Degradation of Activated K-Ras Orthologue via K-Ras-specific Lysine Residues Is Required for Cytokinesis

Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.     

Structures of Human Steroidogenic Cytochrome P450 17A1 with Substrates

The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn²⁰² in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b₅ might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.     

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

Dengue Virus Infections among Haitian and Expatriate Non-governmental Organization Workers — Léogane and Port-au-Prince,Haiti, 2012

In October 2012, the Haitian Ministry of Health and the US CDC were notified of 25 recent dengue cases, confirmed by rapid diagnostic tests (RDTs), among non-governmental organization (NGO) workers. We conducted a serosurvey among NGO workers in Léogane and Port-au-Prince to determine the extent of and risk factors for dengue virus infection. Of the total 776 staff from targeted NGOs in Léogane and Port-au-Prince, 173 (22%; 52 expatriates and 121 Haitians) participated. Anti-dengue virus (DENV) IgM antibody was detected in 8 (15%) expatriates and 9 (7%) Haitians, and DENV non-structural protein 1 in one expatriate. Anti-DENV IgG antibody was detected in 162 (94%) participants (79% of expatriates; 100% of Haitians), and confirmed by microneutralization testing as DENV-specific in 17/34 (50%) expatriates and 42/42 (100%) Haitians. Of 254 pupae collected from 68 containers, 65% were Aedes aegypti; 27% were Ae. albopictus. Few NGO workers reported undertaking mosquito-avoidance action. Our findings underscore the risk of dengue in expatriate workers in Haiti and Haitians themselves.     

Dynamics of Japanese Encephalitis Virus Transmission among Pigs in Northwest Bangladesh and the Potential Impact of Pig Vaccination

Background

Japanese encephalitis (JE) virus infection can cause severe disease in humans, resulting in death or permanent neurologic deficits among survivors. Studies indicate that the incidence of JE is high in northwestern Bangladesh. Pigs are amplifying hosts for JE virus (JEV) and a potentially important source of virus in the environment. The objectives of this study were to describe the transmission dynamics of JEV among pigs in northwestern Bangladesh and estimate the potential impact of vaccination to reduce incidence among pigs.

Methodology/Principal Findings

We conducted a comprehensive census of pigs in three JE endemic districts and tested a sample of them for evidence of previous JEV infection. We built a compartmental model to describe JEV transmission dynamics in this region and to estimate the potential impact of pig vaccination. We identified 11,364 pigs in the study area. Previous JEV infection was identified in 30% of pigs with no spatial differences in the proportion of pigs that were seropositive across the study area. We estimated that JEV infects 20% of susceptible pigs each year and the basic reproductive number among pigs was 1.2. The model suggest that vaccinating 50% of pigs each year resulted in an estimated 82% reduction in annual incidence in pigs.

Conclusions/Significance

The widespread distribution of historic JEV infection in pigs suggests they may play an important role in virus transmission in this area. Future studies are required to understand the contribution of pig infections to JE risk in humans and the potential impact of pig vaccination on human disease.