Sorting Nexin Research Articles

Identification of specific role of SNX family in gastric cancer prognosis evaluation

We here perform a systematic bioinformatic analysis to uncover the role of sorting nexin (SNX) family in clinical outcome of gastric cancer (GC). Comprehensive bioinformatic analysis were realized with online tools such as TCGA, GEO, String, Timer, cBioportal and Kaplan–Meier Plotter. Statistical analysis was conducted with R language or Perl, and artificial neural network (ANN) model was established using Python. Our analysis demonstrated that SNX4/5/6/7/8/10/13/14/15/16/20/22/25/27/30 were higher expressed in GC, whereas SNX1/17/21/24/33 were in the opposite expression profiles. GSE66229 was employed as verification of the differential expression analysis based on TCGA. Clustering results gave the relative transcriptional levels of 30 SNXs in tumor, and it was totally consistent to the inner relevance of SNXs at mRNA level. Protein–Protein Interaction map showed closely and complex connection among 33 SNXs. Tumor immune infiltration analysis asserted that SNX1/3/9/18/19/21/29/33, SNX1/17/18/20/21/29/31/33, SNX1/2/3/6/10/18/29/33, and SNX1/2/6/10/17/18/20/29 were strongly correlated with four kinds of survival related tumor-infiltrating immune cells, including cancer associated fibroblast, endothelial cells, macrophages and Tregs. Kaplan–Meier survival analysis based on GEO presented more satisfactory results than that based on TCGA-STAD did, and all the 29 SNXs were statistically significant, SNX23/26/28 excluded. SNXs alteration contributed to microsatellite instability (MSI) or higher level of MSI-H (hyper-mutated MSI or high level of MSI), and other malignancy encompassing mutation of TP53 and ARID1A, as well as methylation of MLH1.The multivariate cox model, visualized as a nomogram, performed excellently in patients risk classification, for those with higher risk-score suffered from shorter overall survival (OS). Compared to previous researches, our ANN models showed a predictive power at a middle-upper level, with AUC of 0.87/0.72, 0.84/0.72, 0.90/0.71 (GSE84437), 0.98/0.66, 0.86/0.70, 0.98/0.71 (GSE66229), 0.94/0.66, 0.83/0.71, 0.88/0.72 (GSE26253) corresponding to one-, three- and five-year OS and recurrence free survival (RFS) estimation, especially ANN model built with GSE66229 including exclusively SNXs as input data. The SNX family shows great value in postoperative survival evaluation of GC, and ANN models constructed using SNXs transcriptional data manifesting excellent predictive power in both OS and RFS prediction works as convincing verification to that.

Vacuole fragmentation depends on a novel Atg18-containing retromer-complex

ABSTRACT The yeast PROPPIN Atg18 folds as a β-propeller with two binding sites for phosphatidylinositol-3-phosphate (PtdIns3P) and PtdIns(3,5)P2 at its circumference. Membrane insertion of an amphipathic loop of Atg18 leads to membrane tubulation and fission. Atg18 has known functions at the PAS during macroautophagy, but the functional relevance of its endosomal and vacuolar pool is not well understood. Here we show in a proximity-dependent labeling approach and by co-immunoprecipitations that Atg18 interacts with Vps35, a central component of the retromer complex. The binding of Atg18 to Vps35 is competitive with the sorting nexin dimer Vps5 and Vps17. This suggests that Atg18 within the retromer can substitute for both the phosphoinositide binding and the membrane bending capabilities of these sorting nexins. Indeed, we found that Atg18-retromer is required for PtdIns(3,5)P2-dependent vacuolar fragmentation during hyperosmotic stress. The Atg18-retromer is further involved in the normal sorting of the integral membrane protein Atg9. However, PtdIns3P-dependent macroautophagy and the selective cytoplasm-to-vacuole targeting (Cvt) pathway are only partially affected by the Atg18-retromer. We expect that this is due to the plasticity of the different sorting pathways within the endovacuolar system. Abbreviations: BAR: bin/amphiphysin/Rvs; FOA: 5-fluoroorotic acid; PAS: phagophore assembly site; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology.

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.

