Estrogen is a modulator of vascular inflammation

Vascular inflammation underlies the pathogenesis of atherosclerosis. Atherosclerotic changes in the vasculature lead to conditions such as coronary artery disease and stroke, which are the major causes of morbidity and mortality worldwide. Epidemiological studies in premenopausal women suggest a beneficial role for estrogen in preventing vascular inflammation and consequent atherosclerosis. However, the benefits of estrogen areabsent or even reversed in older postmenopausal subjects. The modulation of inflammation by estrogen under different conditions might explain this discrepancy. Estrogen exerts its antiinflammatory effects on the vasculature through different mechanisms such as direct antioxidant effect, generation of nitric oxide, prevention of apoptosis in vascular cells and suppression of cytokines and the renin-angiotensin system. On the other hand, estrogen also elicits proinflammatory changes under certain conditions, which are less completely understood. Some of the mechanisms underlying a possible proinflammatory role for estrogen include increased expression of the proinflammatory receptor for advanced glycation end products, increased tyrosine nitration of cellular proteins, and generation of reactive oxygen species through an uncoupled eNOS. In this review, we have presented evidence for both antiinflammatory and proinflammatory pathways modulated by estrogen and how interactions among such pathways might determine the effects of estrogen on the vascular system.     

Cavities and Atomic Packing in Protein Structures and Interfaces

A comparative analysis of cavities enclosed in a tertiary structure of proteins and interfaces formed by the interaction of two protein subunits in obligate and non-obligate categories (represented by homodimeric molecules and heterocomplexes, respectively) is presented. The total volume of cavities increases with the size of the protein (or the interface), though the exact relationship may vary in different cases. Likewise, for individual cavities also there is quantitative dependence of the volume on the number of atoms (or residues) lining the cavity. The larger cavities tend to be less spherical, solvated, and the interfaces are enriched in these. On average 15 ų of cavity volume is found to accommodate single water, with another 40–45 ų needed for each additional solvent molecule. Polar atoms/residues have a higher propensity to line solvated cavities. Relative to the frequency of occurrence in the whole structure (or interface), residues in β-strands are found more often lining the cavities, and those in turn and loop the least. Any depression in one chain not complemented by a protrusion in the other results in a cavity in the protein–protein interface. Through the use of the Voronoi volume, the packing of residues involved in protein–protein interaction has been compared to that in the protein interior. For a comparable number of atoms the interface has about twice the number of cavities relative to the tertiary structure.     

Extra‐mitochondrial localization and likely reproductive function of a female‐transmitted cytochrome c oxidase subunit II protein

Our previous study documented a reproductive function for the male‐transmitted mitochondrial DNA (mtDNA)‐encoded cytochrome c oxidase subunit II (MCOX2) protein in a unionoid bivalve. Here, immunoblotting, immunohistochemistry and immunoelectron microscopy analyses demonstrate that the female‐transmitted protein (FCOX2) is: (i) expressed in both male and female gonads; (ii) maximally expressed in ovaries just prior to the time of the annual fertilization event; (iii) displayed in the cytoplasm and more strongly in the plasma membrane (microvilli), vitelline matrix and vitelline envelope of mature ovarian eggs; and (iv) strongly localized to the vitelline matrix of some eggs just prior to fertilization. These findings represent evidence for the extra‐mitochondrial localization of an mtDNA‐encoded gene product and are consistent with multifunctionality for FCOX2 in eggs.     

Genetic Characterization of Hepatitis B Virus in Peripheral Blood Leukocytes: Evidence for Selection and Compartmentalization of Viral Variants with the Immune Escape G145R Mutation

