Project description:Sorting nexin 4 (SNX4) is an evolutionary conserved protein that mediates recycling from endosomes back to the plasma membrane in yeast and mammalian cells. SNX4 is expressed in the brain. Altered protein levels are associated with Alzheimer's disease, but the neuronal localization and function of SNX4 have not been addressed. Using a new antibody, endogenous neuronal SNX4 co-localized with both early and recycling endosome markers, similar to the reported localization of SNX4 in non-neuronal cells. Neuronal SNX4 accumulated specifically in synaptic areas, with a predominant localization to presynaptic terminals. Acute depletion of neuronal SNX4 using independent short hairpin RNAs did not affect the levels of the transferrin receptor, a canonical SNX4 cargo. Quantitative mass spectrometry revealed that upon SNX4 knockdown the class of proteins involved in neurotransmission was the most dysregulated. This included integral membrane proteins at both the presynaptic and postsynaptic side of the synapse that participate in diverse synaptic processes such as synapse assembly, neurotransmission and the synaptic vesicle cycle. These data suggest that SNX4 is implicated in a variety of synaptic processes.
Project description:Retromer is a phylogenetically conserved, multisubunit coat complex that controls endosomal protein trafficking and sorting. Mutations in the retromer gene VPS35 cause late-onset Parkinson disease, suggesting that trafficking defects cause neurodegeneration. Sorting nexins assist retromer to guide cell surface proteins to their assigned destinations, and our interest here is sorting nexin 3 (Snx3). Snx3 binds to membranes via a phox homolog (PX) domain that binds phosphatidylinositol 3-phosphate (PI3P), and in human cells its cargo proteins are the transferrin and Wnt receptors and the divalent metal ion transporter, whereas in yeast the best characterized cargo is the iron permease Ftr1. We recently discovered that α-synuclein inhibits Snx3-retromer recycling of Ftr1 in an unexpected way: α-synuclein, which avidly binds to negatively charged lipids, blocks the association of Snx3 to early endosomes. Here, we discuss mechanisms by which α-synuclein can disrupt Snx3-retromer-mediated recycling.
Project description:The retromer complex was first identified more than 20 years ago through studies conducted in the yeast Saccharomyces cerevisiae. Data obtained using many different model systems have revealed that retromer is a key component of the endosomal protein sorting machinery being necessary for recognition of membrane "cargo" proteins and formation of tubular carriers that function as transport intermediates. Naturally, over the course of time and with literally hundreds of papers published on retromer, there have arisen disparities, conflicting observations and some controversies as to how retromer functions in endosomal protein sorting - the most note-worthy being associated with the two activities that define a vesicle coat: cargo selection and vesicle/tubule formation. In this review, we will attempt to chart a course through some of the more fundamental controversies to arrive at a clearer understanding of retromer.
Project description:Glioblastoma is the most malignant primary brain tumor for which the prognosis is still dismal despite 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 to 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 via post-transcriptional regulation of the PDGF receptor 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.
Project description:The chemokine receptor CXCR4, a G protein-coupled receptor, is targeted for lysosomal degradation via a ubiquitin-dependent mechanism that involves the endosomal sorting complex required for transport (ESCRT) machinery. We have reported recently that arrestin-2 also targets CXCR4 for lysosomal degradation; however, the molecular mechanisms by which this occurs remain poorly understood. Here, we show that arrestin-2 interacts with ESCRT-0, a protein complex that recognizes and sorts ubiquitinated cargo into the degradative pathway. Signal-transducing adaptor molecule (STAM)-1, but not related STAM-2, interacts directly with arrestin-2 and colocalizes with CXCR4 on early endosomal antigen 1-positive early endosomes. Depletion of STAM-1 by RNA interference and disruption of the arrestin-2/STAM-1 interaction accelerates agonist promoted degradation of CXCR4, suggesting that STAM-1 via its interaction with arrestin-2 negatively regulates CXCR4 endosomal sorting. Interestingly, disruption of this interaction blocks agonist promoted ubiquitination of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) but not CXCR4 and STAM-1 ubiquitination. Our data suggest a mechanism whereby arrestin-2 via its interaction with STAM-1 modulates CXCR4 sorting by regulating the ubiquitination status of HRS.
Project description:Up to 1 in 3000 individuals in the United States have ?-1 antitrypsin deficiency, and the most common cause of this disease is homozygosity for the antitrypsin-Z variant (ATZ). ATZ is inefficiently secreted, resulting in protein deficiency in the lungs and toxic polymer accumulation in the liver. However, only a subset of patients suffer from liver disease, suggesting that genetic factors predispose individuals to liver disease. To identify candidate factors, we developed a yeast ATZ expression system that recapitulates key features of the disease-causing protein. We then adapted this system to screen the yeast deletion mutant collection to identify conserved genes that affect ATZ secretion and thus may modify the risk for developing liver disease. The results of the screen and associated assays indicate that ATZ is degraded in the vacuole after being routed from the Golgi. In fact, one of the strongest hits from our screen was Vps10, which can serve as a receptor for the delivery of aberrant proteins to the vacuole. Because genome-wide association studies implicate the human Vps10 homolog, sortilin, in cardiovascular disease, and because hepatic cell lines that stably express wild-type or mutant sortilin were recently established, we examined whether ATZ levels and secretion are affected by sortilin. As hypothesized, sortilin function impacts the levels of secreted ATZ in mammalian cells. This study represents the first genome-wide screen for factors that modulate ATZ secretion and has led to the identification of a gene that may modify disease severity or presentation in individuals with ATZ-associated liver disease.
Project description:The G?s subunit is classically involved in the signal transduction of G protein-coupled receptors (GPCRs) at the plasma membrane. Recent evidence has revealed noncanonical roles for G?s in endosomal sorting of receptors to lysosomes. However, the mechanism of action of G?s in this sorting step is still poorly characterized. Here, we report that G?s interacts with ubiquitin to regulate the endosomal sorting of receptors for lysosomal degradation. We reveal that the N-terminal extremity of G?s contains a ubiquitin-interacting motif (UIM), a sorting element usually found in the endosomal sorting complex required for transport (ESCRT) machinery responsible for sorting ubiquitinated receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Mutation of the UIM in G?s confirmed the importance of ubiquitin interaction for the sorting of epidermal growth factor receptor (EGFR) into ILVs for lysosomal degradation. These findings demonstrate a role for G?s as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of G?s in receptor trafficking and signaling for the fine-tuning of the cellular response.
Project description:Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.
Project description:Sorting nexin 3 (SNX3) belongs to a sub-family of sorting nexins that primarily contain a single Phox homology domain capable of binding phosphoinositides and membranes. We report the complete (1)H, (13)C and (15)N resonance assignments of the full-length human SNX3 protein and identification of its secondary structure elements, revealing a canonical fold and unstructured termini.
Project description:Prion propagation has been modeled in vitro; however, the low infectious titer of PrP(Sc) thus generated has cast doubt on the "protein-only" hypothesis. Here we show that prion delivery on suitable nitrocellulose carrier particles abrogates the apparent dissociation of PrP(Sc) and infectivity. Misfolded prion protein generated by protein misfolding cyclic amplification is as infectious as authentic brain-derived PrP(Sc) provided that confounding effects related to differences in the size distribution of prion protein aggregates generated in vitro and consecutive differences in regard to biological clearance are abolished.