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Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection1,2, yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of samples from patients with MIS-C to identify a distinct set of host proteins targeted by patient autoantibodies including a particular autoreactive epitope within SNX8, a protein involved in regulating an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed antibody responses from patients with MIS-C to the complete SARS-CoV-2 proteome and found enriched reactivity against a distinct domain of the SARS-CoV-2 nucleocapsid protein. The immunogenic regions of the viral nucleocapsid and host SNX8 proteins bear remarkable sequence similarity. Consequently, we found that many children with anti-SNX8 autoantibodies also have cross-reactive T cells engaging both the SNX8 and the SARS-CoV-2 nucleocapsid protein epitopes. Together, these findings suggest that patients with MIS-C develop a characteristic immune response to the SARS-CoV-2 nucleocapsid protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link between the infection and the inflammatory syndrome, with implications for better understanding a range of post-infectious autoinflammatory diseases.

The time course of signaling by peptide hormones, neural peptides, and other neuromodulators depends on their storage inside dense core vesicles (DCVs). Adaptor protein 3 (AP-3) assembles the membrane proteins that confer regulated release of DCVs and is thought to promote their trafficking from endosomes directly to maturing DCVs. We now find that regulated monoamine release from DCVs requires sorting nexin 5 (SNX5). Loss of SNX5 disrupts trafficking of the vesicular monoamine transporter (VMAT) to DCVs. The mechanism involves a role for SNX5 in retrograde transport of VMAT from endosomes to the TGN. However, this role for SNX5 conflicts with the proposed function of AP-3 in trafficking from endosomes directly to DCVs. We now identify a transient role for AP-3 at the TGN, where it associates with DCV cargo. Thus, retrograde transport from endosomes by SNX5 enables DCV assembly at the TGN by AP-3, resolving the apparent antagonism. A novel role for AP-3 at the TGN has implications for other organelles that also depend on this adaptor.

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. [ABSTRACT FROM AUTHOR]

Kinetoplastea are free living and parasitic protists with unique features among Eukaryota. Pathogenic Kinetoplastea parasites (i.e., Trypanosoma and Leishmania spp.) undergo several developmental transitions essential for survival in their hosts. These transitions require membrane and cytoskeleton reorganizations that involve phosphoinositides (PIs). Phospholipids like PIs are key regulators of vital functions in all eukaryotes including signal transduction, protein transport and sorting, membrane trafficking, and cytoskeleton and membrane remodeling. A large repertoire of PI-metabolizing enzymes and PI-binding proteins/effectors carrying distinct PI-binding modules like the PX (phox homology) module could play significant roles in the life and virulence of pathogenic Kinetoplastea. The aim of this study was to retrieve the entire spectrum of Kinetoplastea protein sequences containing the PX module (PX-proteins), predict their structures, and identify in them evolutionary conserved and unique traits. Using a large array of bioinformatics tools, protein IDs from two searches (based on PFam's pHMM for PX domain (PF00787)) were combined, aligned, and utilized for the construction of a new Kinetoplastea_PX pHMM. This three-step search retrieved 170 PX-protein sequences. Structural domain configuration analysis identified PX, Pkinase, Lipocalin_5, and Vps5/BAR3-WASP domains and clustered them into five distinct subfamilies. Phylogenetic tree and domain architecture analysis showed that some domain architectures exist in proteomes of all Kinetoplastea spp., while others are genus-specific. Finally, amino acid conservation logos of the Kinetoplastea spp. and Homo sapiens PX domains revealed high evolutionary conservation in residues forming the critical structural motifs for PtdIns3P recognition. This study highlights the PX-Pkinase domain architecture as unique within Trypanosoma spp. and forms the basis for a targeted functional analysis of Kinetoplastea PX-proteins as putative targets for a rational design of anti-parasitic drugs. [ABSTRACT FROM AUTHOR]

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

Lipid droplets (LDs) are hubs for lipid metabolism that form membrane contact sites with multiple organelles. In this issue, Hariri et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201808119) reveal the functions of Mdm1-mediated endoplasmic reticulum (ER)-LD tethering in yeast and Datta et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201808133) identify a role for the Mdm1 orthologue, Snx14, as an ER-LD tether that regulates lipid metabolism in human cells.

