Rab GTPase-activating Protein Research Articles

A pathogen effector co-opts a host RabGAP protein to remodel pathogen interface and subvert defense-related secretion.

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant-derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TOPGAP, to remodel the host-pathogen interface. The effector, PiE354, hijacks TOPGAP as a susceptibility factor to usurp its GAP activity on Rab8a, a key Rab GTPase crucial for defense-related secretion. By hijacking TOPGAP, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co-opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.

Editing and genome-wide analysis upstream open reading frames contributes to enhancing salt tolerance in tomato.

The salinization of soil constitutes a substantial hindrance to the advancement of sustainable agriculture. Our research seeks to elucidate the role of a Rab GTPase-activating protein (RabGAP) family member, SlRabGAP22, in salt tolerance and its translational regulation under salt stress in tomatoes, employing gene-editing techniques and ribosome profiling methodologies. Findings demonstrate that SlRabGAP22 acts as a positive regulator of tomato salt tolerance, with four predicted upstream open reading frames (uORFs) classified into three categories. Functional uORFs were found to be negative regulation. Editing these uORFs along with altering their classifications and characteristics mitigated the inhibitory effects on primary ORFs and fine-tuned gene expression. Enhanced tomato salt tolerance was attributed to improved scavenging of reactive oxygen species, reduced toxicity Na+, and diminished osmotic stress effects. Furthermore, we conducted genome-wide analysis of ORFs to lay the foundation for further research on uORFs in tomatoes. In summary, our findings offer novel perspectives and important data for the enhancement of genetic traits via uORF-based strategies and translational regulation against the backdrop of salt stress.

TBC1D1 is an energy-responsive polarization regulator of macrophages via governing ROS production in obesity.

Energy status is linked to the production of reactive oxygen species (ROS) in macrophages, which is elevated in obesity. However, it is unclear how ROS production is upregulated in macrophages in response to energy overload for mediating the development of obesity. Here, we show that the Rab-GTPase activating protein (RabGAP) TBC1D1, a substrate of the energy sensor AMP-activated protein kinase (AMPK), is a critical regulator of macrophage ROS production and consequent adipose inflammation for obesity development. TBC1D1 deletion decreases, whereas an energy overload-mimetic non-phosphorylatable TBC1D1S231A mutation increases, ROS production and M1-like polarization in macrophages. Mechanistically, TBC1D1 and its downstream target Rab8a form an energy-responsive complex with NOX2 for ROS generation. Transplantation of TBC1D1S231A bone marrow aggravates diet-induced obesity whereas treatment with an ultra-stable TtSOD for removal of ROS selectively in macrophages alleviates both TBC1D1S231A mutation- and diet-induced obesity. Our findings therefore have implications for drug discovery to combat obesity.

Depletion of TBC1D4 Improves the Metabolic Exercise Response by Overcoming Genetically Induced Peripheral Insulin Resistance.

The RabGTPase-activating protein (RabGAP) TBC1D4 (=AS160) represents a key component in the regulation of glucose transport into skeletal muscle and white adipose tissue (WAT) and is therefore crucial during the development of insulin resistance and type-2 diabetes. Increased daily activity has been shown to be associated with improved postprandial hyperglycemia in allele carriers of a loss-of-function variant in the human TBC1D4 gene. Using conventional Tbc1d4-deficient mice (D4KO) fed a high-fat diet (HFD), we show that already a moderate endurance exercise training leads to substantially improved glucose and insulin tolerance and enhanced expression levels of markers for mitochondrial activity and browning in WAT from D4KO animals. Importantly, in vivo and ex vivo analyses of glucose uptake revealed increased glucose clearance in interscapular brown adipose tissue (iBAT) and WAT from trained D4KO mice. Thus, chronic exercise is able to overcome the genetically induced insulin resistance caused by the Tbc1d4-depletion. Gene variants in TBC1D4 may be relevant in future precision medicine as determinants of exercise response.

Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.

DNAJB3 attenuates ER stress through direct interaction with AKT.