CD14 recycling modulates LPS‐induced inflammatory responses of murine macrophages

TLR4 is activated by the bacterial endotoxin lipopolysaccharide (LPS) and triggers two proinflammatory signaling cascades: a MyD88-dependent one in the plasma membrane, and the following TRIF-dependent one in endosomes. An inadequate inflammatory reaction can be detrimental for the organism by leading to sepsis. Therefore, novel approaches to therapeutic modulation of TLR4 signaling are being sought after. The TLR4 activity is tightly connected with the presence of CD14, a GPI-anchored protein that transfers LPS monomers to the receptor and controls its endocytosis. In this study we focused on CD14 trafficking as a still poorly understood factor affecting TLR4 activity. Two independent assays were used to show that after endocytosis CD14 can recycle back to the plasma membrane in both unstimulated and stimulated cells. This route of CD14 trafficking can be controlled by sorting nexins (SNX) 1, 2 and 6, and is important for maintaining the surface level and the total level of CD14, but can also affect the amount of TLR4. Silencing of these SNXs attenuated especially the CD14-dependent endosomal signaling of TLR4, making them a new target for therapeutic regulation of the inflammatory response of macrophages to LPS.

Sorting Nexin 5 Plays an Important Role in Promoting Ferroptosis in Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disease in the elderly, which is related to brain iron metabolism disorders. Ferroptosis is a newly discovered iron-dependent programmed cell death mode, which has been considered an essential mechanism of PD pathogenesis in recent years. However, its underlying mechanisms have not been fully understood. In the present study, the PD rat model and PD cell model were induced by 6-hydroyxdopamine (6-OHDA). The results showed that the expression of Sorting Nexin 5 (SNX5) and the level of ferroptosis will increase after treatment with 6-OHDA. Consistent with these results, ferroptosis inducer erastin synergistically reduced the expression of glutathione peroxidase 4 (GPX4) and increased the expression of SNX5 in the PD cell model, while ferroptosis inhibitor ferrostatin-1 (Fer-1) inhibited the decrease of GPX4 and the increase of SNX5 in the PD cell model. Knockdown of SNX5 in PC-12 cells could reduce intracellular lipid peroxidation and accumulation of Fe2+ and significantly inhibit the occurrence of ferroptosis. In conclusion, the present study suggested that SNX5 promotes ferroptosis in the PD model, thus providing new insights and potential for research on the pharmacological targets of PD.

SORTING NEXIN2 proteins mediate stomatal movement and the response to drought stress by modulating trafficking and protein levels of the ABA exporter ABCG25.

The phytohormone abscisic acid (ABA) regulates ion channel activity and stomatal movement in response to drought stress. Cellular ABA levels change depending on cellular and environmental conditions via modulation of its biosynthesis, catabolism and transport. Although factors involved in ABA biosynthesis and degradation have been studied extensively, how ABA transporters are modulated to fine-tune ABA levels, especially under drought stress, remains elusive. Here, we show that Arabidopsis thaliana SORTING NEXIN 2 (SNX2) proteins play a critical role in endosomal trafficking of the ABA exporter ATP BINDING CASETTE G25 (ABCG25) via direct interaction at endosomes, leading to its degradation in the vacuole. In agreement, snx2a and snx2b mutant plants showed enhanced recycling of GFP-ABCG25 from early endosomes to the plasma membrane and higher accumulation of GFP-ABCG25. Phenotypically, snx2a and snx2b plants were highly sensitive to exogenous ABA and displayed enhanced ABA-mediated inhibition of inward K+ currents and ABA-mediated activation of slow anion currents in guard cells, resulting in an increased tolerance to drought stress. Based on these results, we propose that SNX2 proteins play a crucial role in stomatal movement and tolerance to drought stress by modulating the endosomal trafficking of ABCG25 and thus cellular ABA levels.

The emerging roles of retromer and sorting nexins in the life cycle of viruses.

Retromer and sorting nexins (SNXs) transport cargoes from endosomes to the trans-Golgi network or plasma membrane. Recent studies have unveiled the emerging roles for retromer and SNXs in the life cycle of viruses, including members of Coronaviridae, Flaviviridae and Retroviridae. Key components of retromer/SNXs, such as Vps35, Vps26, SNX5 and SNX27, can affect multiple steps of the viral life cycle, including facilitating the entry of viruses into cells, participating in viral replication, and promoting the assembly of virions. Here we present a comprehensive updated review on the interplay between retromer/SNXs and virus, which will shed mechanistic insights into controlling virus infection.

