27 specifically interacts with DRA via its PDZ domain. The knockdown (KD) of SNX27 reduced DRA activity by 50% but was not accompanied by a decrease of DRA surface expression. This indicates that DRA is trafficked to specific functional domains in the plasma membrane in which DRA is particularly active. Consistently, the disruption of lipid raft integrity by methyl-ß-cyclodextrin has an inhibitory effect on DRA activity that was strongly reduced after SNX27 KD. In differentiated intestinal Caco2 cells, superresolution microscopy and a novel quantitative axial approach revealed that DRA and SNX27 colocalize in rab5-positive early endosomes at the apical pole. SNX27 regulates the activity of DRA in the apical plasma membrane through binding with its PDZ domain. This interaction occurs in rab5-positive early endosomes at the apical pole of differentiated intestinal Caco2 cells. SNX27 is involved in the direct recycling of DRA to the plasma membrane where it is inserted into lipid rafts facilitating increased activity.NEW & NOTEWORTHY SNX27 has a PDZ domain and is involved in the regulation and recycling of transmembrane proteins. The role of SNX27 on the activity and recycling of the intestinal Cl-/HCO3- exchanger DRA has not yet been studied. This study shows that SNX27 directly interacts with DRA in early endosomes at the apical pole of intestinal Caco2 cells and mediates its direct recycling to facilitate high activity in lipid rafts in the apical plasma membrane.

Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane.

Cholera toxin (CT) causes severe diarrhea by increasing intracellular cAMP leading to a PKA-dependent increase in Cl- secretion through CFTR and decreased Na+ absorption through inhibition of Na+/H+ exchanger 3 (NHE3; also known as SLC9A3). The mechanism(s) by which CT inhibits NHE3 is partially understood, although no drug therapy has been successful at reversing this inhibition. We now describe that CT phosphorylates an amino acid in the PDZ domain of SNX27, which inhibits SNX27-mediated trafficking of NHE3 from the early endosomes to the plasma membrane (PM), and contributes to reduced basal NHE3 activity through a mechanism that involves reduced PM expression and reduced endocytic recycling. Importantly, mutagenesis studies (Ser to Asp) showed that the effect of this phosphorylation of SNX27 phenocopies the effects seen upon loss of SNX27 function, affecting PM trafficking of cargo proteins that bind SNX27-retromer. Additionally, CT destabilizes retromer function by decreasing the amount of core retromer proteins. These effects of CT can be partially rescued by enhancing retromer stability by using 'pharmacological chaperones'. Moreover, pharmacological chaperones can be used to increase basal and cholera toxin-inhibited NHE3 activity and fluid absorption by intestinal epithelial cells.This article has an associated First Person interview with the first author of the paper.

Sorting nexin 27 (SNX27) variants associated with seizures, developmental delay, behavioral disturbance, and subcortical brain abnormalities.

Sorting nexin 27 (SNX27) influences the composition of the cellular membrane via regulation of selective endosomal recycling. Molecular analysis indicates that SNX27 regulates numerous cellular processes through promiscuous interactions with its receptor cargos. SNX27 deficient (Snx27 -/- ) mice exhibit reduced embryonic survival, marked postnatal growth restriction and lethality. Haploinsufficient mice (Snx27 +/- ) show a less severe phenotype, with deficits in learning, memory, synaptic transmission and neuronal plasticity. One family previously reported with a homozygous SNX27 frameshift variant (c.515_516del;p.His172Argfs*6), exhibited infantile intractable myoclonic epilepsy, axial hypotonia, startle-like movements, cardiac septal defects, global developmental delay, failure to thrive, recurrent chest infections, persistent hypoxemia and early death secondary to respiratory failure. Here, we report two additional patients with compound heterozygous SNX27 variants, that are predicted to be damaging: (a) c.510C>G;p.Tyr170* and c.1295G>A;p.Cys432Tyr, and (b) c.782dupT;p.Leu262Profs*6 and c.989G>A;p.Arg330His. They exhibit global developmental delay, behavioral disturbance, epilepsy, some dysmorphic features and subcortical white matter abnormalities. In addition, possible connective tissue involvement was noted. Epilepsy, developmental delays and subcortical white matter abnormalities appear to be core features of SNX27-related disorders. We correlate the observed phenotype with available in vitro, in vivo and proteomic data and suggest additional possible molecular mediators of SNX27-related pathology.

Sorting nexin 27 rescues neuroligin 2 from lysosomal degradation to control inhibitory synapse number.

