Circadian rhythms of sorting nexin 25 in the mouse suprachiasmatic nucleus.

Entrainment of mammalian circadian rhythms requires receptor-mediated signaling in the hypothalamic suprachiasmatic nucleus (SCN), the site of the master circadian pacemaker. Receptor-mediated signaling is regulated by endocytosis, indicating that endocytosis-related proteins contribute to SCN pacemaking. Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. In this study, we showed that Snx25 mRNA and SNX25 protein are highly expressed and exhibit remarkable circadian rhythms in the SCN of adult mice. Expression was maximal at about zeitgeber time (ZT) 16 in the subjective night and minimal at ZT8 in the subjective day. Prominent SNX25 immunoreactivity was found in the arginine vasopressin-positive neurons of the SCN. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the circadian pacemaking system.

SNX17 produces anti-arrhythmic effects by preserving functional SERCA2a protein in myocardial infarction.

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.

Expression and purification of the SNX1/SNX6 complex.

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.

SNX10 mediates alcohol-induced liver injury and steatosis by regulating the activation of chaperone-mediated autophagy.

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.

Overexpression of SNX3 Decreases Amyloid-ß Peptide Production by Reducing Internalization of Amyloid Precursor Protein.

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)ß peptide production. OBJECTIVE: To examine the role of SNX3 in Aß production and APP processing. METHODS: The effect of increased expression of SNX3 was studied in HEK293T cells. Aß 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 Aß and soluble N-terminal APP fragments (sAPPß). 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 Aß production by influencing the internalization of APP.

Reduced expression of SETD2 and SNX9 proteins in chemically induced mammary tumorigenesis in Wistar rats: a prognostic histological and proteomic study.

Breast cancer is a major global health concern, appealing for precise prognostic approaches. Thus, the need is to have studies focusing on the identification and recognition of preliminary events leading to the disease. The present study reports the tracing of precancerous progression and serum proteomic analysis in a breast cancer model developed as a result of 7,12-dimethylbenz[a]anthracene (DMBA) administration. Mammary gland histological changes of prime importance were examined by histopathology, and immunohistochemical analysis with Ki-67 was performed to monitor enhanced cell proliferation, right from the onset of hyperplasia till neoplasia. Serum proteomics (one-dimensional (1D) and two-dimensional (2D) electrophoresis, followed by MALDI-TOF MS characterization) was performed to decipher the differentially expressed serum proteins in animals undergoing tumorigenesis vis-à-vis controls. The significance of our study lies in reporting the significantly reduced expression of two proteins: histone-lysine N-methyltransferase (SETD2) and sorting nexin-9 (SNX9) at very early stage (13 weeks) of tumorigenesis, while the full-fledged tumors developed after 6 months. The reduced expression of SETD2 and SNX9 was validated by western blotting and relative expression analysis using quantitative real-time PCR. These proteins may hence prove as potentially useful tools in search for prognostic markers for the early detection of mammary cancer.

Impaired SNX9 Expression in Immune Cells during Chronic Inflammation: Prognostic and Diagnostic Implications.

Chronic inflammation is associated with immunosuppression and downregulated expression of the TCR CD247. In searching for new biomarkers that could validate the impaired host immune status under chronic inflammatory conditions, we discovered that sorting nexin 9 (SNX9), a protein that participates in early stages of clathrin-mediated endocytosis, is downregulated as well under such conditions. SNX9 expression was affected earlier than CD247 by the generated harmful environment, suggesting that it is a potential marker sensing the generated immunosuppressive condition. We found that myeloid-derived suppressor cells, which are elevated in the course of chronic inflammation, are responsible for the observed SNX9 reduced expression. Moreover, SNX9 downregulation is reversible, as its expression levels return to normal and immune functions are restored when the inflammatory response and/or myeloid-derived suppressor cells are neutralized. SNX9 downregulation was detected in numerous mouse models for pathologies characterized by chronic inflammation such as chronic infection (Leishmania donovani), cancer (melanoma and colorectal carcinoma), and an autoimmune disease (rheumatoid arthritis). Interestingly, reduced levels of SNX9 were also observed in blood samples from colorectal cancer patients, emphasizing the feasibility of its use as a diagnostic and prognostic biomarker sensing the host's immune status and inflammatory stage. Our new discovery of SNX9 as being regulated by chronic inflammation and its association with immunosuppression, in addition to the CD247 regulation under such conditions, show the global impact of chronic inflammation and the generated immune environment on different cellular pathways in a diverse spectrum of diseases.

Abnormal Expression of miR-21 and miR-95 in Cancer Stem-Like Cells is Associated with Radioresistance of Lung Cancer.