Kinetoplastea are free living and parasitic protists with unique features among Eukaryota. Pathogenic Kinetoplastea parasites (i.e., Trypanosoma and Leishmania spp.) undergo several developmental transitions essential for survival in their hosts. These transitions require membrane and cytoskeleton reorganizations that involve phosphoinositides (PIs). Phospholipids like PIs are key regulators of vital functions in all eukaryotes including signal transduction, protein transport and sorting, membrane trafficking, and cytoskeleton and membrane remodeling. A large repertoire of PI-metabolizing enzymes and PI-binding proteins/effectors carrying distinct PI-binding modules like the PX (phox homology) module could play significant roles in the life and virulence of pathogenic Kinetoplastea. The aim of this study was to retrieve the entire spectrum of Kinetoplastea protein sequences containing the PX module (PX-proteins), predict their structures, and identify in them evolutionary conserved and unique traits. Using a large array of bioinformatics tools, protein IDs from two searches (based on PFam’s pHMM for PX domain (PF00787)) were combined, aligned, and utilized for the construction of a new Kinetoplastea_PX pHMM. This three-step search retrieved 170 PX-protein sequences. Structural domain configuration analysis identified PX, Pkinase, Lipocalin_5, and Vps5/BAR3-WASP domains and clustered them into five distinct subfamilies. Phylogenetic tree and domain architecture analysis showed that some domain architectures exist in proteomes of all Kinetoplastea spp., while others are genus-specific. Finally, amino acid conservation logos of the Kinetoplastea spp. and Homo sapiens PX domains revealed high evolutionary conservation in residues forming the critical structural motifs for PtdIns3P recognition. This study highlights the PX-Pkinase domain architecture as unique within Trypanosoma spp. and forms the basis for a targeted functional analysis of Kinetoplastea PX-proteins as putative targets for a rational design of anti-parasitic drugs.

Chlamydia trachomatis is an obligate intracellular pathogen that resides in a membrane-bound compartment, the inclusion. The bacteria secrete a unique class of proteins, Incs, which insert into the inclusion membrane and modulate the host-bacterium interface. We previously reported that IncE binds specifically to the Sorting Nexin 5 Phox domain (SNX5-PX) and disrupts retromer trafficking. Here, we present the crystal structure of the SNX5-PX:IncE complex, showing IncE bound to a unique and highly conserved hydrophobic groove on SNX5. Mutagenesis of the SNX5-PX:IncE binding surface disrupts a previously unsuspected interaction between SNX5 and the cation-independent mannose-6-phosphate receptor (CI-MPR). Addition of IncE peptide inhibits the interaction of CI-MPR with SNX5. Finally, C. trachomatis infection interferes with the SNX5:CI-MPR interaction, suggesting that IncE and CI-MPR are dependent on the same binding surface on SNX5. Our results provide new insights into retromer assembly and underscore the power of using pathogens to discover disease-related cell biology.

BACKGROUND & AIMS: The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohns disease (CD) cause significant morbidity and are increasing in prevalence among all populations, including African Americans. More than 200 susceptibility loci have been identified in populations of predominantly European ancestry, but few loci have been associated with IBD in other ethnicities. METHODS: We performed 2 high-density, genome-wide scans comprising 2345 cases of African Americans with IBD (1646 with CD, 583 with UC, and 116 inflammatory bowel disease unclassified) and 5002 individuals without IBD (controls, identified from the Health Retirement Study and Kaiser Permanente database). Single-nucleotide polymorphisms (SNPs) associated at P

Abstract Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia. Sorting nexin 10 (SNX10) has been reported to be an important regulator in embryonic development and human diseases, however, little is known about its role in cardiac disease. The aim of this study was to investigate the clinical significance of SNX10 expression in AF. Methods: Nineteen valvular heart disease patients with AF and nine valvular heart disease patients with sinus rhythm (SR) were enrolled. Atrial tissue samples from patients undergoing open heart surgery were examined. Atrial tissues of normal hearts were obtained from two cases’ autopsies. The SNX10 expression and its associations with the degree of fibrosis were analyzed by immunohistochemistry and Masson’s trichrome staining. Results: SNX10 expression was detected in the cytoplasm of cardiac cells in human myocardial tissue. The SNX10 expression level was higher in the SR group than in the AF group (P=0.023). SNX10 expression was negatively associated with the degree of fibrosis (P=0.017, Spearman rho=-0.447), the New York Heart Association degree (P=0.003, Spearman rho=-0.545), left atrial diameter (P=0.038, Spearman rho=-0.393), right atrial diameter (P=0.043, Spearman rho=-0.386), and the brain natriuretic peptide (BNP) level 24 hours after surgery (P=0.030, Spearman rho=-0.426), but not the BNP level before surgery and 72 hours after surgery. No statistical significance was observed between SNX10 and the level of troponin T and C-reactive protein. Conclusion: Decreased SNX10 might serve as a potential risk factor in AF of the valvular heart disease.