Circulating Exosomal Proteins are linked to Neuropathogenesis in SIV‐infected Rhesus Macaque: A Proteomic Approach

BackgroundCombined antiretroviral therapy suppresses HIV replication, but 30‐60% of patients suffer from HIV‐1 associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in HIV CNS infection. Using proteomics, we investigated possible circulating exosomal protein links to neuropathogenesis in SIV‐infected rhesus macaque (RM).MethodsExosomes were isolated from sera of SIV‐infected (SIV‐Exo) and uninfected (CTL‐Exo) RM (N = 3/group) by QIAGEN exoEasy kit and characterized by the qNano‐IZON system. Proteomic analysis of the isolated exosomes was performed using liquid chromatography/mass spectrometry (LC‐MS/MS).ResultsqNano‐IZON analysis indicated that isolated EVs were predominantly exosomes (particle size < 150 nm). In the LC‐MS/MS study, 5,654 proteins were quantified, with 236 proteins (~ 4%) significantly differentially expressed (DE) between CTL‐Exo and SIV‐Exo. Two or more unique peptides were detected in 85% (4777/5654) of quantified proteins, and in 89% (211/236) of significant DE proteins, indicating the depth of analysis. We quantified most of the exosome‐associated proteins (tetraspanins, enzymes, lipid rafts, cytoskeletal, and endosome‐specific proteins) reported in previous studies. The heat‐maps and hierarchical clustering indicated that proteins involved in latent viral reactivation (heat shock transcription factor 1), inflammation (complement factor H, antioxidants, glycoproteins), unfolded protein response (UPR) (proteasome activators, cochaperones), neuropathology (amyloid beta [Aβ] precursor, chromogranin‐A and ‐B), and signaling (cytoskeleton regulators, cyclin‐H, mTOR complex 2, CD74) were expressed at significantly higher levels in SIV‐Exo than CTL‐Exo. However, proteins involved in mitochondrial (Mt) fission (Mt‐fission 1, ‐fission factor, and fission regulator 1), and ATP production (Mt Complex‐I, ‐IV, and ‐V), that play a critical role in the brain energy supply, were significantly decreased in SIV‐Exo. Moreover, exosomal‐proteins involved in autophagy‐mediated degradation (autophagy related 9A, ‐2B, lysosomal associated membrane protein 2), endosomal recycling (sorting nexin 4) and exocytosis (synaptogyrin), sprouting angiogenesis (jumonij domain‐containing 6), and cytoskeleton organization (calponin) were also expressed at significantly lower levels in SIV‐Exo than CTL‐Exo.ConclusionsOur novel findings suggest that circulating exosomal proteins are associated with viral reactivation, inflammation, UPR, mitochondrial dysfunction, defective autophagy, and Aβ and Tau pathology that may elucidate the etiology of HAND, and possibly provide novel therapeutic targets.

Sorting Nexin1 negatively modulates phosphate uptake by facilitating Phosphate Transporter1;1 degradation in Arabidopsis.

High-affinity phosphate (Pi) transporters (PHTs) PHT1;1 and PHT1;4 are necessary for plant root Pi uptake especially under Pi-deficient conditions, but how their protein stability is modulated remains elusive. Here, we identified a Ttransfer DNA insertion mutant of Sorting Nexin1 (SNX1), which had more Pi content and less anthocyanin accumulation than the wild type under deficient Pi. By contrast, the snx1-2 mutant displayed higher sensitivity to exogenous arsenate in terms of seed germination and root elongation, revealing higher Pi uptake rates. Further study showed that SNX1 could co-localize and interact with PHT1;1 and PHT1;4 in vesicles and at the plasma membrane. Genetic analysis showed that increased Pi content in the snx1-2 mutant under low Pi conditions could be extensively compromised by mutating PHT1;1 in the double mutant snx1-2 pht1;1, revealing that SNX1 is epistatic to PHT1;1. In addition, SNX1 negatively controls PHT1;1 protein stability; therefore, PHT1;1 protein abundance in the plasma membrane was increased in the snx1-2 mutant compared with the wild type under either sufficient or deficient Pi. Together, our study (i) identifies SNX1 as a key modulator of the plant response to low Pi and (ii) unravels its role in the modulation of PHT1;1 protein stability, PHT1;1 accumulation at the plasma membrane, and root Pi uptake.

Interactome and Ubiquitinome Analyses Identify Functional Targets of Herpes Simplex Virus 1 Infected Cell Protein 0.

