Expression,phosphorylation and function of the Rab-GTPase activating protein TBC1D1 in pancreatic beta-cells

The Rab-GTPase activating protein TBC1D1 is a paralog of AS160/TBC1D4. AS160/TBC1D4, a downstream effector of Akt, has been shown to play a central role in beta-cell function and survival. The two proteins have overlapping function in insulin signalling in muscle cells. However, the expression and the potential role of TBC1D1 in beta-cells remain unknown. Therefore, the aim of this study is to investigate whether TBC1D1 is expressed in beta-cells and whether it plays, as AS160/TBC1D4, a role in beta-cell function and survival. Using human and rat beta-cells, this study shows for the first time that TBC1D1 is expressed and phosphorylated in response to glucose in these cells. Knockdown of TBC1D1 in beta-cells resulted in increased basal and glucose-stimulated insulin release, decreased proliferation but no change in apoptosis.     

TBC1D14 sets the TRAPP for ATG9

Amino acid withdrawal induces the formation of autophagosomes, which results in dozens of these large double-membrane vesicles appearing in the starved cell within 10–15 min, and the initiation of autophagy. This vesicle-mediated response clearly requires an adequate supply of membrane and a tight molecular regulation creating a substantial challenge for the cell in terms of vesicle trafficking pathways. Several membrane sources, which contribute to autophagosome initiation and formation, have been identified including the ER, Golgi, plasma membrane, mitochondria and recycling endosomes. How contributions from these organelles are regulated is an intensive area of study. Members of several families of membrane traffic regulators, including small GTPases, such as RAB proteins, and their regulators, SNARE proteins and BAR domain-containing proteins, have recently been shown to support autophagosome formation.     

Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

Retromer is an endosomal multi‐protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans‐Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer‐associated RAB7‐specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer‐dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin‐mediated mitophagy.     

TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy

The RabGAP protein TBC1D5 controls cellular endomembrane trafficking processes and binds the retromer subunit VPS29 and the ubiquitin‐like protein ATG8 (LC3). Here, we describe that TBC1D5 also associates with ATG9 and the active ULK1 complex during autophagy. Moreover, ATG9 and TBC1D5 interact with clathrin and the AP2 complex. Depletion of TBC1D5 leads to missorting of ATG9 to late endosomes upon activation of autophagy, whereas inhibition of clathrin‐mediated endocytosis or AP2 depletion alters ATG9 trafficking and its association with TBC1D5. Taken together, our data show that TBC1D5 and the AP2 complex are important novel regulators of the rerouting of ATG9‐containing vesicular carriers toward sites of autophagosome formation.     

TBC domain family, member 15 is a novel mammalian Rab GTPase-activating protein with substrate preference for Rab7

Ypt/Rabs are Ras-related GTPases that function as key regulators of intracellular vesicular trafficking. Their slow intrinsic rates of GTP hydrolysis are catalyzed by GTPase-activating proteins (GAPs). Ypt/Rab-GAPs constitute a family of proteins that contain a TBC (Tre-2/Bub2/Cdc16) domain. Only three of the 51 family members predicted in the human genome are confirmed Ypt/Rab-GAPs. Here, we report the identification and characterization of a novel mammalian Ypt/Rab-GAP, TBC domain family, member 15 (TBC1D15). TBC1D15 is ubiquitously expressed and localized predominantly to the cytosol. The TBC domain of TBC1D15 exhibits relatively high homology with that of Gyp7p, a yeast Ypt/Rab-GAP. Furthermore, TBC1D15 stimulates the intrinsic GTPase activity of Rab7, and to a lesser extent Rab11, but is essentially inactive towards Rab4 or Rab6. These data increase the number of mammalian TBC domain family members with demonstrated Rab-GAP activity to four, and suggest that TBC1D15 may be involved in Rab7-mediated late endosomal trafficking.     

Discovery of TBC1D7 as a Potential Driver for Melanoma Cell Invasion

Metastasis is the leading cause for mortality in melanoma patients. Here, an unbiased mass spectrometry‐based quantitative proteomic method is utilized to assess differential protein expression in a matched pair of primary/metastatic melanoma cell lines (i.e., WM‐115/WM‐266‐4) derived from the same patient. It is found that TBC1D7 is overexpressed in metastatic over primary melanoma cells, and elevated expression of TBC1D7 promotes the invasion of these melanoma cells in vitro, partly through modulating the activities of secreted matrix metalloproteinases 2 and 9. Additionally, interrogation of publicly available data show that higher mRNA expression of TBC1D7 predicts poorer survival in melanoma patients. Together, the results suggest TBC1D7 as a driver for melanoma cell invasion, which is an important element in melanoma metastasis. The proteomic data generated from this study may also be useful for exploring the roles of other proteins in melanoma metastasis.     