Endocytosis is a fundamental process that controls protein/lipid composition of the plasma membrane, thereby shaping cellular metabolism, sensing, adhesion, signaling, and nutrient uptake. Endocytosis is essential for the cell to adapt to its surrounding environment, and a tight regulation of the endocytic mechanisms is required to maintain cell function and survival. This is particularly significant in the central nervous system (CNS), where composition of neuronal cell surface is crucial for synaptic functioning. In fact, distinct pathologies of the CNS are tightly linked to abnormal endolysosomal function, and several genome wide association analysis (GWAS) and biochemical studies have identified intracellular trafficking regulators as genetic risk factors for such pathologies. The sorting nexins (SNXs) are a family of proteins involved in protein trafficking regulation and signaling. SNXs dysregulation occurs in patients with Alzheimer's disease (AD), Down's syndrome (DS), schizophrenia, ataxia and epilepsy, among others, establishing clear roles for this protein family in pathology. Interestingly, restoration of SNXs levels has been shown to trigger synaptic plasticity recovery in a DS mouse model. This review encompasses an historical and evolutionary overview of SNXs protein family, focusing on its organization, phyla conservation, and evolution throughout the development of the nervous system during speciation. We will also survey SNXs molecular interactions and highlight how defects on SNXs underlie distinct pathologies of the CNS. Ultimately, we discuss possible strategies of intervention, surveying how our knowledge about the fundamental processes regulated by SNXs can be applied to the identification of novel therapeutic avenues for SNXs-related disorders. [ABSTRACT FROM AUTHOR]

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.

Sorting nexins (SNXs) are a diverse group of cytoplasmic‐ and membrane‐associated phosphoinositide‐binding proteins containing the PX domain proteins. The function of SNX proteins in regulating intracellular protein trafficking consists of endocytosis, endosomal sorting, and endosomal signaling. Dysfunctions of SNX proteins are demonstrated to be involved in several cancerous/neoplastic diseases. Here, we review the accumulated evidence of the molecular structure and biological function of SNX proteins and discuss the regulatory role of SNX proteins in distinct cancerous/neoplastic diseases. SNX family proteins may be a valuable potential biomarker and therapeutic strategy for diagnostics and treatment of cancerous/neoplastic diseases. [ABSTRACT FROM AUTHOR]

Genetic and environmental components as well as their interaction contribute to the risk of schizophrenia, making it highly relevant to include environmental factors in genetic studies of schizophrenia. This study comprises genome-wide association (GWA) and follow-up analyses of all individuals born in Denmark since 1981 and diagnosed with schizophrenia as well as controls from the same birth cohort. Furthermore, we present the first genome-wide interaction survey of single nucleotide polymorphisms (SNPs) and maternal cytomegalovirus (CMV) infection. The GWA analysis included 888 cases and 882 controls, and the follow-up investigation of the top GWA results was performed in independent Danish (1396 cases and 1803 controls) and German-Dutch (1169 cases, 3714 controls) samples. The SNPs most strongly associated in the single-marker analysis of the combined Danish samples were rs4757144 in ARNTL (P=3.78 × 10(-6)) and rs8057927 in CDH13 (P=1.39 × 10(-5)). Both genes have previously been linked to schizophrenia or other psychiatric disorders. The strongest associated SNP in the combined analysis, including Danish and German-Dutch samples, was rs12922317 in RUNDC2A (P=9.04 × 10(-7)). A region-based analysis summarizing independent signals in segments of 100 kb identified a new region-based genome-wide significant locus overlapping the gene ZEB1 (P=7.0 × 10(-7)). This signal was replicated in the follow-up analysis (P=2.3 × 10(-2)). Significant interaction with maternal CMV infection was found for rs7902091 (P(SNP × CMV)=7.3 × 10(-7)) in CTNNA3, a gene not previously implicated in schizophrenia, stressing the importance of including environmental factors in genetic studies.