Herpes simplex virus 1 (HSV-1) can productively infect multiple cell types and establish latent infection in neurons. Infected cell protein 0 (ICP0) is an HSV-1 E3 ubiquitin ligase crucial for productive infection and reactivation from latency. However, our knowledge about its targets especially in neuronal cells is limited. We confirmed that, like in non-neuronal cells, ICP0-null virus exhibited major replication defects in primary mouse neurons and Neuro-2a cells. We identified many ICP0-interacting proteins in Neuro-2a cells, 293T cells, and human foreskin fibroblasts by mass spectrometry–based interactome analysis. Co-immunoprecipitation assays validated ICP0 interactions with acyl-coenzyme A thioesterase 8 (ACOT8), complement C1q binding protein (C1QBP), ovarian tumour domain-containing protein 4 (OTUD4), sorting nexin 9 (SNX9), and vimentin (VIM) in both Neuro-2a and 293T cells. Overexpression and knockdown experiments showed that SNX9 restricted replication of an ICP0-null but not wild-type virus in Neuro-2a cells. Ubiquitinome analysis by immunoprecipitating the trypsin-digested ubiquitin reminant followed by mass spectrometry identified numerous candidate ubiquitination substrates of ICP0 in infected Neuro-2a cells, among which OTUD4 and VIM were novel substrates confirmed to be ubiquitinated by transfected ICP0 in Neuro-2a cells despite no evidence of their degradation by ICP0. Expression of OTUD4 was induced independently of ICP0 during HSV-1 infection. Overexpressed OTUD4 enhanced type I interferon expression during infection with the ICP0-null but not wild-type virus. In summary, by combining two proteomic approaches followed by confirmatory and functional experiments, we identified and validated multiple novel targets of ICP0 and revealed potential restrictive activities of SNX9 and OTUD4 in neuronal cells.

A Single Amino Acid Residue R144 of SNX16 Affects Its Ability to Inhibit the Replication of Influenza A Virus.

Influenza A virus (IAV) is an important zoonotic pathogen, posing a severe burden for the health of both animals and humans. Many host factors are involved in the life cycle of IAV to regulate its replication. Herein, we identified sorting nexin-16 (SNX16) as a new host factor that negatively modulates the replication of IAV. When transiently overexpressed in cells, SNX16 appears to be expressed as two obvious bands. Mutagenesis analysis indicated that the amino acid residue R144 of SNX16 was responsible for its two-band expression phenotype. We found that the R144A mutation of SNX16 changed its cellular distribution in A549 cells and partially weakened the inhibitory effect of SNX16 on IAV replication. Further investigation revealed that SNX16 could negatively regulate the early stage of the replication cycle of IAV. Taken together, our results demonstrated that SNX16 is a novel restriction host factor for the replication of IAV by engaging in the early stage of IAV life cycle, and a single amino acid residue at position 144 plays an important role in the cellular distribution and anti-influenza function of SNX16.

SNX5 targets a monoamine transporter to the TGN for assembly into dense core vesicles by AP-3.

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.

A Comparative Analysis of the Membrane Binding and Remodeling Properties of Two Related Sorting Nexin Complexes Involved in Autophagy.

The sorting nexin (SNX) proteins, Atg20 and Atg24, are involved in nonselective autophagy, are necessary for efficient selective autophagy, and are required for the cytoplasm-to-vacuole transport pathway. However, the specific roles of these proteins in autophagy are not well understood. Atg20 and Atg24 each contain a Phox homology domain that facilitates phosphoinositide binding. They also each contain an SNX-Bin/Amphiphysin/Rvs domain that forms a cup-shaped dimer, capable of binding to curved membranes and remodeling those membranes in some cases. Atg20 and Atg24 form two distinct complexes, an Atg24/Atg24 homodimer and an Atg20/Atg24 heterodimer. Despite the presence of Atg24 in both complexes, it is currently unclear if these complexes have different membrane binding and remodeling properties. Therefore, in this study, we explored the membrane binding and shaping properties of these two dimeric complexes. We found that Atg24/Atg24 and Atg20/Atg24 have distinct membrane binding preferences. Both dimers recognized membranes containing phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 3,5-bisphosphate, but Atg20/Atg24 bound to a broader array of liposomes, including those lacking phosphorylated phosphatidylinositol. In addition, we discovered that while both complexes bound to autophagosomal-like liposomes containing at least 5% PI(3)P, Atg20/Atg24 was capable of binding to autophagosomal-like liposomes lacking PI(3)P. Lastly, we observed that the Atg20/Atg24 heterodimer tubulates PI(3)P-containing and autophagosomal-like liposomes, but the Atg24/Atg24 homodimer could not tubulate these liposomes. Our findings suggest that these two dimers contain distinct membrane binding and shaping properties.

