Succinate dehydrogenase (SDH)‐deficient renal cell carcinoma (RCC) is mainly associated with a mutation in the SDHB gene and sometimes with mutations in the SDHC or SDHD genes. However, only three ...cases of succinate dehydrogenase A (SDHA)‐deficient RCC have been reported, and the relation between SDHA mutations and RCC has not been clarified. This study assessed the role of SDHA gene mutations in human RCC. We investigated SDHA/B/C/D gene mutations in 129 human RCCs. Targeted next‐generation sequencing and direct Sanger sequencing revealed single nucleotide variants (SNVs) of the SDHA gene with amino acid sequence variations in 11/129 tumors, while no SDHB/C/D gene mutations were found. Tumor cells with SNVs of the SDHA gene were characterized by eosinophilic cytoplasm and various patterns of proliferation. Immunohistochemistry examination found that the 11 tumors with SNVs of the SDHA gene showed significant reduction of SDHA protein and SDHB protein expression compared to the 19 tumors without SDHA or SDHB mutations (both P < .0001). Western blotting showed a greater decrease in the expression of SDHA and SDHB proteins in the 11 tumors with SNVs of the SDHA gene than in the 19 tumors without (both P < .0001). There was a positive correlation between SDHA and SDHB protein levels (P < .0001). On immunohistochemistry and Western blotting, the 11 tumors with SNVs of the SDHA gene had higher protein expression for nuclear factor E2‐related factor 2 (Nrf2) compared to the 19 tumors without the mutation (P < .01). These observations suggest that SDHA gene mutations might be associated with a subset of RCC.
Succinate dehydrogenase (SDH) A gene mutation was identified in 11 out of 129 human renal cell carcinoma (RCC) samples by targeted next‐generation sequencing and direct Sanger sequencing, while no SDHB/C/D gene mutations were detected. Immunohistochemistry and western blotting identified a reduction of SDHA and SDHB protein expression in the tumors with SDHA gene mutations. These findings suggest that SDHA gene mutations might be linked to a subset of RCC.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in cell proliferation by promotion of metabolic activity. It is also the major regulator of antioxidants and has a pivotal role in tumor ...cell proliferation and resistance to chemotherapy. Accordingly, we investigated the role of Nrf2 in renal cell carcinoma (RCC).
In 50 patients who had metastatic RCC and received cytoreductive nephrectomy, we performed Nrf2 gene mutation analysis using targeted next-generation sequencing, as well as investigating a specific single nucleotide polymorphism (SNP; rs6721961) in the Nrf2 promoter region and Nrf2 protein expression.
Targeted next-generation sequencing revealed that five tumors had SNPs of Nrf2 associated with amino acid sequence variation, while 11 tumors had SNPs of Kelch-like ECH-associated protein 1 gene, 35 had SNPs of von Hippel-Lindau gene, and none had SNPs of fumarate hydratase gene. The three genotypes of rs6721961 showed the following frequencies: 60% for C/C, 34% for C/A, and 6% for A/A. Nrf2 mutation and the C/A or A/A genotypes were significantly associated with increased Nrf2 protein expression (p = 0.0184 and p = 0.0005, respectively). When the primary tumor showed Nrf2 gene mutation, the C/A or A/A genotype, or elevated Nrf2 protein expression, the response of metastases to vascular endothelial growth factor-targeting therapy was significantly worse (p = 0.0142, p = 0.0018, and p < 0.0001, respectively), and overall survival was significantly reduced (p = 0.0343, p = 0.0421, and p < 0.0001, respectively). Elevated Nrf2 protein expression was also associated with shorter survival according to multivariate Cox proportional analysis.
These findings suggest an associated between progression of RCC and Nrf2 signaling.