Previous studies have identified an interaction between the human papillomavirus (HPV) L2 minor capsid protein and sorting nexins 17 and 27 (SNX17 and SNX27) during virus infection. Further studies show the involvement of both retromer and retriever complexes in this process since knockdown of proteins from either complex impairs infection. In this study, we show that HPV L2 and 5-ethynyl-29-deoxyuridine (EdU)-labeled pseudovirions colocalize with both retromer and retriever, with components of each complex being bound by L2 during infection. We also show that both sorting nexins may interact with either of the recycling complexes but that the interaction between SNX17 and HPV16 L2 is not responsible for retriever recruitment during infection, instead being required for retromer recruitment. Furthermore, we show that retriever recruitment most likely involves a direct interaction between L2 and the C16orf62 subunit of the retriever, in a manner similar to that of its interaction with the VPS35 subunit of retromer. IMPORTANCE Previous studies identified sorting nexins 17 and 27, as well as the retromer complex, as playing a role in HPV infection. This study shows that the newly identified retriever complex also plays an important role and begins to shed light on how both sorting nexins contribute to retromer and retriever recruitment during the infection process. [ABSTRACT FROM AUTHOR]

Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia. Sorting nexin 10 (SNX10) has been reported to be an important regulator in embryonic development and human diseases, however, little is known about its role in cardiac disease. The aim of this study was to investigate the clinical significance of SNX10 expression in AF. Methods: Nineteen valvular heart disease patients with AF and nine valvular heart disease patients with sinus rhythm (SR) were enrolled. Atrial tissue samples from patients undergoing open heart surgery were examined. Atrial tissues of normal hearts were obtained from two cases’ autopsies. The SNX10 expression and its associations with the degree of fibrosis were analyzed by immunohistochemistry and Masson’s trichrome staining. Results: SNX10 expression was detected in the cytoplasm of cardiac cells in human myocardial tissue. The SNX10 expression level was higher in the SR group than in the AF group (P=0.023). SNX10 expression was negatively associated with the degree of fibrosis (P=0.017, Spearman rho=-0.447), the New York Heart Association degree (P=0.003, Spearman rho=-0.545), left atrial diameter (P=0.038, Spearman rho=-0.393), right atrial diameter (P=0.043, Spearman rho=-0.386), and the brain natriuretic peptide (BNP) level 24 hours after surgery (P=0.030, Spearman rho=-0.426), but not the BNP level before surgery and 72 hours after surgery. No statistical significance was observed between SNX10 and the level of troponin T and C-reactive protein. Conclusion: Decreased SNX10 might serve as a potential risk factor in AF of the valvular heart disease. [ABSTRACT FROM AUTHOR]

In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR., (© 2024 Wu et al.)

Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.

Postsynaptic density 95/discs large/zonus occludens-1 (PDZ) domain-interacting motifs, in addition to their well-established roles in protein scaffolding at the cell surface, are proposed to act as cis-acting determinants directing the molecular sorting of transmembrane cargo from endosomes to the plasma membrane. This hypothesis requires the existence of a specific trans-acting PDZ protein that mediates the proposed sorting operation in the endosome membrane. Here, we show that sorting nexin 27 (SNX27) is required for efficient PDZ-directed recycling of the beta(2)-adrenoreceptor (beta(2)AR) from early endosomes. SNX27 mediates this sorting function when expressed at endogenous levels, and its recycling activity requires both PDZ domain-dependent recognition of the beta(2)AR cytoplasmic tail and Phox homology (PX) domain-dependent association with the endosome membrane. These results identify a discrete role of SNX27 in PDZ-directed recycling of a physiologically important signaling receptor, and extend the concept of cargo-specific molecular sorting in the recycling pathway.

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo. [ABSTRACT FROM AUTHOR]

Dysregulation of various APP trafficking components in the endosome has been previously implicated in Alzheimer’s disease (AD). Although single nucleotide polymorphisms within the gene locus encoding the endosomal component, SNX8 have been previously associated with AD, how SNX8 levels are altered and its contribution to AD onset is currently unknown. Here, we observe decreased expression of SNX8 in human AD and AD mouse brain. SNX8 predominantly localized to early and late endosomes, where SNX8 overexpression enhanced total APP levels, cell surface APP distribution and consequent soluble APPα cleavage. SNX8 depletion resulted in elevated β-amyloid (Aβ) levels, while SNX8 overexpression reduced Aβ levels in cells and in an APP/PS1 AD mouse model. Importantly, SNX8 overexpression rescued cognitive impairment in APP/PS1 mice. Together, these results implicate a neuroprotective role for SNX8 in enhancing non-amyloidogenic APP trafficking and processing pathways. Given that endosomal dysfunction is an early event in AD, restoration of dysfunctional endosomal components such as SNX8 may be beneficial in future therapeutic strategies.