Differential gene expression analysis for osteosarcoma lung metastases.

To explore the exact molecular mechanisms underline osteosarcoma (OS) patients with lung metastases. The differentially expressed gene (DEG) as well as differentially expressed miRNAs (DEMs) for OS lung metastases were deeply investigated with two independent sources of databases (GEO dataset and clinical participants); The enriched biological processes and signaling pathways were explored; the miRNAs-mRNAs network was constructed; the functions of potential DEGs and DEMs were also verified with external analysis. The OS patients with lung metastases displayed 323 DEGs as C-C motif chemokine ligand 3 (CCL3), sorting nexin 10 (SNX10), alpha-2-macroglobulin (A2M), carboxypeptidase E (CPE), Rap guanine nucleotide exchange factor 4 (RAPGEF4), PDZ domain containing 2 (PDZD2), calpain 10 (CAPN10), four and a half LIM domains 2 (FHL2), alkaline phosphatase, biomineralization associated (ALPL), interleukin 6 (IL6), solute carrier family 26 member 1 (SLC26A1) as well as smoothened, frizzled class receptor (SMO) were significant differentially expressed. At the same time, 21 DEMs were potential for the progress of OS lung metastasis with hsa-miR-638, hsa-miR-451, hsa-miR-486-5p, hsa-miR-134 and hsa-miR-648 were significant distinct. It could been shown that hsa-miR-638 manipulated the largest number of target genes. The functions of hsa-miR-638 and target mRNAs for the development of lung metastasis in OS could be confirmed by quantitative Real-time PCR analysis. This integrated study hypothesized several miRNA dependent signaling pathway for OS patients with lung metastases and initiated a potential strategy for better understanding the lung metastases in clinic.

Protein Kinase A Regulates Autophagy-Associated Proteins Impacting Growth and Virulence of Aspergillus fumigatus.

Cellular recycling via autophagy-associated proteins is a key catabolic pathway critical to invasive fungal pathogen growth and virulence in the nutrient-limited host environment. Protein kinase A (PKA) is vital for the growth and virulence of numerous fungal pathogens. However, the underlying basis for its regulation of pathogenesis remains poorly understood in any species. Our Aspergillus fumigatus PKA-dependent whole proteome and phosphoproteome studies employing advanced mass spectroscopic approaches identified numerous previously undefined PKA-regulated proteins in catabolic pathways. Here, we demonstrate reciprocal inhibition of autophagy and PKA activity, and identify 16 autophagy-associated proteins as likely novel PKA-regulated effectors. We characterize the novel PKA-phosphoregulated sorting nexin Atg20, and demonstrate its importance for growth, cell wall stress response, and virulence of A. fumigatus in a murine infection model. Additionally, we identify physical and functional interaction of Atg20 with previously characterized sorting nexin Atg24. Furthermore, we demonstrate the importance of additional uncharacterized PKA-regulated putative autophagy-associated proteins to hyphal growth. Our data presented here indicate that PKA regulates the autophagy pathway much more extensively than previously known, including targeting of novel effector proteins with fungal-specific functions important for invasive disease.

Early Endosomal Vps34-Derived Phosphatidylinositol-3-Phosphate Is Indispensable for the Biogenesis of the Endosomal Recycling Compartment.

