SNX16 is required for hepatocellular carcinoma survival via modulating the EGFR-AKT signaling pathway.

Sorting nexin 16 (SNX16), a pivotal sorting nexin, emerges in tumor progression complexity, fueling research interest. However, SNX16's biological impact and molecular underpinnings in hepatocellular carcinoma (HCC) remain elusive. This study probes SNX16's function, clinical relevance via mRNA, and protein expression in HCC. Overexpression/knockdown assays of SNX16 were employed to elucidate impacts on HCC cell invasion, proliferation, and EMT. Additionally, the study delved into SNX16's regulation of the EGFR-AKT signaling cascade mechanism. SNX16 overexpression in HCC correlates with poor patient survival; enhancing proliferation, migration, invasion, and tumorigenicity, while SNX16 knockdown suppresses these processes. SNX16 downregulation curbs phospho-EGFR, dampening AKT signaling. EGFR suppression counters SNX16-overexpression-induced HCC proliferation, motility, and invasiveness. Our findings delineate SNX16's regulatory role in HCC, implicating it as a prospective therapeutic target.

ITGB5 facilitates gastric cancer metastasis by promoting TGFBR2 endosomal recycling.

TGFBR2, a key regulator of the TGFß signaling pathway, plays a crucial role in gastric cancer (GC) metastasis through its endosomal recycling process. Despite its importance, the mechanisms governing this process remain unclear. Here, we identify integrin ß5 (ITGB5) as a critical mediator that promotes TGFBR2 endosomal recycling. Our study reveals elevated expression of ITGB5 in GC, particularly in metastatic cases, correlating with poor patient outcomes. Knockdown of ITGB5 impairs GC cell metastasis both in vitro and in vivo. Mechanistically, ITGB5 facilitates epithelial-mesenchymal transition mediated by TGFß signaling, thereby enhancing GC metastasis. Acting as a scaffold, ITGB5 interacts with TGFBR2 and SNX17, facilitating SNX17-mediated endosomal recycling of TGFBR2 and preventing lysosomal degradation, thereby maintaining its surface distribution on tumor cells. Notably, TGFß signaling directly upregulates ITGB5 expression, establishing a positive feedback loop that exacerbates GC metastasis. Our findings shed light on the role of ITGB5 in promoting GC metastasis through SNX17-mediated endosomal recycling of TGFBR2, providing insights for the development of targeted cancer therapies.

Altered lipid homeostasis is associated with cerebellar neurodegeneration in SNX14 deficiency.

Dysregulated lipid homeostasis is emerging as a potential cause of neurodegenerative disorders. However, evidence of errors in lipid homeostasis as a pathogenic mechanism of neurodegeneration remains limited. Here, we show that cerebellar neurodegeneration caused by Sorting Nexin 14 (SNX14) deficiency is associated with lipid homeostasis defects. Recent studies indicate that SNX14 is an interorganelle lipid transfer protein that regulates lipid transport, lipid droplet (LD) biogenesis, and fatty acid desaturation, suggesting that human SNX14 deficiency belongs to an expanding class of cerebellar neurodegenerative disorders caused by altered cellular lipid homeostasis. To test this hypothesis, we generated a mouse model that recapitulates human SNX14 deficiency at a genetic and phenotypic level. We demonstrate that cerebellar Purkinje cells (PCs) are selectively vulnerable to SNX14 deficiency while forebrain regions preserve their neuronal content. Ultrastructure and lipidomic studies reveal widespread lipid storage and metabolism defects in SNX14-deficient mice. However, predegenerating SNX14-deficient cerebella show a unique accumulation of acylcarnitines and depletion of triglycerides. Furthermore, defects in LD content and telolysosome enlargement in predegenerating PCs suggest lipotoxicity as a pathogenic mechanism of SNX14 deficiency. Our work shows a selective cerebellar vulnerability to altered lipid homeostasis and provides a mouse model for future therapeutic studies.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ∼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

N-terminal signals in the SNX-BAR paralogs Vps5 and Vin1 guide endosomal coat complex formation.