Retromer is an evolutionarily conserved endosomal trafficking complex that mediates the retrieval of cargo proteins from a degradative pathway for sorting back to the cell surface. To promote cargo recycling, the core retromer trimer of VPS (vacuolar protein sorting)26, VPS29 and VPS35 recognises cargo either directly, or through an adaptor protein, the most well characterised of which is the PDZ [postsynaptic density 95 (PSD95), disk large, zona occludens] domain-containing sorting nexin SNX27. Neuroligins (NLGs) are postsynaptic trans-synaptic scaffold proteins that function in the clustering of postsynaptic proteins to maintain synaptic stability. Here, we show that each of the NLGs (NLG1-3) bind to SNX27 in a direct PDZ ligand-dependent manner. Depletion of SNX27 from neurons leads to a decrease in levels of each NLG protein and, for NLG2, this occurs as a result of enhanced lysosomal degradation. Notably, while depletion of the core retromer component VPS35 leads to a decrease in NLG1 and NLG3 levels, NLG2 is unaffected, suggesting that, for this cargo, SNX27 acts independently of retromer. Consistent with loss of SNX27 leading to enhanced lysosomal degradation of NLG2, knockdown of SNX27 results in fewer NLG2 clusters in cultured neurons, and loss of SNX27 or VPS35 reduces the size and number of gephyrin clusters. Together, these data indicate that NLGs are SNX27-retromer cargoes and suggest that SNX27-retromer controls inhibitory synapse number, at least in part through trafficking of NLG2.

Interplay Between SNX27 and DAG Metabolism in the Control of Trafficking and Signaling at the IS.

Recognition of antigens displayed on the surface of an antigen-presenting cell (APC) by T-cell receptors (TCR) of a T lymphocyte leads to the formation of a specialized contact between both cells named the immune synapse (IS). This highly organized structure ensures cell-cell communication and sustained T-cell activation. An essential lipid regulating T-cell activation is diacylglycerol (DAG), which accumulates at the cell-cell interface and mediates recruitment and activation of proteins involved in signaling and polarization. Formation of the IS requires rearrangement of the cytoskeleton, translocation of the microtubule-organizing center (MTOC) and vesicular compartments, and reorganization of signaling and adhesion molecules within the cell-cell junction. Among the multiple players involved in this polarized intracellular trafficking, we find sorting nexin 27 (SNX27). This protein translocates to the T cell-APC interface upon TCR activation, and it is suggested to facilitate the transport of cargoes toward this structure. Furthermore, its interaction with diacylglycerol kinase ζ (DGKζ), a negative regulator of DAG, sustains the precise modulation of this lipid and, thus, facilitates IS organization and signaling. Here, we review the role of SNX27, DAG metabolism, and their interplay in the control of T-cell activation and establishment of the IS.

Retromer- and WASH-dependent sorting of nutrient transporters requires a multivalent interaction network with ANKRD50.

Retromer and the associated actin-polymerizing WASH complex are essential for the endocytic recycling of a wide range of integral membrane proteins. A hereditary Parkinson's-disease-causing point mutation (D620N) in the retromer subunit VPS35 perturbs retromer's association with the WASH complex and also with the uncharacterized protein ankyrin-repeat-domain-containing protein 50 (ANKRD50). Here, we firmly establish ANKRD50 as a new and essential component of the SNX27-retromer-WASH super complex. Depletion of ANKRD50 in HeLa or U2OS cells phenocopied the loss of endosome-to-cell-surface recycling of multiple transmembrane proteins seen upon suppression of SNX27, retromer or WASH-complex components. Mass-spectrometry-based quantification of the cell surface proteome of ANKRD50-depleted cells identified amino acid transporters of the SLC1A family, among them SLC1A4, as additional cargo molecules that depend on ANKRD50 and retromer for their endocytic recycling. Mechanistically, we show that ANKRD50 simultaneously engages multiple parts of the SNX27-retromer-WASH complex machinery in a direct and co-operative interaction network that is needed to efficiently recycle the nutrient transporters GLUT1 (also known as SLC2A1) and SLC1A4, and potentially many other surface proteins.

SNX27 and SORLA Interact to Reduce Amyloidogenic Subcellular Distribution and Processing of Amyloid Precursor Protein.