The autophosphorylation of specific tyrosine residues occurs in the cytoplasmic region of the insulin receptor (IR) upon insulin binding, and this in turn initiates signal transduction. The R3 ...subfamily (Ptprb, Ptprh, Ptprj and Ptpro) of receptor-like protein tyrosine phosphatases (RPTPs) is characterized by an extracellular region with 6-17 fibronectin type III-like repeats and a cytoplasmic region with a single phosphatase domain. We herein identified the IR as a substrate for R3 RPTPs by using the substrate-trapping mutants of R3 RPTPs. The co-expression of R3 RPTPs with the IR in HEK293T cells suppressed insulin-induced tyrosine phosphorylation of the IR. In vitro assays using synthetic phosphopeptides revealed that R3 RPTPs preferentially dephosphorylated a particular phosphorylation site of the IR: Y960 in the juxtamembrane region and Y1146 in the activation loop. Among four R3 members, only Ptprj was co-expressed with the IR in major insulin target tissues, such as the skeletal muscle, liver and adipose tissue. Importantly, the activation of IR and Akt by insulin was enhanced, and glucose and insulin tolerance was improved in Ptprj-deficient mice. These results demonstrated Ptprj as a physiological enzyme that attenuates insulin signalling in vivo, and indicate that an inhibitor of Ptprj may be an insulin-sensitizing agent.
Zn2‐Cys6 binuclear transcription factors Upc2p and Ecm22p regulate the expression of genes involved in ergosterol biosynthesis and exogenous sterol uptake in Saccharomyces cerevisiae. We identified ...two UPC2/ECM22 homologues in the pathogenic fungus Candida glabrata which we designated CgUPC2A and CgUPC2B. The contribution of these two genes to sterol homeostasis was investigated. Cells that lack CgUPC2A (upc2AΔ) exhibited enhanced susceptibility to the sterol biosynthesis inhibitors, fluconazole and lovastatin, whereas upc2BΔ‐mutant cells were as susceptible to the drugs as wild‐type cells. The growth of upc2AΔ cells was also severely attenuated under anaerobic conditions. Lovastatin treatment enhanced the expression of ergosterol biosynthetic genes, ERG2 and ERG3 in wild‐type and upc2BΔ but not in upc2AΔ cells. Similarly, serum‐induced expression of ERG2 and ERG3 was completely impaired in upc2AΔ cells but was unaffected in upc2BΔ cells, whereas serum‐induced expression of the sterol transporter gene CgAUS1 was impaired in both upc2AΔ and upc2BΔ cells. These results suggest that in C. glabrata CgUPC2A but not in CgUPC2B is the main transcriptional regulator of the genes responsible for maintaining sterol homeostasis as well as susceptibility to sterol inhibitors.
In clathrin-independent endocytosis, Hook1, a microtubule- and cargo-tethering protein, participates in sorting of cargo proteins such as CD98 (encoded by SLC3A2) and CD147 (encoded by BSG) into ...recycling endosomes. However, the molecular mechanism that regulates Hook1-mediated endosomal sorting is not fully understood. In the present study, we found that γ-taxilin is a novel regulator of Hook1-mediated endosomal sorting. γ-Taxilin depletion promoted both CD98-positive tubular formation and CD98 recycling. Conversely, overexpression of γ-taxilin inhibited the CD98-positive tubular formation. Depletion of Hook1, or Rab10 or Rab22a (which are both involved in Hook1-mediated endosomal sorting), attenuated the effect of γ-taxilin depletion on the CD98-positive tubular formation. γ-Taxilin depletion promoted CD147-mediated spreading of HeLa cells, suggesting that γ-taxilin might be a pivotal player in various cellular functions in which Hook1-mediated cargo proteins are involved. γ-Taxilin bound to the C-terminal region of Hook1 and inhibited its interaction with CD98; the latter interaction is necessary for sorting CD98. We suggest that γ-taxilin negatively regulates the sorting of Hook1-mediated cargo proteins into recycling endosomes by interfering with the interactions between Hook1 and the cargo proteins.
α-Taxilin is a member of the taxilin family, a binding partner of the syntaxin family involved in intracellular vesicle traffic. The taxilin family members share a long coiled-coil structure, which ...is homologous to that of Uso1, a yeast homologue of p115. It has been revealed that a parallel homodimerization of p115, an elongated polypeptide, is required for tethering of the transport vesicles to the acceptor membrane. In this study we examined whether α-taxilin has similar biochemical properties to p115. Gel filtration and sedimentation analyses suggest that α-taxilin is an elongated polypeptide containing a long coiled-coil structure. A pull-down assay revealed that His6-α-taxilin binds to GST-α-taxilin, and not to GST in dose-dependent and saturable manners. α-Taxilin formed a parallel homodimer through at least two independent regions. Together, α-taxilin may be involved in tethering of the transport vesicles to the acceptor membranes in a similar way to p115.