The G-protein coupled receptor (GPCR) signaling was long believed to involve activation of receptor exclusively at the cell surface, followed by its binding to heterotrimeric G-proteins and arrestins to trigger various intracellular signaling cascades, and termination of signaling by internalization of the receptor. It is now accepted that many GPCRs continue to signal after internalization in the endosomes. Since the breakthrough discoveries of nuclear binding sites for their ligands in 1980s, several GPCRs have been detected at cell nuclei. But mechanisms of nuclear localization of GPCRs, many of whom contain putative nuclear localization signals, remain poorly understood to date. Nevertheless, it is known that subcellular trafficking of GPCRs is regulated by members of Ras superfamily of small GTPases, most notably by Rab and Arf GTPases. In this commentary, we highlight several recent studies which suggest novel roles of small GTPases, importins and sorting nexin proteins in the nuclear translocation of GPCRs via vesicular transport pathways. Taken together with increasing evidence for in vivo functionality of the nuclear GPCRs, better understanding of their trafficking will provide valuable clues in cell biology. [ABSTRACT FROM AUTHOR]

Endosomes are dynamic intracellular compartments that control the sorting of a constant stream of different transmembrane cargos either for ESCRT‐mediated degradation or for egress and recycling to compartments such as the Golgi and the plasma membrane. The recycling of cargos occurs within tubulovesicular membrane domains and is facilitated by peripheral membrane protein machineries that control both membrane remodelling and selection of specific transmembrane cargos. One of the primary sorting machineries is the Retromer complex, which controls the recycling of a large array of different cargo molecules in cooperation with various sorting nexin (SNX) adaptor proteins. Recently a Retromer‐like complex was also identified that controls plasma membrane recycling of cargos including integrins and lipoprotein receptors. Termed "Retriever," this complex uses a different SNX family member SNX17 for cargo recognition, and cooperates with the COMMD/CCDC93/CCDC22 (CCC) complex to form a larger assembly called "Commander" to mediate endosomal trafficking. In this review we focus on recent advances that have begun to provide a molecular understanding of these two distantly related transport machineries. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Sorting nexin 17 (SNX17) is a critical cytoplasmic adaptor protein that regulates endosomal trafficking of membrane proteins to determine their recycling and/or degradation. The potential role of SNX17 in cardiovascular pathophysiology has not been reported. Methods and results Cardiac arrhythmias were monitored using standard limb lead II electrocardiograph, and cardiac performances were determined by echocardiography in a rat model of myocardial infarction (MI) created by left anterior descending coronary artery ligation. We found that SNX17 was substantially downregulated in ischemic myocardium. The downregulation contributed to the cardiac electrical disturbances and contractile dysfunction as SNX17 replacement mitigated the detrimental alterations of MI hearts. Specifically, silence of SNX17 expression using RNA interference caused intracellular Ca2+ overload as revealed by the abnormal rise of resting [Ca2+] i and deceleration of Ca2+ decay, whereas SNX17 overexpression using vectors elicited the opposite effects. Moreover, the protein level of SERCA2a was significantly decreased by silencing SNX17. Immunohistochemistry indicated that SNX17 and SERCA2a were co-localized, and co-immunoprecipitation revealed the binding between the phox-homology domain of SNX17 and SERCA2a protein. Furthermore, lysosome inhibitor chloroquine prevented SNX17 silencing-induced reduction of SERCA2a protein level. Conclusion Abnormal downregulation of SNX17 contributes to ischemic damages of cardiac electrophysiology and contractile function. SNX17 is an endogenous anti-arrhythmic factor acting by preserving functional SERCA2a protein in MI thereby offering a new strategy for the management of MI to alleviate ischemic myocardial injuries. Highlights • SNX17, a member of SNXs family, has been established playing a fundamental role in endosomes recycling and lysosomal degradation relevance to diversity of human diseases. • Our findings unexpectedly reveal that a unique aspect of the SNX17-dependent management is the judgement of cardiomyocyte fate. • The deficiency of SNX17 initiates SERCR2a protein downregulation by disturbing intracellular proper transport of SERCA2a. • A plausible underlying mechanism arouse in this study is supplement of SNX17 stabilize the intracellular calcium disorders by recovering the SERCA2a protein level in setting of myocardial infarction. • Considering that SNX17 is the guarantee of SERCA2a performance, aiming directly at SNX17 might offer a safe and efficient intervention and therapeutic strategy for SERCA2a-associated cardiovascular diseases. [ABSTRACT FROM AUTHOR]