Identification of TBC7 having TBC domain as a novel binding protein to TSC1-TSC2 complex

TBC7, a TBC (Tre-2/Bub2/Cdc16) 1 domain protein, was identified as a novel binding protein to the TSC1-TSC2 tumor suppressor complex by peptide mass fingerprinting analysis of the proteins immunoprecipitated with FLAG-epitope tagged TSC1 and TSC2 from the transfected mammalian cells. The in vivo and in vitro association of TBC7 and the TSC1-TSC2 complex was confirmed by the co-immunoprecipitation and pull-down analysis, respectively, and TBC7 was revealed to bind to the C-terminal half region of TSC1, which is distinct from the binding site with TSC2. The immunofluorescence microscopy and subcellular fractionation showed that TBC7 co-localizes with the tumor suppressor complex in the endomembrane. Overexpression of TBC7 enhanced ubiquitination of TSC1 and increased phosphorylation of S6 protein by S6 kinase, that is located in the mTOR-signaling pathway. These results indicate TBC7 could take a part in the negative regulation of the tumor suppressor complex through facilitating the downregulation of TSC1.     

Crystal structure of TBC1D15 GTPase‐activating protein (GAP) domain and its activity on Rab GTPases

TBC1D15 belongs to the TBC (Tre‐2/Bub2/Cdc16) domain family and functions as a GTPase‐activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark‐TBC1D15 and Sus‐TBC1D15 belong to the same subfamily of TBC domain‐containing proteins, and their GAP‐domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost.     

TBC1D25 alleviates nonalcoholic steatohepatitis by inhibiting abnormal lipid accumulation and inflammation

Nonalcoholic fatty liver disease (NAFLD) is a strong stimulant of cardiovascular diseases, affecting one-quarter of the world's population. TBC1 domain family member 25 (TBC1D25) regulates the development of myocardial hypertrophy and cerebral ischemia–reperfusion injury; however, its effect on NAFLD/nonalcoholic steatohepatitis (NASH) has not been reported. In this study, we demonstrated that TBC1D25 expression is upregulated in NASH. TBC1D25 deficiency aggravated hepatic steatosis, inflammation, and fibrosis in NASH. In vitro tests revealed that TBC1D25 overexpression restrained NASH responses. Subsequent mechanistic validation experiments demonstrated that TBC1D25 interfered with NASH progression by inhibiting abnormal lipid accumulation and inflammation. TBC1D25 deficiency significantly promoted NASH occurrence and development. Therefore, TBC1D25 may potentially be used as a clinical therapeutic target for NASH treatment.     

TBC1D20 mediates autophagy as a key regulator of autophagosome maturation

In humans, loss of TBC1D20 (TBC1 domain family, member 20) protein function causes Warburg Micro syndrome 4 (WARBM4), an autosomal recessive disorder characterized by congenital eye, brain, and genital abnormalities. TBC1D20-deficient mice exhibit ocular abnormalities and male infertility. TBC1D20 is a ubiquitously expressed member of the family of GTPase-activating proteins (GAPs) that increase the intrinsically slow GTP-hydrolysis rate of small RAB-GTPases when bound to GTP. Biochemical studies have established TBC1D20 as a GAP for RAB1B and RAB2A. However, the cellular role of TBC1D20 still remains elusive, and there is little information about how the functional loss of TBC1D20 causes clinical manifestations in WARBM4-affected children. Here we evaluate the role of TBC1D20 in cells carrying a null mutant allele, as well as TBC1D20-deficient mice, which display eye and testicular abnormalities. We demonstrate that TBC1D20, via its RAB1B GAP function, is a key regulator of autophagosome maturation, a process required for maintenance of autophagic flux and degradation of autophagic cargo. Our results provide evidence that TBC1D20-mediated autophagosome maturation maintains lens transparency by mediating the removal of damaged proteins and organelles from lens fiber cells. Additionally, our results show that in the testes TBC1D20-mediated maturation of autophagosomes is required for autophagic flux, but is also required for the formation of acrosomes. Furthermore TBC1D20-deficient mice, while not mimicking severe developmental brain abnormalities identified in WARBM4 affected children, display disrupted neuronal autophagic flux resulting in adult-onset motor dysfunction. In summary, we show that TBC1D20 has an essential role in the maturation of autophagosomes and a defect in TBC1D20 function results in eye, testicular, and neuronal abnormalities in mice implicating disrupted autophagy as a mechanism that contributes to WARBM4 pathogenesis.     