Endosomal coats incorporate membrane-binding subunits such as sorting nexin (SNX) proteins. The Saccharomyces cerevisiae SNX-BAR paralogs Vin1 and Vps5 are respective subunits of the endosomal VINE and retromer complexes whose dimerizing BAR domains are required for complex assembly and membrane association. However, a degree of promiscuity is predicted for yeast BAR-BAR pairings, and recent work has implicated the unstructured N-terminal domains of Vin1 and Vps5 in coat formation. Here, we map N-terminal signals in both SNX-BAR paralogs that contribute to the assembly and function of two distinct endosomal coats in vivo. Whereas Vin1 leverages a polybasic region and adjacent hydrophobic motif to bind Vrl1 and form VINE, the N-terminus of Vps5 interacts with the retromer subunit Vps29 at two sites, including a conserved hydrophobic pocket in Vps29 that engages other accessory proteins in humans. We also examined the sole isoform of Vps5 from the milk yeast Kluyveromyces lactis and found that ancestral yeasts may have used a nested N-terminal signal to form both VINE and retromer. Our results suggest that the specific assembly of Vps5-family SNX-BAR coats depends on inputs from unique N-terminal sequence features in addition to BAR domain coupling, expanding our understanding of endosomal coat biology.

Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.

SNX8 enables lysosome reformation and reverses lysosomal storage disorder.

Lysosomal Storage Disorders (LSDs), which share common phenotypes, including enlarged lysosomes and defective lysosomal storage, are caused by mutations in lysosome-related genes. Although gene therapies and enzyme replacement therapies have been explored, there are currently no effective routine therapies against LSDs. During lysosome reformation, which occurs when the functional lysosome pool is reduced, lysosomal lipids and proteins are recycled to restore lysosome functions. Here we report that the sorting nexin protein SNX8 promotes lysosome tubulation, a process that is required for lysosome reformation, and that loss of SNX8 leads to phenotypes characteristic of LSDs in human cells. SNX8 overexpression rescued features of LSDs in cells, and AAV-based delivery of SNX8 to the brain rescued LSD phenotypes in mice. Importantly, by screening a natural compound library, we identified three small molecules that enhanced SNX8-lysosome binding and reversed LSD phenotypes in human cells and in mice. Altogether, our results provide a potential solution for the treatment of LSDs.

Association analysis of the sorting nexin 29 (SNX29) gene copy number variations with growth traits in Diannan small-ear (DSE) pigs.

SNX29 is a potential functional gene associated with meat production traits. Previous studies have shown that SNX29 copy number variation (CNV) could be implicated with phenotype in goats. However, in Diannan small-ear (DSE) pigs, the genetic impact of SNX29 CNV on growth traits remains unclear. Therefore, this study investigated the associations between SNX29 CNVs (CNV10810 and CNV10811) and growth traits in 415 DSE pigs. The results revealed that the CNV10810 mutation was significantly associated with backfat thickness in DSE pigs at 12 and 15 months old (P < 0.05), while the CNV10811 mutation had significant effects on various growth traits at 6 and 12 months old, particularly for body weight, body height, back height and backfat thickness (P < 0.05 or P < 0.001). In conclusion, our results confirm that SNX29 CNV plays a role in regulating growth and development in pigs, thus suggesting its potential application for pig breeding programmes.

SNX10 promoted liver IR injury by facilitating macrophage M1 polarization via NLRP3 inflammasome activation.

BACKGROUND: Liver ischemia reperfusion (IR) injury is a common cause of liver dysfunction in patients post liver partial resection and liver transplantation. However, the cellular defense mechanisms underlying IR are not well understood. Macrophage mediated sterile inflammation plays critical roles in liver IR injury. Sorting nexin (SNX) 10, a member of the SNX family which functions in regulation of endosomal sorting. This study aimed to explore the role of sorting nexin 10 (SNX10) during liver IR injury with a focus on regulating macrophage function. METHODS: Both the gene and protein expression levels of SNX10 were analyzed in human specimens from 10 patients undergoing liver partial resection with ischemic insult and in a mouse model of liver IR. The in vivo effects of SNX10 in liver IR injury and sterile inflammation in mice were investigated. Bone marrow derived macrophages (BMDMs) were used to determine the role of SNX10 in modulating macrophage function in vitro. RESULTS: Increased expression of SNX10 was observed both in human specimens and mice livers post IR. SNX10 knockdown alleviated IR induced sterile inflammation and liver damage in mice. SNX10 promoted M1 polarization of macrophage treated with LPS and facilitated inflammatory response by activating NLRP3 inflammasome. CONCLUSIONS: We report for the first time that SNX10 is upregulated in IR-stressed livers. SNX10 activation aggravates liver IR injury and sterile inflammation by facilitating macrophage M1 polarization and inflammatory response suggesting SNX10 as a potential therapeutic target for liver IR injury.

SNX17 Mediates Dendritic Spine Maturation via p140Cap.