The sorting nexin (SNX) family proteins play an essential role in vesicular transport, cell signaling, and membrane remodeling. The SNX members SNX1/2 and SNX5/6 form dimers, and mediate endosome-to-trans Golgi network (TGN) transport through coordinating cargo selection and membrane remodeling. It is well-known how a SNX-BAR protein forms a homodimer; however, it is less clear how a heterodimer is formed. Here a detailed expression and purification protocol of the SNX1/SNX6 complex, from both worm and human, is described. Keys to the successful protein production include co-expression of both genes, and inclusion of glycerol in the protein buffer. Solution studies suggest that SNX1 and SNX6 form a 1:1 heterodimer. The production of a large amount, high quality of the SNX1/SNX6 complex provides a basis for future biochemical and structural studies of the complex, and in vitro reconstitution of SNX1/SNX6-mediated transport. [ABSTRACT FROM AUTHOR]

Group I metabotropic glutamate receptors (mGluRs) function as modulators of neuronal physiology and they have also been implicated in various neuropsychiatric disorders. Trafficking of mGluRs plays important roles in controlling the precise localization of these receptors at specific region of the cell, as well as it regulates the activity of these receptors. Despite this obvious significance, we know very little about the cellular machineries that control the trafficking of these receptors in the CNS. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of few cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. Using "molecular replacement" approach in hippocampal neurons derived from mice of both sexes, we show here that SNX1 plays critical role in the trafficking of mGluR1, a member of the group I mGluR family. Overexpression of dominant-negative SNX1 or knockdown of endogenous SNX1 resulted in the rapid recycling of the receptor. Importantly, recycling via the rapid recycling route, did not allow the resensitization of the receptors. Our data suggest that both, N-terminal and C-terminal region of SNX1 play critical role in the normal trafficking of the receptor. In addition, we also show here that SNX1 regulates the trafficking of mGluR1 through the interaction with Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), a protein that has been implicated in both signaling and vesicular trafficking. Thus, these studies reveal a mechanistic role of SNX1 in the trafficking of group I mGluRs and its physiological implications. [ABSTRACT FROM AUTHOR]

Protein homeostasis is maintained by removing misfolded, damaged, or excess proteins and damaged organelles from the cell by three major pathways; the ubiquitin-proteasome system, the autophagy-lysosomal pathway, and the endo-lysosomal pathway. The requirement for ubiquitin provides a link between all three pathways. Sorting nexins are a highly conserved and diverse family of membrane-associated proteins that not only traffic proteins throughout the cells but also provide a second common thread between protein homeostasis pathways. In this review, we will discuss the connections between sorting nexins, ubiquitin, and the interconnected roles they play in maintaining protein quality control mechanisms. Underlying their importance, genetic defects in sorting nexins are linked with a variety of human diseases including neurodegenerative, cardiovascular diseases, viral infections, and cancer. This serves to emphasize the critical roles sorting nexins play in many aspects of cellular function.

Intracellular pathogens have developed elegant mechanisms to modulate host endosomal trafficking. The highly conserved retromer pathway has emerged as an important target of viruses and intravacuolar bacteria. Some pathogens require retromer function to survive. For others, retromer activity restricts intracellular growth; these pathogens must disrupt retromer function to survive. In this review, we discuss recent paradigm changes to the current model for retromer assembly and cargo selection. We highlight how the study of pathogen effectors has contributed to these fundamental insights, with a special focus on the biology and structure of two recently described bacterial effectors, Chlamydia trachomatis IncE and Legionella pneumophila RidL. These two pathogens employ distinct strategies to target retromer components and overcome restriction of intracellular growth imposed by retromer. [ABSTRACT FROM AUTHOR]