UNLABELLED: Proteolytic generation of amyloidogenic amyloid ß (Aß) fragments from the amyloid precursor protein (APP) significantly contributes to Alzheimer's disease (AD). Although amyloidogenic APP proteolysis can be affected by trafficking through genetically associated AD components such as SORLA, how SORLA functionally interacts with other trafficking components is yet unclear. Here, we report that SNX27, an endosomal trafficking/recycling factor and a negative regulator of the γ-secretase complex, binds to the SORLA cytosolic tail to form a ternary complex with APP. SNX27 enhances cell surface SORLA and APP levels in human cell lines and mouse primary neurons, and depletion of SNX27 or SORLA reduces APP endosome-to-cell surface recycling kinetics. SNX27 overexpression enhances the generation of cell surface APP cleavage products such as soluble alpha-APP C-terminal fragment (CTFα) in a SORLA-dependent manner. SORLA-mediated Aß reduction is attenuated by downregulation of SNX27. This indicates that an SNX27/SORLA complex functionally interacts to limit APP distribution to amyloidogenic compartments, forming a non-amyloidogenic shunt to promote APP recycling to the cell surface. SIGNIFICANCE STATEMENT: Many genes have been identified as risk factors for Alzheimer's disease (AD), and a large proportion of these genes function to limit production or toxicity of the AD-associated amyloid ß (Aß) peptide. Whether and how these genes precisely operate to limit AD onset remains an important question. We identify binding and trafficking interactions between two of these factors, SORLA and SNX27, and demonstrate that SNX27 can direct trafficking of SORLA and the Aß precursor APP to the cell surface to limit the production of Aß. Diversion APP to the cell surface through modulation of this molecular complex may represent a complimentary strategy for future development in AD treatment.

SNX27 Deletion Causes Hydrocephalus by Impairing Ependymal Cell Differentiation and Ciliogenesis.

Hydrocephalus is a brain disorder derived from CSF accumulation due to defects in CSF clearance. Although dysfunctional apical cilia in the ependymal cell layer are causal to the onset of hydrocephalus, mechanisms underlying proper ependymal cell differentiation are largely unclear. SNX27 is a trafficking component required for normal brain function and was shown previously to suppress γ-secretase-dependent amyloid precursor protein and Notch cleavage. However, it was unclear how SNX27-dependent γ-secretase inhibition could contribute to brain development and pathophysiology. Here, we describe and characterize an Snx27-deleted mouse model for the ependymal layer defects of deciliation and hydrocephalus. SNX27 deficiency results in reductions in ependymal cells and cilia density, as well as severe postnatal hydrocephalus. Inhibition of Notch intracellular domain signaling with γ-secretase inhibitors reversed ependymal cells/cilia loss and dilation of lateral ventricles in Snx27-deficient mice, giving strong indication that Snx27 deletion triggers defects in ependymal layer formation and ciliogenesis through Notch hyperactivation. Together, these results suggest that SNX27 is essential for ependymal cell differentiation and ciliogenesis, and its deletion can promote hydrocephalus pathogenesis. SIGNIFICANCE STATEMENT: Down's syndrome (DS) in humans and mouse models has been shown previously to confer a high risk for the development of pathological hydrocephalus. Because we have previously described SNX27 as a component that is consistently downregulated in DS, we present here a robust Snx27-deleted mouse model that produces hydrocephalus and associated ciliary defects with complete penetrance. In addition, we find that γ-secretase/Notch modulation may be a candidate drug target in SNX27-associated hydrocephalus such as that observed in DS. Based on these findings, we anticipate that future study will determine whether modulation of a SNX27/Notch/γ-secretase pathway can also be of therapeutic interest to congenital hydrocephalus.

VPS26A-SNX27 Interaction-Dependent mGluR5 Recycling in Dorsal Horn Neurons Mediates Neuropathic Pain in Rats.