Sorting nexin17 (SNX17) is a member of the sorting nexin family, which plays a crucial role in endosomal trafficking. Previous research has shown that SNX17 is involved in the recycling or degradation of various proteins associated with neurodevelopmental and neurological diseases in cell models. However, the significance of SNX17 in neurological function in the mouse brain has not been thoroughly investigated. In this study, we generated Snx17 knockout mice and observed that the homozygous deletion of Snx17 (Snx17-/-) resulted in lethality. On the other hand, heterozygous mutant mice (Snx17+/-) exhibited anxiety-like behavior with a reduced preference for social novelty. Furthermore, Snx17 haploinsufficiency led to impaired synaptic transmission and reduced maturation of dendritic spines. Through GST pulldown and interactome analysis, we identified the SRC kinase inhibitor, p140Cap, as a potential downstream target of SNX17. We also demonstrated that the interaction between p140Cap and SNX17 is crucial for dendritic spine maturation. Together, this study provides the first in vivo evidence highlighting the important role of SNX17 in maintaining neuronal function, as well as regulating social novelty and anxiety-like behaviors.

Ropivacaine inhibits the proliferation and metastasis of gastric cancer cells via the SNX10/SRC/STAT3 pathway.

Gastric cancer currently has no effective treatment due to its high metastasis and heterogeneity. It has been reported that ropivacaine (Rop) can inhibit the growth, migration, and invasion of gastric cancer. However, the therapeutic mechanism of Rop still needs to be further explored to provide insights for its clinical application. This study aimed to explore the effects of Rop on the growth, migration, and invasion of gastric cancer cells and the underlying mechanisms. The expression levels of SNX10 were assessed in gastric cancer tissues and cell line AGS by qRT-PCR. Cell Counting Kit-8 (CCK8) assay, wound-healing assay, and transwell assay were then used to examine the effects of Rop on the AGS cell viability, migration, invasion, and proliferation, respectively. Additionally, colony formation assay was used to measure cell proliferation ability, and flow cytometry was used to detect apoptosis level. Protein levels of SNX10, SRC, and STAT3 were detected by western blot. According to the experimental results, the decreased SNX10 mRNA expression was observed in gastric cancer tissue and cell line AGS. Rop inhibited the proliferation, migration, and invasion of AGS cells, but promoted apoptosis and upregulated SNX10 expression. Moreover, Rop inhibited the expression of MMP-2 and MMP-9, phosphorylation of SRC and STAT3. SNX10 knockdown could reverse Rop-induced anticancer effects. Collectively, Rop showed a potential role in preventing proliferation and metastasis of gastric cancer. The action mechanism of Rop may be related to the upregulation of SNX10 expression and further inhibition of SRC/STAT3 signaling pathway. Our findings provide new insights into the anticancer properties of Rop.

Inhibiting sorting nexin 10 promotes mucosal healing through SREBP2-mediated stemness restoration of intestinal stem cells.

Intestinal stem cell (ISC) is a promising therapeutic target for inflammatory bowel disease. Cholesterol availability is critical for ISC stemness. Low plasma cholesterol is a typical feature of Crohn's disease (CD); however, its impact on mucosal healing remains unclear. Here, we identified an essential role of sorting nexin 10 (SNX10) in maintaining the stemness of ISCs. SNX10 expression in intestinal tissues positively correlates with the severity of human CD and mouse colitis. Conditional SNX10 knockout in intestinal epithelial cells or ISCs promotes intestinal mucosal repair by maintaining the ISC population associated with increased intracellular cholesterol synthesis. Disassociation of ERLIN2 with SCAP by SNX10 deletion enhances the activation of SREBP2, resulting in increased cholesterol biosynthesis. DC-SX029, a small-molecule inhibitor of SNX10, was used to verify the druggable potential of SNX10 for the treatment of patients with CD. Our study provides a strategy for mucosal healing through SREBP2-mediated stemness restoration of ISCs.

The plant-unique protein DRIF1 coordinates with sorting nexin 1 to regulate membrane protein homeostasis.