Background & Aims Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. However, the cellular defense mechanisms underlying ALD are not well understood. Recent studies highlighted the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Sorting nexin (SNX)-10 has a regulatory function in endolysosomal trafficking and stabilisation. Here, we investigated the roles of SNX10 in CMA activation and in the pathogenesis of alcohol-induced liver injury and steatosis. Methods Snx10 knockout ( Snx10 KO) mice and their wild-type (WT) littermates fed either the Lieber–DeCarli liquid alcohol diet or a control liquid diet, and primary cultured WT and Snx10 KO hepatocytes stimulated with ethanol, were used as in vivo and in vitro ALD models, respectively. Activation of CMA, liver injury parameters, inflammatory cytokines, oxidative stress and lipid metabolism were measured. Results Compared with WT littermates, Snx10 KO mice exhibited a significant amelioration in ethanol-induced liver injury and hepatic steatosis. Both in vivo and in vitro studies showed that SNX10 deficiency upregulated lysosome-associated membrane protein type 2A (LAMP-2A) expression and CMA activation, which could be reversed by SNX10 overexpression in vitro . LAMP-2A interference confirmed that the upregulation of Nrf2 and AMPK signalling pathways induced by SNX10 deficiency relied on CMA activation. Pull-down assays revealed an interaction between SNX10 and cathepsin A (CTSA), a key enzyme involved in LAMP-2A degradation. Deficiency in SNX10 inhibited CTSA maturation and increased the stability of LAMP-2A, resulting in an increase in CMA activity. Conclusions SNX10 controls CMA activity by mediating CTSA maturation, and, thus, has an essential role in alcohol-induced liver injury and steatosis. Our results provide evidence for SNX10 as a potential promising therapeutic target for preventing or ameliorating liver injury in ALD. Lay summary Alcoholic liver disease is a major cause of morbidity and mortality worldwide. Recent studies highlight the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Our study reveals that deficiency of sorting nexin (SNX) 10 increases the stability of LAMP-2A by inhibiting cathepsin A maturation, resulting in the increase of CMA activity and, thus, alleviates alcohol-induced liver injury and steatosis. [ABSTRACT FROM AUTHOR]

The sorting nexins family of proteins (SNXs) plays pleiotropic functions in protein trafficking and intracellular signaling and has been associated with several disorders, namely Alzheimer’s disease and Down’s syndrome. Despite the growing association of SNXs with neurodegeneration, not much is known about their function in the nervous system. The aim of this work was to use the nematode Caenorhabditis elegans that encodes in its genome eight SNXs orthologs, to dissect the role of distinct SNXs, particularly in the nervous system. By screening the C. elegans SNXs deletion mutants for morphological, developmental and behavioral alterations, we show here that snx-3 gene mutation leads to an array of developmental defects, such as delayed hatching, decreased brood size and life span and reduced body length. Additionally, ∆snx-3 worms present increased susceptibility to osmotic, thermo and oxidative stress and distinct behavioral deficits, namely, a chemotaxis defect which is independent of the described snx-3 role in Wnt secretion. ∆snx-3 animals also display abnormal GABAergic neuronal architecture and wiring and altered AIY interneuron structure. Pan-neuronal expression of C. elegans snx-3 cDNA in the ∆snx-3 mutant is able to rescue its locomotion defects, as well as its chemotaxis toward isoamyl alcohol. Altogether, the present work provides the first in vivo evidence of the SNX-3 role in the nervous system. [ABSTRACT FROM AUTHOR]

ADAM9 is an active member of the family of transmembrane ADAMs (a disintegrin and metalloproteases). It plays a role in processes such as bone formation and retinal neovascularization, and importantly, its expression in human cancers correlates with disease stage and poor prognosis. Functionally, ADAM9 can cleave several transmembrane proteins, thereby shedding their ectodomains from the cell surface. Moreover, ADAM9 regulates cell behavior by binding cell-surface receptors such as integrin and membrane-type matrix metalloproteases. Because these functions are mainly restricted to the cell surface, understanding the mechanisms regulating ADAM9 localization and activity at this site is highly important. To this end, we here investigated how intracellular trafficking regulates ADAM9 availability at the cell surface. We found that ADAM9 undergoes constitutive clathrin-dependent internalization and subsequent degradation or recycling to the plasma membrane. We confirmed previous findings of an interaction between ADAM9 and the intracellular sorting protein, sorting nexin 9 (SNX9), as well as its close homolog SNX18. Knockdown of either SNX9 or SNX18 had no apparent effects on ADAM9 internalization or recycling. However, double knockdown of SNX9 and SNX18 decreased ADAM9 internalization significantly, demonstrating a redundant role in this process. Moreover, SNX9 knockdown revealed a nonredundant effect on overall ADAM9 protein levels, resulting in increased ADAM9 levels at the cell surface, and a corresponding increase in the shedding of Ephrin receptor B4, a well-knownADAM9substrate. Together, our findings demonstrate that intracellular SNX9-mediated trafficking constitutes an important ADAM9 regulatory pathway. [ABSTRACT FROM AUTHOR]