Chronic kidney disease (CKD) is a worldwide health problem, and prevention of CKD is important for preservation of renal function after kidney surgery. There is evidence that transcription factor ...nuclear factor erythroid 2-related factor 2 (Nrf2) has a vital antioxidant and detoxifying role in protecting the kidneys against various diseases. Impaired activation of Nrf2 is associated with oxidative stress related to CKD, and Nrf2 is also a key player in the development of cancer. However, the clinical impact of Nrf2 has not been investigated in patients with renal cell carcinoma (RCC). A retrospective study was performed in 89 patients undergoing nephrectomy for RCC. The estimated glomerular filtration rate (eGFR) and serum uric acid (SUA) were investigated over time after surgery. We investigated Nrf2 protein expression in all tumors and single nucleotide polymorphisms (SNPs) of the Nrf2 gene in 7 tumors. In patients whose tumors showed higher Nrf2 expression, there was a more rapid decrease of eGFR and increase of SUA after nephrectomy. Multivariate analysis confirmed that increased Nrf2 expression was an independent poor prognostic factor related to shorter overall survival. Among the 7 tumor samples, an SNP on exon 5 of the Nrf2 gene in one tumor and three genotypes (C/C, C/A, and A/A) of rs6721961 at the promoter region of the Nrf2 gene were observed. Although the mechanisms underlying the influence of Nrf2 are still unclear, our findings suggested that elevated tumor expression of Nrf2 was associated with postoperative CKD and biologically aggressive RCC with an unfavorable prognosis.
Never in mitosis A-related kinase 2A (Nek2A), a centrosomal serine/threonine kinase, is involved in mitotic progression by regulating the centrosome cycle. Particularly, Nek2A is necessary for ...dissolution of the intercentriole linkage between the duplicated centrosomes prior to mitosis. Nek2A activity roughly parallels its cell cycle-dependent expression levels, but the precise mechanism regulating its activity remains unclear. In this study, we found that γ-taxilin co-localized with Nek2A at the centrosome during interphase and interacted with Nek2A in yeast two-hybrid and pull-down assays and that γ-taxilin regulated centrosome disjunction in a Nek2A-dependent manner. γ-Taxilin depletion increased the number of cells with striking splitting of centrosomes. The precocious splitting of centrosomes induced by γ-taxilin depletion was attenuated by Nek2A depletion, suggesting that γ-taxilin depletion induces the Nek2A-mediated dissolution of the intercentriole linkage between the duplicated centrosomes nevertheless mitosis does not yet begin. Taken together with the result that γ-taxilin protein expression levels were decreased at the onset of mitosis, we propose that γ-taxilin participates in Nek2A-mediated centrosome disjunction as a negative regulator through its interaction with Nek2A.
•γ-Taxilin co-localizes with Nek2A at the centrosome during interphase.•γ-Taxilin interacts with Nek2A in yeast two-hybrid and pull-down assays.•γ-Taxilin protein expression levels decrease at the onset of mitosis.•γ-Taxilin depletion induces immature splitting of centrosomes.•The immature splitting of centrosomes induced by γ-taxilin depletion depends on Nek2A.
α-Taxilin is a member of the taxilin family, a binding partner of the syntaxin family involved in intracellular vesicle traffic. The taxilin family members share a long coiled-coil structure, which ...is homologous to that of Uso1, a yeast homologue of p115. It has been revealed that a parallel homodimerization of p115, an elongated polypeptide, is required for tethering of the transport vesicles to the acceptor membrane. In this study we examined whether α-taxilin has similar biochemical properties to p115. Gel filtration and sedimentation analyses suggest that α-taxilin is an elongated polypeptide containing a long coiled-coil structure. A pull-down assay revealed that His6-α-taxilin binds to GST-α-taxilin, and not to GST in dose-dependent and saturable manners. α-Taxilin formed a parallel homodimer through at least two independent regions. Together, α-taxilin may be involved in tethering of the transport vesicles to the acceptor membranes in a similar way to p115.
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