Metabolic stress involved in several dysregulation disorders such as type 2 diabetes mellitus (T2DM) results in down regulation of several heat shock proteins (HSPs) including DNAJB3. This down regulation of HSPs is associated with insulin resistance (IR) and interventions which induce the heat shock response (HSR) help to increase the insulin sensitivity. Metabolic stress leads to changes in signaling pathways through increased activation of both c-jun N-terminal kinase-1 (JNK1) and the inhibitor of κB inflammatory kinase (IKKβ) which in turn leads to inactivation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2). DNAJB3 interacts with both JNK1 and IKKβ kinases to mitigate metabolic stress. In addition DNAJB3 also activates the PI3K-PKB/AKT pathway through increased phosphorylation of AKT1 and its substrate AS160, a Rab GTPase-activating protein, which results in mobilization of GLUT4 transporter protein and improved glucose uptake. We show through pull down that AK T1 is an interacting partner of DNAJB3, further confirmed by isothermal titration calorimetry (ITC) which quantified the avidity of AKT1 for DNAJB3. The binding interface was identified by combining protein modelling with docking of the AKT1-DNAJB3 complex. DNAJB3 is localized in the cytoplasm and ER, where it interacts directly with AKT1 and mobilizes AS160 for glucose transport. Inhibition of AKT1 resulted in loss of GLUT4 translocation activity mediated by DNAJB3 and also abolished the protective effect of DNAJB3 on tunicamycin-induced ER stress. Taken together, our findings provide evidence for a direct protein-protein interaction between DNAJB3 and AKT1 upon which DNAJB3 alleviates ER stress and promotes GLUT4 translocation.

RAB5C is a TBC1D21 interactor involved in sperm morphology formation

<p>背景與目的: 不孕是重要的公眾健康議題，造成不孕的因素其中一半與男性相關。其中造成男性不孕的一項重要病因是畸型精子症。在先前的研究中，我們以cDNA微晶片比較造精功能正常與異常的睪丸組織中各別基因的表現量。其中發現一個與不孕相關Rab GTPase激活蛋白，名為TBC1D21蛋白。TBC1D21特異的表現於減數分裂後期精細胞中。而於Tbc1d21基因剔除小鼠模式中發現嚴重精子尾部的中節區域損壞。進一步我們以蛋白質質體學方法鑑定出與TBC1D21結合蛋白。於本研究中我們聚焦於其中一個TBC1D21結合蛋白RAB5C。研究方法與結果: RAB5C蛋白表現於小鼠睪丸組織切片中的精子細胞的頂體與尾部。於成熟精子中，RAB5C蛋白濃縮於頭部頂體與散落於尾部中節區域。其中有趣的是精子經過頂體反應後，於頂體的RAB5C蛋白量明顯下降消失。最後我們利用共同免疫染色法確認RAB5C與TBC1D21共同散落於成熟精子尾部中節區域。結論:基於以上結果，我們發現TBC1D21 交互作用蛋白RAB5C參與於精子頭部頂體與尾部中節的形成。</p> <p> </p><p>Background and purpose: Infertility is a critical public health issue, and half of its causes are related to male factors. Teratozoospermia is one of major cause of male infertility. In our previous studies, we identified the TBC1 domain family member 21 (TBC1D21) gene, a Rab GTPase-activating protein (GAP), as a sterility-related gene in cDNA microarray compared to normal and spermatogenic testicular tissues. TBC1D21 is specifically expressed in post-meiotic male germ cells. Furthermore, the loss of Tbc1d21 allele in mice results in severe defects in the midpiece of the sperm’s tail. Additionally, TBC1D21 interactors were selected using proteomic assays. Herein, we focused on RAB5C, which is a TBC1D21 interactor. Methods and Results: RAB5C was localized in the acrosomal region and elongating tail in spermatids of murine testicular sections. In a mature sperm cell, RAB5C signals were concentrated in the acrosome and were scattered around the midpiece. Interestingly, RAB5C pro-tein levels decreased in the acrosome after triggering acrosomal reaction. Finally, we confirmed that the interspersed RAB5C signals were co-localized with TBC1D21 within the midpieces of the mature sperm cells. Conclusion: Based on these results, we suggest RAB5C, a TBC1D21 interactor, was involved in acrosomal and midpiece formation.</p> <p> </p>

Deletion of Tbc1d4/As160 abrogates cardiac glucose uptake and increases myocardial damage after ischemia/reperfusion

