Racial differences in protein S Tokushima and two protein C variants as genetic risk factors for venous thromboembolism.

BACKGROUND: Racial differences in genetic risk factors for venous thromboembolism (VTE) are elucidated, with factor V Leiden and prothrombin G20210A being prevalent among the Caucasian population but rare among non-Caucasians. OBJECTIVES: To assess the worldwide distribution of three gene polymorphisms previously identified as genetic risk factors among East Asian subpopulations: protein S (PS) Tokushima (p.Lys196Glu), protein C (PC) p.Arg189Trp, and PC p.Lys193del. METHODS: An international collaborative study group of seven centers in five countries-Japan, South Korea, Singapore, Hungary, and Brazil-was created, and genotype analyses were performed. A total of 2850 unrelated individuals (1061 patients with VTE and 1789 controls) were included. RESULTS: PS Tokushima was confined to Japanese patients with VTE (allele frequency, 2.35%) and controls (1.12%), with an odds ratio (OR) of 2.15 (95% confidence interval, 1.16-3.99). PC p.Arg189Trp carriers were prevalent among Chinese and Malay patients with VTE in Singapore, with allele frequencies of 10.53% and 22.73%, respectively. Carriers of PC p.Lys193del were identified among Japanese and Korean patients with VTE (0.87% and 2.35%, respectively) and controls (0.36% and 1.07%, respectively), with the OR for VTE not being significant, and Chinese patients with VTE in Singapore (5.26%). In contrast, no carriers of PS Tokushima and two PC gene variants were found among patients with VTE or controls from Hungary, Brazil, or Indians in Singapore. CONCLUSION: The three variants were prevalent among East and Southeast Asians, having some differences in geographic distribution, but were absent among Caucasian subpopulations and Brazilians.

Plasma phenotypes of protein S Lys196Glu and protein C Lys193del variants prevalent among young Japanese women.

: Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations, respectively; however, their diagnosis by plasma analyses is difficult because of the type II deficiency phenotype. Three gene variant genotypes were examined in young Japanese women (n = 231). Plasma total protein S activity and total protein S antigen levels were measured using a total protein S assay system, protein C and protein S activities by clot-based methods, and protein C and free protein S antigen levels by latex agglutination methods. protein S Tokushima (p.Lys196Glu) and protein C p.Lys193del variants were prevalent among participants with allele frequencies of 1.08 and 0.86%, respectively, whereas any carrier of protein C p.Arg189Trp variant was not identified. The plasma phenotype of the type II deficiency of protein S Tokushima heterozygotes was demonstrated by decreased total protein S activity with a normal total protein S antigen level; however, the protein C activities of protein C p.Lys193del heterozygotes were within reference intervals, whereas their protein C antigen levels were elevated. We compared the diagnostic accuracy of the total protein S activity/total protein S antigen ratio for identifying protein S Tokushima heterozygotes with that of the clot-based protein S activity/free protein S antigen ratio and found that sensitivity and specificity of 100% each was only achieved by the former. Protein S Tokushima and protein C p.Lys193del are prevalent among young Japanese women, and a plasma analysis using the total protein S assay system is more accurate than the clot-based protein S activity/free protein S antigen ratio for diagnosing protein S Tokushima carriers.

Relationship between plasma protein S levels and apolipoprotein C-II in Japanese middle-aged obese women and young nonobese women.

: Protein S, a nonenzymatic cofactor to activated protein C, presents in two forms in plasma, free form and in a complex with C4b-binding protein. The aim of this study was to determine the association of plasma protein S levels with the variables related to cardiovascular disease risk. The relationships between plasma protein S levels with lipids, inflammation markers, and adiposity were first examined on middle-aged obese women (n = 62), then on young nonobese women (n = 160) to verify the findings in the obese women. Total and free protein S antigen levels in middle-aged obese women, approximately half being in a postmenopausal state and suffered from dyslipidemia, correlated negatively with estradiol and positively with triglycerides, total cholesterol, LDL cholesterol, apoA-II, apoB, apoC-II, apoC-III, apoE, hemoglobin A1c, and protein C, whereas there was no correlation with HDL cholesterol, apoA-I, BMI, visceral fat area, blood pressure, or factor VII activity. Multiple linear regression analyses revealed that protein C, apoC-II, and fibrinogen were significant predictors of total protein S antigen levels, accounting for 51.9% of variance, and apoC-II as a singular significant predictor for free protein S antigen levels (12.3% of variance). In young nonobese women, most being normolipidemic, apoC-II was also selected as a significant predictor of total protein S antigen levels, but not of free protein S antigen levels. The positive relationship between plasma protein S levels and apoC-II, a key regulator of triglycerides hydrolysis, may contribute to the pathogenesis of increased concentrations of plasma protein S.

TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells.

Recycling endosomes are generally thought to play a central role in endocytic recycling, but recent evidence has indicated that they also participate in other cellular events, including cytokinesis, autophagy, and neurite outgrowth. Rab small GTPases are key regulators in membrane trafficking, and although several Rab isoforms, e.g., Rab11, have been shown to regulate recycling endosomal trafficking, the precise mechanism by which these Rabs regulate recycling endosomes is not fully understood. In this study, we focused on a Rab-GTPase-activating protein (Rab-GAP), one of the key regulators of Rabs, and comprehensively screened 43 mammalian Tre-2/Bub2/Cdc16 (TBC)/Rab-GAP-domain-containing proteins (TBC proteins) for proteins that specifically localize on recycling endosomes in mouse embryonic fibroblasts (MEFs). Four of the 43 mammalian TBC proteins screened, i.e., TBC1D11, TBC1D12, TBC1D14, and EVI5, were found to colocalize well with transferrin receptor, a well-known recycling endosome marker. We further investigated the biochemical properties of TBC1D12, a previously uncharacterized TBC protein. The results showed that TBC1D12 interacted with active Rab11 through its middle region and that it did not display Rab11-GAP activity in vitro. The recycling endosomal localization of TBC1D12 was found to depend on the expression of Rab11. We also found that TBC1D12 expression had no effect on common Rab11-dependent cellular events, e.g., transferrin recycling, in MEFs and that it promoted neurite outgrowth, a specialized Rab11-dependent cellular event, of PC12 cells independently of its GAP activity. These findings indicated that TBC1D12 is a novel Rab11-binding protein that modulates neurite outgrowth of PC12 cells.

Rab12 Regulates Retrograde Transport of Mast Cell Secretory Granules by Interacting with the RILP-Dynein Complex.

Secretory granule (SG) transport is a critical step in regulated exocytosis including degranulation of activated mast cells. The latter process results in the release of multiple inflammatory mediators that play key roles in innate immunity, as well as in allergic responses. In this study, we identified the small GTPase Rab12 as a novel regulator of mast cell SG transport, and we provide mechanistic insights into its mode of action. We show that Rab12 is activated in a stimulus-dependent fashion and promotes microtubule-dependent retrograde transport of the SGs in the activated cells. We also show that this minus end transport of the SGs is mediated by the RILP-dynein complex and identify RILP as a novel effector of Rab12. Finally, we show that Rab12 negatively regulates mast cell degranulation. Taken together, our results identify Rab12 as a novel regulator of mast cell responses and disclose for the first time, to our knowledge, the mechanism of retrograde transport of the mast cell SGs.

Dennd3 functions as a guanine nucleotide exchange factor for small GTPase Rab12 in mouse embryonic fibroblasts.

Small GTPase Rab12 regulates mTORC1 (mammalian target of rapamycin complex 1) activity and autophagy through controlling PAT4 (proton/amino acid transporter 4) trafficking from recycling endosomes to lysosomes, where PAT4 is degraded. However, the precise regulatory mechanism of the Rab12-mediated membrane trafficking pathway remained to be determined because a physiological Rab12-GEF (guanine nucleotide exchange factor) had yet to be identified. In this study we performed functional analyses of Dennd3, which has recently been shown to possess a GEF activity toward Rab12 in vitro. The results showed that knockdown of Dennd3 in mouse embryonic fibroblast cells caused an increase in the amount of PAT4 protein, the same as Rab12 knockdown did, and knockdown of Dennd3 and overexpression of Dennd3 were found to result in an increase and a decrease, respectively, in the intracellular amino acid concentration. Dennd3 overexpression was also found to reduce mTORC1 activity and promoted autophagy in a Rab12-dependent manner. Unexpectedly, however, Dennd3 knockdown had no effect on mTORC1 activity or autophagy despite increasing the intracellular amino acid concentration. Further study showed that Dennd3 knockdown reduced Akt activity, and the reduction in Akt activity is likely to have canceled out amino acid-induced mTORC1 activation through PAT4. These findings indicated that Dennd3 not only functions as a Rab12-GEF but also modulates Akt signaling in mouse embryonic fibroblast cells.