Membrane protein homeostasis is fine-tuned by the cellular pathways for vacuolar degradation and recycling, which ultimately facilitate plant growth and cell-environment interactions. The endosomal sorting complex required for transport (ESCRT) machinery plays important roles in regulating intraluminal vesicle (ILV) formation and membrane protein sorting to vacuoles. We previously showed that the plant-specific ESCRT component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FREE1) performs multiple functions in plants, although the underlying mechanisms remain elusive. In this study, we performed a suppressor screen of the FREE1-RNAi mutant and identified and characterized 2 suppressor of free1 (sof) mutants in Arabidopsis (Arabidopsis thaliana). These mutants, sof10 and sof641, result in a premature stop codon or a missense mutation in AT5G10370, respectively. This gene was named DEAH and RING domain-containing protein as FREE1 suppressor 1 (DRIF1). DRIF1 has a homologous gene, DRIF2, in the Arabidopsis genome with 95% identity to DRIF1. The embryos of drif1 drif2 mutants arrested at the globular stage and formed enlarged multivesicular bodies (MVBs) with an increased number of ILVs. DRIF1 is a membrane-associated protein that coordinates with retromer component sorting nexin 1 to regulate PIN-FORMED2 recycling to the plasma membrane. Altogether, our data demonstrate that DRIF1 is a unique retromer interactor that orchestrates FREE1-mediated ILV formation of MVBs and vacuolar sorting of membrane proteins for degradation in plants.

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

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

α-hederin regulates glucose metabolism in intestinal epithelial cells by increasing SNX10 expression.

BACKGROUND: Sorting nexin 10 (SNX10) has recently been identified as a critical regulator of colorectal carcinogenesis, whose deletion promoted cell proliferation and survival in human CRC cells, and promoted colorectal tumor growth and upregulated amino-acid metabolism in mice. However, what happens when silencing SNX10 in normal human intestinal epithelial cells (IECs) remains unknown, and no drugs targeting SNX10 have been reported. Here, we first investigated the biological function and underlying mechanisms of SNX10 in normal human IECs, and found that α-hederin, a pentacyclic triterpenoid saponin, has a regulatory effect on SNX10 expression. PURPOSE: This study aimed to explore the function of SNX10 in IECs to provide a new target for the prevention and treatment of malignant transformation and the intervention mechanism of α-hederin for further development of potential novel agents targeting SNX10. METHODS: The transfection approach was used to construct SNX10 stable knockdown cells. Cell proliferation was detected by CCK8, clone formation, EdU, flow cytometry, and wound healing assays. Enzyme activity assays for glucose metabolism, qRT-PCR, western blotting, and immunofluorescence staining were performed to investigate the protein expression of signaling pathways. RESULTS: Silencing SNX10 promoted cell proliferation and cycle transition in IECs and increased the activity of key enzymes involved in glucose metabolism. Moreover, DEPDC5 expression was significantly decreased following SNX10 knockdown, followed by activation of the mTORC1 pathway. α-hederin reversed the accelerated cell proliferation, cycle progression, and glucose metabolic activity, as well as the activated mTORC1 pathway caused by SNX10 knockdown, by notably increasing SNX10 expression in a dose-dependent manner. CONCLUSION: We first reported that knockdown of SNX10 in normal human IECs promoted cell proliferation and activated glucose metabolism by activating the mTORC1 pathway. Meanwhile, we first found that α-hederin down-regulated glucose metabolism activity and slowed cell proliferation by increasing SNX10 expression in IECs.

Genetics of sexually dimorphic adipose distribution in humans.

Obesity-associated morbidity is exacerbated by abdominal obesity, which can be measured as the waist-to-hip ratio adjusted for the body mass index (WHRadjBMI). Here we identify genes associated with obesity and WHRadjBMI and characterize allele-sensitive enhancers that are predicted to regulate WHRadjBMI genes in women. We found that several waist-to-hip ratio-associated variants map within primate-specific Alu retrotransposons harboring a DNA motif associated with adipocyte differentiation. This suggests that a genetic component of adipose distribution in humans may involve co-option of retrotransposons as adipose enhancers. We evaluated the role of the strongest female WHRadjBMI-associated gene, SNX10, in adipose biology. We determined that it is required for human adipocyte differentiation and function and participates in diet-induced adipose expansion in female mice, but not males. Our data identify genes and regulatory mechanisms that underlie female-specific adipose distribution and mediate metabolic dysfunction in women.

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

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

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

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

Sorting nexins: role in the regulation of blood pressure.

Sorting nexins (SNXs) are a family of proteins that regulate cellular cargo sorting and trafficking, maintain intracellular protein homeostasis, and participate in intracellular signaling. SNXs are also important in the regulation of blood pressure via several mechanisms. Aberrant expression and dysfunction of SNXs participate in the dysregulation of blood pressure. Genetic studies show a correlation between SNX gene variants and the response to antihypertensive drugs. In this review, we summarize the progress in SNX-mediated regulation of blood pressure, discuss the potential role of SNXs in the pathophysiology and treatment of hypertension, and propose novel strategies for the medical therapy of hypertension.