siRNA抑制哺乳动物细胞内Rab9 GTPase表达的研究

构建Rab9 GTPase siRNA表达载体,观察siRNA对哺乳动物细胞内Rab9 GTPase表达的抑制作用,为应用Rab9 GTPase siRNA表达载体进行抗麻疹病毒感染研究奠定基础。根据Rab9 GTPase基因的mRNA序列设计合成2对靶向Rab9 GTPase基因的寡核苷酸,克隆到表达载体pSUPER.neo EGFP,通过双酶切和序列分析对重组表达载体进行鉴定。将鉴定为阳性的重组表达载体转染B95a细胞株,通过逆转录聚合酶链反应(RT-PCR)和免疫印迹技术(Western-blot)检测B95a细胞内Rab9 GTPase mRNA和蛋白的表达水平。双酶切和序列分析表明成功构建了Rab9 GTPase siRNA表达载体,RT-PCR和Western-blot技术实验表明重组表达载体可显著抑制B95a细胞内Rab9 GTPase mRNA和蛋白的表达(最高抑制率分别为89.4%±0.5%和87.6%±0.7%),而对照组则没有变化。结果表明,成功构建了Rab9 GTPase siRNA表达载体,该表达载体可有效地抑制Rab9 GTPase在哺乳动物细胞内的表达。     

Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

MAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.     

Immunofluorescent localization of the Rab-GAP protein TBC1D4 (AS160) in mouse kidney

TBC1D4 (or AS160) was identified as a Rab-GTPase activating protein (Rab-GAP) that controls insulin-dependent trafficking of the glucose transporter GLUT4 in skeletal muscle cells and in adipocytes. Recent in vitro cell culture studies suggest that TBC1D4 may also regulate the intracellular trafficking of kidney proteins such as the vasopressin-dependent water channel AQP2, the aldosterone-regulated epithelial sodium channel ENaC, and the Na(+)-K(+)-ATPase. To study the possible role of TBC1D4 in the kidney in vivo, we raised a rabbit polyclonal antibody against TBC1D4 to be used for immunoblotting and immunohistochemical studies. In immunoblots on mouse kidney homogenates, the antibody recognizes specific bands at the expected size of 160 kDa and at lower molecular weights, which are absent in kidneys of TBC1D4 deficient mice. Using a variety of nephron-segment-specific marker proteins, immunohistochemistry reveals TBC1D4 in the cytoplasm of the parietal epithelial cells of Bowman's capsule, the thin and thick limbs of Henle's loop, the distal convoluted tubule, the connecting tubule, and the collecting duct. In the latter, both principal as well as intercalated cells are TBC1D4-positive. Thus, with the exception of the proximal tubule, TBC1D4 is highly expressed along the nephron and the collecting duct, where it may interfere with the intracellular trafficking of many renal transport proteins including AQP2, ENaC and Na(+)-K(+)-ATPase. Hence, TBC1D4 may play an important role for the control of renal ion and water handling and hence for the control of extracellular fluid homeostasis.     

Crystal structures of human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating protein (RabGAP) domains reveal critical elements for GLUT4 translocation

We have solved the x-ray crystal structures of the RabGAP domains of human TBC1D1 and human TBC1D4 (AS160), at 2.2 and 3.5 Å resolution, respectively. Like the yeast Gyp1p RabGAP domain, whose structure was solved previously in complex with mouse Rab33B, the human TBC1D1 and TBC1D4 domains both have 16 α-helices and no β-sheet elements. We expected the yeast Gyp1p RabGAP/mouse Rab33B structure to predict the corresponding interfaces between cognate mammalian RabGAPs and Rabs, but found that residues were poorly conserved. We further tested the relevance of this model by Ala-scanning mutagenesis, but only one of five substitutions within the inferred binding site of the TBC1D1 RabGAP significantly perturbed catalytic efficiency. In addition, substitution of TBC1D1 residues with corresponding residues from Gyp1p did not enhance catalytic efficiency. We hypothesized that biologically relevant RabGAP/Rab partners utilize additional contacts not described in the yeast Gyp1p/mouse Rab33B structure, which we predicted using our two new human TBC1D1 and TBC1D4 structures. Ala substitution of TBC1D1 Met⁹³⁰, corresponding to a residue outside of the Gyp1p/Rab33B contact, substantially reduced catalytic activity. GLUT4 translocation assays confirmed the biological relevance of our findings. Substitutions with lowest RabGAP activity, including catalytically dead RK and Met⁹³⁰ and Leu¹⁰¹⁹ predicted to perturb Rab binding, confirmed that biological activity requires contacts between cognate RabGAPs and Rabs beyond those in the yeast Gyp1p RabGAP/mouse Rab33B structure.     

Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella‐containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane‐bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP‐bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63‐containing late endosomes. Nischarin is recruited to the SCV in a Rab14‐dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners—Rac1, Rab14 and Rab9 GTPases—reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.     

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.