BackgroundType 2 Diabetes mellitus (T2DM) is a major risk factor for cardiovascular disease and associated with poor outcome after myocardial infarction (MI). In T2DM, cardiac metabolic flexibility, i.e. the switch between carbohydrates and lipids as energy source, is disturbed. The RabGTPase-activating protein TBC1D4 represents a crucial regulator of insulin-stimulated glucose uptake in skeletal muscle by controlling glucose transporter GLUT4 translocation. A human loss-of-function mutation in TBC1D4 is associated with impaired glycemic control and elevated T2DM risk. The study’s aim was to investigate TBC1D4 function in cardiac substrate metabolism and adaptation to MI.MethodsCardiac glucose metabolism of male Tbc1d4-deficient (D4KO) and wild type (WT) mice was characterized using in vivo [18F]-FDG PET imaging after glucose injection and ex vivo basal/insulin-stimulated [3H]-2-deoxyglucose uptake in left ventricular (LV) papillary muscle. Mice were subjected to cardiac ischemia/reperfusion (I/R). Heart structure and function were analyzed until 3 weeks post-MI using echocardiography, morphometric and ultrastructural analysis of heart sections, complemented by whole heart transcriptome and protein measurements.ResultsTbc1d4-knockout abolished insulin-stimulated glucose uptake in ex vivo LV papillary muscle and in vivo cardiac glucose uptake after glucose injection, accompanied by a marked reduction of GLUT4. Basal cardiac glucose uptake and GLUT1 abundance were not changed compared to WT controls. D4KO mice showed mild impairments in glycemia but normal cardiac function. However, after I/R D4KO mice showed progressively increased LV endsystolic volume and substantially increased infarction area compared to WT controls. Cardiac transcriptome analysis revealed upregulation of the unfolded protein response via ATF4/eIF2α in D4KO mice at baseline. Transmission electron microscopy revealed largely increased extracellular matrix (ECM) area, in line with decreased cardiac expression of matrix metalloproteinases of D4KO mice.ConclusionsTBC1D4 is essential for insulin-stimulated cardiac glucose uptake and metabolic flexibility. Tbc1d4-deficiency results in elevated cardiac endoplasmic reticulum (ER)-stress response, increased deposition of ECM and aggravated cardiac damage following MI. Hence, impaired TBC1D4 signaling contributes to poor outcome after MI.

Improving Lipophagy by Restoring Rab7 Cycle: Protective Effects of Quercetin on Ethanol-Induced Liver Steatosis.

Chronic alcohol consumption retards lipophagy, which contributes to the pathogenesis of liver steatosis. Lipophagy-related Rab7 has been presumed as a crucial regulator in the progression of alcohol liver disease despite elusive mechanisms. More importantly, whether or not hepatoprotective quercetin targets Rab7-associated lipophagy disorder is unknown. Herein, alcoholic fatty liver induced by chronic-plus-single-binge ethanol feeding to male C57BL/6J mice was manifested by hampering autophagosomes formation with lipid droplets and fusion with lysosomes compared with the normal control, which was normalized partially by quercetin. The GST-RILP pulldown assay of Rab7 indicated an improved GTP-Rab7 as the quercetin treatment for ethanol-feeding mice. HepG2 cells transfected with CYP2E1 showed similar lipophagy dysfunction when exposed to ethanol, which was blocked when cells were transfected with siRNA-Rab7 in advance. Ethanol-induced steatosis and autophagic flux disruption were aggravated by the Rab7-specific inhibitor CID1067700 while alleviated by transfecting with the Rab7Wt plasmid, which was visualized by immunofluorescence co-localization analysis and mCherry-GFP-LC3 transfection. Furthermore, TBC1D5, a Rab GTPase-activating protein for the subsequent normal circulation of Rab7, was downregulated after alcohol administration but regained by quercetin. Rab7 circulation retarded by ethanol and corrected by quercetin was further revealed by fluorescence recovery after photobleaching (FRAP). Altogether, quercetin attenuates hepatic steatosis by normalizing ethanol-imposed Rab7 turnover disorders and subsequent lipophagy disturbances, highlighting a novel mechanism and the promising prospect of quercetin-like phytochemicals against the crucial first hit from alcohol.