Phosphatidylinositol-3-phosphate (PI3P), a major identity tag of early endosomes (EEs), provides a platform for the recruitment of numerous cellular proteins containing an FYVE or PX domain that is required for PI3P-dependent maturation of EEs. Most of the PI3P in EEs is generated by the activity of Vps34, a catalytic component of class III phosphatidylinositol-3-phosphate kinase (PI3Ks) complex. In this study, we analyzed the role of Vps34-derived PI3P in the EE recycling circuit of unperturbed cells using VPS34-IN1 (IN1), a highly specific inhibitor of Vps34. IN1-mediated PI3P depletion resulted in the rapid dissociation of recombinant FYVE- and PX-containing PI3P-binding modules and endogenous PI3P-binding proteins, including EEA1 and EE sorting nexins. IN1 treatment triggered the rapid restructuring of EEs into a PI3P-independent functional configuration, and after IN1 washout, EEs were rapidly restored to a PI3P-dependent functional configuration. Analysis of the PI3P-independent configuration showed that the Vps34-derived PI3P is not essential for the pre-EE-associated functions and the fast recycling loop of the EE recycling circuit but contributes to EE maturation toward the degradation circuit, as previously shown in Vps34 knockout and knockdown studies. However, our study shows that Vps34-derived PI3P is also essential for the establishment of the Rab11a-dependent pathway, including recycling cargo sorting in this pathway and membrane flux from EEs to the pericentriolar endosomal recycling compartment (ERC). Rab11a endosomes of PI3P-depleted cells expanded and vacuolized outside the pericentriolar area without the acquisition of internalized transferrin (Tf). These endosomes had high levels of FIP5 and low levels of FIP3, suggesting that their maturation was arrested before the acquisition of FIP3. Consequently, Tf-loaded-, Rab11a/FIP5-, and Rab8a-positive endosomes disappeared from the pericentriolar area, implying that PI3P-associated functions are essential for ERC biogenesis. ERC loss was rapidly reversed after IN1 washout, which coincided with the restoration of FIP3 recruitment to Rab11a-positive endosomes and their dynein-dependent migration to the cell center. Thus, our study shows that Vps34-derived PI3P is indispensable in the recycling circuit to maintain the slow recycling pathway and biogenesis of the ERC.

α-synuclein and phosphoinositide-binding proteins: α-synuclein inhibits the association of PX- but not FYVE-containing proteins with vesicles in vivo

By an unknown mechanism, alpha-synuclein (α-syn) inhibits autophagy in yeast and human cells. Herein, using the yeast Saccharomyces cerevisiae, we tested the hypothesis that α-syn disrupts autophagy by inhibiting the required association of sorting nexin 4 (Snx4) with phagophores. Snx4 contains a phox (PX) homology domain that selectively binds membranes enriched in phosphatidylinositol 3-phosphate (PI3P). Using fluorescence microscopy, we show that upon nitrogen starvation, 70% of the cells exhibited green puncta (phagophores); whereas identically treated cells expressing α-syn exhibited a significantly lower percentage of cells (30%) with such puncta. Our interpretation is that α-syn outcompetes Snx4 for binding to membranes enriched in PI3P, resulting in fewer phagophores and consequently inefficient induction of autophagy. As a control, we tested whether α-syn disrupts the binding of Vps27-GFP to late endosomes/multivesicular bodies (MVBs). Vps27 contains a PI3P-binding domain called FYVE. α-Syn did not disrupt the binding of Vps27-GFP to late endosomes. α-Syn likely inhibits the binding of PX- but not FYVE-containing proteins to PI3P because FYVE domains bind more than two-orders of magnitude tighter than PX domains. We propose that in all cells, whether yeast or human, α-syn has the potential to inhibit protein trafficking pathways that are dependent on PX-domain proteins such as sorting nexins.

Structural Predictions of the SNX-RGS Proteins Suggest They Belong to a New Class of Lipid Transfer Proteins.

Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key example of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins.

Lifespan Increase of Podospora anserina by Oleic Acid Is Linked to Alterations in Energy Metabolism, Membrane Trafficking and Autophagy.

The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.

Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to regulate TORC1 signaling.

The sorting nexins (SNXs), constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility and autophagy. In particular, the SNX‐Bin‐Amphiphysin‐Rvs (BAR) proteins, a SNX subfamily characterized by a C‐terminal dimeric BAR lipid curvature domain and a conserved Phox‐homology domain, are of great interest. In budding yeast, many SNX‐BAR proteins have well‐characterized endo‐vacuolar trafficking roles. Phylogenetic analyses allowed us to identify an additional SNX‐BAR protein, Vps501, with a novel endo‐vacuolar role. We report that Vps501 uniquely localizes to the vacuolar membrane and has physical and genetic interactions with the SEA complex to regulate TORC1 inactivation. We found cells displayed a severe deficiency in starvation‐induced/nonselective autophagy only when SEA complex subunits are ablated in combination with Vps501, indicating a cooperative role with the SEA complex during TORC1 signaling during autophagy induction. Additionally, we found the SEACIT complex becomes destabilized in vps501Δsea1Δ cells, which resulted in aberrant endosomal TORC1 activity and subsequent Atg13 hyperphosphorylation. We have also discovered that the vacuolar localization of Vps501 is dependent upon a direct interaction with Sea1 and a unique lipid binding specificity that is also required for its function.