High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.     

Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling.     

Mechanism by which cAMP activates PI3-kinase and increases bile acid secretion in WIF-B9 cells

Previous studies in rat bile canalicular membrane vesicles and WIF-B9 cells revealed that cAMP-induced trafficking of ATP-binding cassette (ABC) transporters to the canalicular membrane and their activation require phosphoinositide 3-kinase (PI3-K) products. In the present studies, canalicular secretion of fluorescein isothiocyanate-glycocholate in WIF-B9 cells was increased by cAMP and a decapeptide that enhances PI3-K activity; these effects were inhibited by wortmannin. To determine the mechanism(s) whereby cAMP activates PI3-K, we examined signal transduction pathways in WIF-B9 and COS-7 cells. cAMP activated PI3-K in both cell lines in a phosphotyrosine-independent manner. PI3-K activity increased in association with p110 beta in both cell lines. The effect of cAMP was KT-5720 sensitive, suggesting involvement of protein kinase A. Expression of a dominant-negative beta-adrenergic receptor kinase COOH terminus (beta-ARKct), which blocks G beta gamma signaling, decreased PI3-K activation in both cell lines. cAMP increased GTP-bound Ras in COS-7 but not WIF-B9 cells. Expression of dominant-negative Ras abolished cAMP-mediated PI3-K, which suggests that the effect is downstream of Ras and G beta gamma. These data indicate that cAMP activates PI3-K in a cell type-specific manner and provide insight regarding mechanisms of PI3-K activation required for bile acid secretion.     

Burden of Infectious Diseases,Substance Use Disorders,and Mental Illness among Ukrainian Prisoners Transitioning to the Community

Background

The epidemics of incarceration, substance use disorders (SUDs), and infectious diseases are inextricably intertwined, especially in the Former Soviet Union (FSU). Few objective data documenting this relationship regionally are available. We therefore conducted a comprehensive, representative country-wide prison health survey in Ukraine, where one of the world’s most volatile HIV epidemics persists, in order to address HIV prevention and treatment needs.

Methods

A nation-wide, multi-site randomly sampled biobehavioral health survey was conducted in four Ukrainian regions in 13 prisons among individuals being released within six months. After consent, participants underwent standardized health assessment surveys and serological testing for HIV, viral hepatitis, and syphilis.

Results

Of the 402 participants (mean age = 31.9 years), 20.1% were female. Prevalence of HIV, HCV, HBV, and syphilis was 19.4% (95% CI = 15.5%–23.3%), 60.2% (95% CI = 55.1%–65.4%), 5.2% (95% CI = 3.3%–7.2%), and 10% (95% CI = 7.4%–13.2%), respectively, with regional differences observed; HIV prevalence in the south was 28.6%. Among the 78 HIV-infected inmates, 50.7% were unaware of their HIV status and 44 (56.4%) had CD4<350 cells/mL, of which only five (11%) antiretroviral-eligible inmates were receiving it. Nearly half of the participants (48.7%) reported pre-incarcertion drug injection, primarily of opioids, yet multiple substance use (31.6%) and alcohol use disorders (56.6%) were common and 40.3% met screening criteria for depression.

Conclusions

This is the only such representative health study of prisoners in the FSU. This study has important implications for regional prevention and treatment because, unlike elsewhere, there is no recent evidence for reduction in HIV incidence and mortality in the region. The prevalence of infectious diseases and SUDs is high among this sample of prisoners transitioning to the community. It is critical to address pre- and post-release prevention and treatment needs with the development of linkage programs for the continuity of care in the community after release.     

Role of TRPC3 in the formation of receptor-and store-operated calcium channels in carcinoma A431 cells

Activation of phospholipase C (PLC)-linked signaling cascades in nonexcitable cells stimulates Ca²⁺ release from inositol-1,4,5-trisphosphate (IP₃)-sensitive intracellular Ca²⁺ stores and activation of Ca²⁺ entry via plasma membrane Ca²⁺ channels. The attention of investigators is currently focused on the properties and molecular basis of channels involved in Ca²⁺ entry into nonexcitable cells. According to current views, mammalian TRP proteins are involved in receptor-and store-dependent influx of Ca²⁺; however, little is known about the linkage between specific TRP proteins and endogenous channels responsible for Ca²⁺ entry. The aim of the present study was to elucidate the role of TRPC3 in the formation of store-dependent or receptor-operated pathways of Ca²⁺ entry into A431 cells. Registration of Ca²⁺ influx based on fluorescence measurements of intracellular Ca²⁺ concentrations and analysis of integral membrane currents revealed that partial inhibition of TRPC3 expression by small interfering RNA (siRNA) results in suppression of store-dependent Ca²⁺ entry without any effect on receptor-operated Ca²⁺ influx. In-depth studies of single channels revealed that TRPC3 suppression in A431 cells results in the disappearance of one type of store-operated channels and formation of a novel type of store-independent Ca²⁺-permeable channels. This, in turn, testifies to the crucial role of TRPC3 in normal functioning of store-operated Ca²⁺ channels in A431 cells.     

Individual variability and an error in the method of determining protein thermostability]

The individual variability of the heat resistance of two enzymes (aldolase and actomyosine) of the m. gastrochemius of the grass frog Rana temporaria has been quantitatively evaluated by means of analysis of variance. 37--41% of the total variability of the character may be due to errors in the methods of investigation, whereas 59--63% of the variability is due to phenotypic differences between individuals of the same population. The extent of error is primarily associated with the initial stages of the experiment, i. e. the isolation of the enzyme preparations. The individual variability of the heat resistance of proteins is lower than that of the cells or the organisms from which they are derived.     

Changes in the properties of the enzymes of energy allowance resulting from heat shock in lake frogs

A study was made of the activity and heat resistance of preparations of Na, K-ATPase, Mg-ATPase and succinate dehydrogenase of the lake frog R. ridibunda caused by the heat shock of animals. A decrease in the activity and an increase in the heat resistance of all the three enzymes studied were observed. The level of individual correlations between these parameters remained unchanged. An increase in the heat resistance of Na, K-ATPase occurs at the expense of a higher threshold of its thermal inactivation without changes in the affinity of this enzyme to Na+ and K+. Under discussion is the question of the functional significance of the changes observed.     

Activity and thermal stability of the enzymes of energy support in the common frog

The activity and heat resistance of succinate dehydrogenase (SDG), Na, K-ATPase and Mg-ATPase of the grass frog (39 specimens) have been determined. No correlation was found between individual levels of heat resistance both of either enzyme examined and of the same enzyme but taken from different tissues (SDG of liver and muscles). The average level of heat resistance of SDG in liver is significantly higher than that in m. gastrocnemius. A statistically significant correlation was observed between individual levels of enzyme activity in the internal organs (SDG of liver, Na, K-ATPase and Mg-ATPase of kidney). The activity of SDG of muscles does not correlate with that of either partner.