GP73 is a TBC-domain Rab GTPase-activating protein contributing to the pathogenesis of non-alcoholic fatty liver disease without obesity

The prevalence of non-obese nonalcoholic fatty liver disease (NAFLD) is increasing worldwide with unclear etiology and pathogenesis. Here, we show GP73, a Golgi protein upregulated in livers from patients with a variety of liver diseases, exhibits Rab GTPase-activating protein (GAP) activity regulating ApoB export. Upon regular-diet feeding, liver-GP73-high mice display non-obese NAFLD phenotype, characterized by reduced body weight, intrahepatic lipid accumulation, and gradual insulin resistance development, none of which can be recapitulated in liver-GAP inactive GP73-high mice. Common and specific gene expression signatures associated with GP73-induced non-obese NAFLD and high-fat diet (HFD)-induced obese NAFLD are revealed. Notably, metformin inactivates the GAP activity of GP73 and alleviates GP73-induced non-obese NAFLD. GP73 is pathologically elevated in NAFLD individuals without obesity, and GP73 blockade improves whole-body metabolism in non-obese NAFLD mouse model. These findings reveal a pathophysiological role of GP73 in triggering non-obese NAFLD and may offer an opportunity for clinical intervention.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

25-OR: Role for TBC1D30 in Secretion of Mature Insulin

Genome-wide association studies (GWAS) have identified loci associated with the processing and secretion of mature insulin. Increased proinsulin (PI) secretion due to incomplete processing of insulin precursors has been linked to pancreatic beta-cell stress. At one PI GWAS locus, rs150781447-T is associated with increased PI secretion in Finns (p=5.5X10-11) and causes an amino acid (AA) change (R279C) at a conserved position in exon 4 of TBC1D30, a putative Rab GTPase Activating Protein (GAP), while no GWAS proxy variants (r2>0.8, 1000G Finns) are located in transcribed or enhancer regions. We hypothesized that TBC1D30 is involved in PI processing and that missense variant rs150781447, predicted to be damaging, has a negative impact on gene function. We used genome editing to generate four sets of TBC1D30 mutants in 832/13-INS1 cells. Compared to mock-edited control cells, apparent loss-of-function in frame insertion-deletion cells (106% expression) secreted 2.1-fold more PI (p=0.0001), and heterozygous truncation knockout cells (44% expression) secreted 1.9-fold more PI (p=0.005). Cells heterozygous for rs150781447, generated by prime editing, secreted 1.2-fold more PI (p=0.89), suggesting that the heterozygous variant has subtle or no effect on gene function. Unexpectedly, when exon 4 (42 AA) was deleted in frame, the cells secreted 3.2-fold less PI (p=0.02), consistent with a gain-of-function effect and suggesting that exon 4, part of the conserved Rab GAP protein domain, has a critical function. Preliminary insulin secretion data, while variable, demonstrate trends similar to proinsulin secretion, consistent with a stronger effect of TBC1D30 on processing than secretion. Together, these data demonstrate TBC1D30 as the likely target of the nearby PI GWAS locus and suggest that Rab GAP TBC1D30 plays a critical role in the processing of mature insulin. Disclosure V. A. Parsons: None. S. Vadlamudi: None. A. H. Moxley: None. K. L. Mohlke: None. Funding National Institutes of Health (1T32GM135128-01)

The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.

AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase

TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ∼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis–Menten kinetics using in vitro phosphorylation assays with purified kinases and stable isotope-labeled γ-[18O4]-ATP. These data revealed that Ser324 (KM ∼6 μM) and Thr649 (KM ∼25 μM) were preferential sites for phosphorylation by AKT, whereas Ser348, Ser577, Ser595 (KM ∼10 μM), Ser711 (KM ∼79 μM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.

AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP

In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

TBC1D21 Potentially Interacts with and Regulates Rap1 during Murine Spermatogenesis.

Few papers have focused on small guanosine triphosphate (GTP)-binding proteins and their regulation during spermatogenesis. TBC1D21 genes (also known as male germ cell RAB GTPase-activating protein MGCRABGAP) are related to sterility, as determined through cDNA microarray testing of human testicular tissues exhibiting spermatogenic defects. TBC1D21 is a protein specifically expressed in the testes that exhibits specific localizations of elongating and elongated spermatids during mammalian spermiogenesis. Furthermore, through co-immunoprecipitation (co-IP) and nano liquid chromatography–tandem mass spectrometry (nano LC–MS/MS), Rap1 has been recognized as a potential TBC1D21 interactor. This study determined the possible roles of Rap1 and TBC1D21 during mammalian spermiogenesis. First, the binding ability between Rap1 and TBC1D21 was verified using co-IP. Second, the stronger signals of Rap1 expressed in elongating and elongated murine spermatids extracted from testicular sections, namely spermatogonia, spermatocytes, and round spermatids, were compared. Third, Rap1 and TBC1D21 exhibited similar localizations at postacrosomal regions of spermatids and at the midpieces of mature sperms, through isolated male germ cells. Fourth, the results of an activating Rap1 pull-down assay indicated that TBC1D21 overexpression inactivates Rap1 activity in cell models. In conclusion, TBC1D21 may interact with and potentially regulate Rap1 during murine spermatogenesis.

Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation

Ehrlichia chaffeensis, an obligatory intracellular bacterium, infects monocytes/macrophages by sequestering a regulator of endosomal traffic, the small GTPase RAB5, on its membrane-bound inclusions to avoid routing to host-cell phagolysosomes. How RAB5 is sequestered on ehrlichial inclusions is poorly understood, however. We found that native Ehrlichia translocated factor-2 (Etf-2), a previously predicted effector of the Ehrlichia type IV secretion system, and recombinant Etf-2 (cloned into the Ehrlichia genome) are secreted into the host-cell cytoplasm and localize to ehrlichial inclusions. Ectopically expressed Etf-2-GFP also localized to inclusions and membranes of early endosomes marked with RAB5 and interacted with GTP-bound RAB5 but not with a GDP-bound RAB5. Etf-2, although lacking a RAB GTPase-activating protein (GAP) Tre2-Bub2-Cdc16 (TBC) domain, contains two conserved TBC domain motifs, namely an Arg finger and a Gln finger, and site-directed mutagenesis revealed that both Arg188 and Gln245 are required for Etf-2 localization to early endosomes. The yeast two-hybrid assay and microscale thermophoresis revealed that Etf-2 binds tightly to GTP-bound RAB5 but not to GDP-bound RAB5. However, Etf-2 lacks RAB5-specific GAP activity. Etf-2 localized to bead-containing phagosomes as well as endosomes containing beads coated with the C-terminal fragment of EtpE (entry-triggering protein of Ehrlichia), an Ehrlichia outer-membrane invasin, and significantly delayed RAB5 dissociation from and RAB7 localization to phagosomes/endosomes and RABGAP5 localization to endosomes. Thus, binding of Etf-2 to RAB5-GTP appears to delay RAB5 inactivation by impeding RABGAP5 localization to endosomes. This suggests a unique mechanism by which RAB5 is sequestered on ehrlichial inclusions to benefit bacterial survival and replication.

The Rab GTPase activating protein Gyp2 contributes to UV stress tolerance in Metarhizium acridum.

GTPase activation protein (GAP) for Rab GTPases can accelerate GTP hydrolysis to alter the activity of Rab GTPases. To explore the function of GAP in entomopathogenic fungi, we constructed a deletion mutant of Gyp2 gene, a member of the Gyp (GAP for Ypt/Rab proteins) family in the locust-specific fungal pathogen, Metarhizium acridum. Results showed that the ∆MaGyp2 mutant had dramatically decreased tolerance to ultraviolet irradiation compared to wild type strain. Quantitative real-time PCR revealed that UV irradiation repair related genes Uve1 and WC1 were downregulated in ∆MaGyp2 mutant. Seven of other ten Gyp family members had significantly increased transcription in ∆MaGyp2 mutant compared with wild type, which may partly rescue the deficiency of MaGyp2.

Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal glucose transporter 4 redistribution but not insulin-mediated responses in rats

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.

The carboxy-terminal region of the TBC1D4 (AS160) RabGAP mediates protein homodimerization

TBC1D4 (also known as AS160) is a Rab·GTPase-activating protein (RabGAP) which functions in insulin signaling. TBC1D4 is critical for translocation of glucose transporter 4 (GLUT4), from an inactive, intracellular, vesicle-bound site to the plasma membrane, where it promotes glucose entry into cells. The TBC1D4 protein is structurally subdivided into two N-terminal phosphotyrosine-binding (PTB) domains, a C-terminal catalytic RabGAP domain, and a disordered segment in between containing potential Akt phosphorylation sites. Structural predictions further suggest that a region C-terminal to the RabGAP domain adopts a coiled-coil motif. We show that C-terminal region (CTR) region is largely α-helical and mediates TBC1D4 RabGAP dimerization. RabGAP catalytic activity and thermal stability appear to be independent of CTR-mediated dimerization.