This study demonstrated that miR-21 and miR-95 expression were significantly higher in the ALDH1(+)CD133(+)subpopulation than in the ALDH1(-)CD133(-) subpopulation of lung cancer cells. Combined delivery of anti-miR-21 and anti-miR-95 by calcium phosphate nanoparticles significantly inhibited tumor growth in a xenograft tumor model and sensitized radiotherapy. The anti-miRNAs significantly reduced miR-21 and miR-95 levels, increased PTEN, SNX1, and SGPP1 protein expression, but reduced Akt Ser(473) and Thr(308) phosphorylation. ALDH1(+)CD133(+) subpopulation of NSCLC tumor cells confers radioresistance due to high expression of miR-21 and miR-95. Targeting inhibition of miR-21 and miR-95 can inhibit tumor growth through elevating PTEN, SNX1, and SGPP1 expression and inhibiting Akt phosphorylation.

[Expression, purification and phosphoinositide binding specifity of recombinant human SNX7 expressed in Escherichia coli].

Sorting nexins (SNXs) are a large group of proteins that contain Phox (PX) domain and involve in regulating endocytosis and endosome sorting. SNX7, a member of SNXs family, contains a PX domain and a BAR domain. In zebrafish, SNX7 is a liver-enriched anti-apoptotic protein and indispensible for the liver development. A fragment of SNX7 cDNA ((px-bar)snx7), encoding the PX domain and the BAR domain, was inserted into the expressing vector p28a, transformed into E. coli Rosseta 2 (DE3), and then induced by isopropyl ß-D-1-Thiogalactopyranoside (IPTG). After affinity, ion exchange and gel filtration purification, the purity of (PX-BAR)SNX7 reached over 95%. Dynamic light scattering (DLS) experiment indicated that (PX-BAR)SNX7 was homogeneous in solution. Lipid overlay assay showed that (PX-BAR)SNX7 can bind to PtdIns(5)P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3.

Silencing of SNX1 by siRNA stimulates the ligand-induced endocytosis of EGFR and increases EGFR phosphorylation in gefitinib-resistant human lung cancer cell lines.

Gefitinib is known to suppress the activation of EGFR signaling, which is required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We previously demonstrated that the gefitinib-sensitive NSCLC cell line PC9 shows efficient ligand-induced endocytosis of phosphorylated EGFR (pEGFR). In contrast, the gefitinib-resistant NSCLC cell lines QG56 and A549 showed internalized pEGFR accumulation in the aggregated early endosomes, and this was associated with SNX1, a protein that interacts with and enhances the degradation of EGFR upon EGF stimulation. In the present study, to investigate the role of SNX1 on EGF-stimulated EGFR/pEGFR endocytosis via the endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in human NSCLC cell lines. Using immunofluorescence, we demonstrated that transfection of SNX1 siRNA into gefitinib-resistant NSCLC cells resulted in the disappearance of a large amounts of SNX1 staining. In addition, upon 15 min of EGF stimulation, we observed an efficient EGFR phosphorylation and a rapid endocytic delivery of pEGFR from early endosomes to late endosomes. Further, western blot analysis revealed that silencing of SNX1 expression by siRNA in the gefitinib-resistant cells leads to an accelerated degradation of EGFR along with a dramatic increase in the amounts of pEGFR after EGF stimulation. Based on these findings, we suggest that SNX1 is involved in the negative regulation of ligand-induced EGFR phosphorylation and mediates EGFR/pEGFR trafficking out of early endosomes for targeting to late endosomes/lysosomes via the early/late endocytic pathway in human lung cancer cells.

MicroRNA-95 promotes cell proliferation and targets sorting Nexin 1 in human colorectal carcinoma.

MicroRNAs (miRNAs) are strongly implicated in cancer but their specific roles and functions in the major cancers have yet to be fully elucidated. In this study, we defined the oncogenic significance and function of miR-95, which we found to be elevated in colorectal cancer (CRC) tissues by microarray analysis. Evaluation of an expanded CRC cohort revealed that miR-95 expression was up-regulated in nearly half of the tumors examined (42/87) compared with the corresponding noncancerous tissues. Ectopic overexpression of miR-95 in human CRC cell lines promoted cell growth in vitro and tumorigenicity in vivo, whereas RNAi-mediated silencing of miR-95 decreased cell growth ratio. Mechanistic studies revealed that miR-95 repressed the expression of reporter gene coupled to the 3'-untranslated region of sorting nexin 1 (SNX1), whereas miR-95 silencing up-regulated SNX1 expression. Moreover, miR-95 expression levels correlated inversely with SNX1 protein levels in human CRC tissues. RNAi-mediated knockdown of SNX1 phenocopied the proliferation-promoting effect of miR-95, whereas overexpression of SNX1 blocked miR-95-induced proliferation of CRC cells. Taken together, these results demonstrated that miR-95 increases proliferation by directly targeting SNX1, defining miR-95 as a new oncogenic miRNA in CRC.