Chaperone-mediated autophagy (CMA) characterized by the selective degradation of target proteins has been linked with tumorigenesis in recent years. Here, we explored the function of sorting nexin 10 (SNX10), a protein involved in maintaining endosome/lysosome homeostasis, in mediating CMA activity and its impact on the progression of mouse inflammation-driven colorectal cancer. Our results revealed that SNX10 deficiency increased the activation of CMA by preventing the degradation of lysosomal LAMP-2A. In SNX10 KO cells, we disclosed that p21Cip1/WAF1, a master effector in various tumor suppressor pathways, is a substrate of CMA, and decrease of p21Cip1/WAF1 caused by SNX10-mediated CMA activation contributes to HCT116 cell proliferation and survival. Moreover, we found that SNX10 KO promoted tumorigenesis in the mouse colorectum which could be restored by SNX10 over-expression. Furthermore, SNX10 was remarkably down-regulated in human CRC tissues which showed the increased activity of CMA and decreased expression of p21Cip1/WAF1. These findings suggest that SNX10 acts as a tumor suppressor in the mouse colorectum and drives inflammation-associated colorectal cancer by a chaperone-mediated autophagy mechanism. [ABSTRACT FROM AUTHOR]

Background: Sorting nexins (SNXs) have diverse functions in protein sorting and membrane trafficking. Recently, single-nucleotide polymorphisms in SNX3 were found to be associated with Alzheimer disease. However, it remains unknown whether SNX3 participates in amyloid (A)β peptide production. Objective: To examine the role of SNX3 in Aβ production and APP processing. Methods: The effect of increased expression of SNX3 was studied in HEK293T cells. Aβ peptides were measured by immunoassay. Protein-protein association was analyzed by a bimolecular fluorescence complementation (BiFC) assay. APP uptake was measured with an α-bungarotoxin-binding assay, and flow cytometry was used to measure cell surface APP levels. Results: We found that overexpression of SNX3 in HEK293T cells decreases the levels of secreted Aβ and soluble N-terminal APP fragments (sAPPβ). The reduction correlated with a decreased association of APP with BACE1, as revealed by BiFC. This effect may, in part, be explained by a reduced internalization of APP; SNX3 overexpression reduced APP internalization as determined by an α-bungarotoxin-binding assay, and caused increased APP levels on the cell surface, as shown by flow cytometry. In addition, SNX3 overexpression increased the cellular levels of full-length APP. Conclusion: These results provide evidence that SNX3 regulates Aβ production by influencing the internalization of APP. [ABSTRACT FROM AUTHOR]

Abnormal production of amyloid-β peptides (Aβ) by proteolytic processing of amyloid precursor protein (APP) is thought to be central to the pathogenesis of Alzheimer's disease (AD). Although many efforts have been made to investigate mechanisms that regulate APP processing, many details remain incompletely understood. Sorting nexins (SNXs) are a family of proteins which are involved in many intracellular trafficking events. Several SNXs have been implicated in APP processing and Aβ production. In this study, we extended the investigation to SNX7. We found that overexpression of SNX7 in HEK293T cells reduces the levels of secreted Aβ and β-cleaved N-terminal APP fragments (sAPPβ). Moreover, SNX7 overexpression caused a significant reduction of the steady-state levels of APP as well as of the cell surface APP levels. By using NH 4 Cl and Bafilomycin A1 to inhibit the lysosomal degradative pathway, we found that the reduction of APP induced by SNX7 overexpression was prevented by such inhibition. No change in the cell surface distribution or steady-state levels of BACE1 was detected after overexpression of SNX7. Taken together, these results suggest that SNX7 regulates Aβ production by directing APP for degradation. [ABSTRACT FROM AUTHOR]

Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ~260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation. [ABSTRACT FROM AUTHOR]