Retromer, which crucially contributes to endosomal sorting machinery through the retrieval and recycling of signaling receptors away from degradation, has been identified as a critical element for glutamatergic-receptor-dependent neural plasticity at excitatory synapses. We observed it accompanied by behavioral allodynia; neuropathic injury time-dependently enhanced VPS26A and SNX27 expression; VPS26A-SNX27 coprecipitation; and VPS26A-positive, SNX27-positive, and VPS26A-SNX27 double-labeled immunoreactivity in the dorsal horn of Sprague Dawley rats that were all sufficiently ameliorated through the focal knock-down of spinal VPS26A expression. Although the knock-down of spinal SNX27 expression exhibited similar effects, spinal nerve ligation (SNL)-enhanced VPS26A expression remained unaffected. Moreover, SNL also increased membrane-bound and total mGluR5 abundance, VPS26A-bound SNX27 and mGluR5 and mGluR5-bound VPS26A and SNX27 coprecipitation, and mGluR5-positive and VPS26A/SNX27/mGluR5 triple-labeled immunoreactivity in the dorsal horn, and these effects were all attenuated through the focal knock-down of spinal VPS26A and SNX27 expression. Although administration with MPEP adequately ameliorated SNL-associated allodynia, mGluR5 expression, and membrane insertion, SNL-enhanced VPS26A and SNX27 expression were unaffected. Together, these results suggested a role of spinal VPS26A-SNX27-dependent mGluR5 recycling in the development of neuropathic pain. This is the first study that links retromer-associated sorting machinery with the spinal plasticity underlying pain hypersensitivity and proposes the possible pathophysiological relevance of endocytic recycling in pain pathophysiology through the modification of glutamatergic mGluR5 recycling. SIGNIFICANCE STATEMENT: VPS26A-SNX27-dependent mGluR5 recycling plays a role in the development of neuropathic pain. The regulation of the VPS26A-SNX27 interaction that modifies mGluR5 trafficking and expression in the dorsal horn provides a novel therapeutic strategy for pain relief.

Identification of molecular heterogeneity in SNX27-retromer-mediated endosome-to-plasma-membrane recycling.

Retromer is a protein assembly that orchestrates the sorting of transmembrane cargo proteins into endosome-to-Golgi and endosome-to-plasma-membrane transport pathways. Here, we have employed quantitative proteomics to define the interactome of human VPS35, the core retromer component. This has identified a number of new interacting proteins, including ankyrin-repeat domain 50 (ANKRD50), seriologically defined colon cancer antigen 3 (SDCCAG3) and VPS9-ankyrin-repeat protein (VARP, also known as ANKRD27). Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo-specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma-membrane sorting. Extending this, suppression of the retromer-associated WASH complex did not uniformly affect retromer cargo, thereby confirming cargo-specific functions for retromer-interacting proteins. Further analysis of the retromer-VARP interaction identified a role for retromer in endosome-to-melanosome transport. Suppression of VPS35 led to mistrafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway. Overall, these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex.

Cytoplasmic tail determines the membrane trafficking and localization of SARS-CoV-2 spike protein.

The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry through associating with ACE2 on host cells. Intracellular trafficking and palmitoylation of S protein are required for its function. The short cytoplasmic tail of S protein plays a key role in the intracellular trafficking, which contains the binding site for the host trafficking proteins such as COPI, COPII and SNX27. This cytoplasmic tail also contains the palmitoylation sites of S protein. Protein palmitoylation modification of S protein could be catalyzed by a family of zinc finger DHHC domain-containing protein palmitoyltransferases (ZDHHCs). The intracellular trafficking and membrane location facilitate surface expression of S protein and assembly of progeny virions. In this review, we summarize the function of S protein cytoplasmic tail in transportation and localization. S protein relies on intracellular trafficking pathways and palmitoylation modification to facilitate the life cycle of SARS-CoV-2, meanwhile it could interfere with the host transport pathways. The interplay between S protein and intracellular trafficking proteins could partially explain the acute symptoms or Long-COVID complications in multiple organs of COVID-19 patients.

SNX27-driven membrane localisation of OTULIN antagonises linear ubiquitination and NF-κB signalling activation.

BACKGROUND: Linear ubiquitination is a novel type of ubiquitination that plays important physiological roles in signalling pathways such as tumour necrosis factor (TNF) signalling. However, little is known about the regulatory mechanisms of linear ubiquitination, except the well-described enzymatic regulators E3 ligase linear ubiquitin chain assembly complex (LUBAC) and deubiquitinase OTULIN. RESULTS: Previously, we identified SNX27, a member of the sorting nexin family protein, as a selective linear ubiquitin chain interactor in mass spectrometry-based ubiquitin interaction screening. Here, we demonstrated that the interaction between the linear ubiquitin chain and SNX27 is mediated by the OTULIN. Furthermore, we found that SNX27 inhibits LUBAC-mediated linear ubiquitin chain formation and TNFα-induced signalling activation. Mechanistic studies showed that, upon TNFα stimulation, OTULIN-SNX27 is localised to membrane-associated TNF receptor complex, where OTULIN deubiquitinates the linear polyubiquitin chain that formed by the LUBAC complex. Significantly, chemical inhibition of SNX27-retromer translocation by cholera toxin inhibits OTULIN membrane localization. CONCLUSIONS: In conclusion, our study demonstrated that SNX27 inhibits TNFα induced NF-κB signalling activation via facilitating OTULIN to localize to TNF receptor complex.