The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and classically has been described to be hepatotropic, causing a wide range of clinical and subclinical manifestations of liver disease (57). Nevertheless, studies of HBV-infected human subjects and woodchucks infected with Woodchuck hepatitis virus (WHV; an animal model of hepadnaviral infection) have reported different molecular forms of replicative intermediates in the lymphatic cells and have established that hepadnaviruses are strongly lymphotropic in nature (29). Moreover, the results of studies of human subjects as well as with animal models have revealed that the life-long occult persistence of replication- and transmission-competent viruses in lymphatic cells is a strict consequence of hepadnaviral infections (29).More interestingly, in animal models, lymphatic system-restricted occult hepadnaviral infection has been found to be transmissible vertically as an asymptomatic, serologically occult infection exclusively confined to the lymphatic system (29). Earlier we provided evidence that occult HBV persisting in the lymphatic cells are transmissible, specifically to the PBL through horizontal intrafamilial modes (9). These observations clearly indicate important immunological, pathogenic, and epidemiological implications of lymphatic system-restricted hepadnaviral infections. Although the involvement of specific viral variants has been suggested to explain this lymphatic system-restricted hepadnaviral infection and transmission (29), the classical belief that hepatocytes are the primary target and only reservoir of HBV has precluded the genetic characterization of hepadnaviruses from extrahepatic sites.Fascinatingly, despite being classically considered a hepatotropic virus, hepatitis C virus (HCV), belonging to the family Flaviviridae, also shows occult persistence and lymphotropism very similar to that of hepadnaviruses (37). Similarly to WHV, HBV, and HCV, other viruses, including HIV (human immunodeficiency virus), small ruminant lentivirus, and Epstein-Barr virus, also have been shown to infect and persist in different anatomical compartments of the body in addition to their classical target cells (38, 40, 43, 45, 50). Furthermore, recent molecular evolutionary analyses based on envelope sequences of these viruses (e.g., HIV, HCV, small ruminant lentivirus, Epstein-Barr virus, etc.) have established clearly that these viruses undergo selection and independent evolution in diverse tissues, leading to the tissue-specific compartmentalization of viral populations (38, 40, 43, 45, 50). In contrast to other viruses, to the best of our knowledge, methodical molecular evolutionary studies to characterize HBV sequences isolated from extrahepatic sites of HBV-infected subjects have not been reported in the literature.We hypothesized that similar to other viruses, HBV also undergo independent evolution in different compartments of the body under the influence of differential immune pressure. To examine our hypothesis, we used the most easily available lymphatic cells, the peripheral blood leukocytes (PBL), determined the HBV envelope sequences from HBV DNA isolated from these cells, and performed advanced genetic, phylogenetic, and mutational analysis. The results of this work demonstrate a highly compartment-specific preponderance of HBV genetic variants in serum and PBL of the same study population, providing evidence in favor of the compartmentalization of HBV genetic variants. The results and important implications of these findings are discussed in this work.     

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, and anion SO₄⁻ increased enzyme activity while strong inhibition was observed in the presence of Hg²⁺, Cl⁻, HCO₃⁻, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V_max and K_m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K_i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.     

Accessibility and partner number of protein residues, their relationship and a webserver, ContPlot for their display

Background  

Depending on chemical features residues have preferred locations – interior or exterior – in protein structures, which also determine how many other residues are found around them. The close packing of residues is the hallmark of protein interior and protein-protein interaction sites.     

Dual PPAR-alpha and -gamma activators derived from novel benzoxazinone containing thiazolidinediones having antidiabetic and hypolipidemic potential

2,4-Thiazolidinedione derivatives of 1,3-benzoxazinone were synthesized and evaluated for their PPAR-alpha and -gamma dual activation. DRF-2519, a compound obtained through SAR of TZD derivatives of benzoxazinone, has shown potent dual PPAR activation. In ob/ob mice, it showed better efficacy than the comparator molecules. In fat fed rat model, it showed significant improvement in lipid parameters, which was better than fibrates.     

Regulation of RhoGEF activity by intramolecular and intermolecular SH3 domain interactions

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members.     

Structural and Functional Roles of Coevolved Sites in Proteins

Background

Understanding the residue covariations between multiple positions in protein families is very crucial and can be helpful for designing protein engineering experiments. These simultaneous changes or residue coevolution allow protein to maintain its overall structural-functional integrity while enabling it to acquire specific functional modifications. Despite the significant efforts in the field there is still controversy in terms of the preferable locations of coevolved residues on different regions of protein molecules, the strength of coevolutionary signal and role of coevolution in functional diversification.

Methodology

In this paper we study the scale and nature of residue coevolution in maintaining the overall functionality and structural integrity of proteins. We employed a large scale study to investigate the structural and functional aspects of coevolved residues. We found that the networks representing the coevolutionary residue connections within our dataset are in general of ‘small-world’ type as they have clustering coefficient values higher than random networks and also show smaller mean shortest path lengths similar and/or lower than random and regular networks. We also found that altogether 11% of functionally important sites are coevolved with any other sites. Active sites are found more frequently to coevolve with any other sites (15%) compared to protein (11%) and ligand (9%) binding sites. Metal binding and active sites are also found to be more frequently coevolved with other metal binding and active sites, respectively. Analysis of the coupling between coevolutionary processes and the spatial distribution of coevolved sites reveals that a high fraction of coevolved sites are located close to each other. Moreover, ∼80% of charge compensatory substitutions within coevolved sites are found at very close spatial proximity (< = 5Å), pointing to the possible preservation of salt bridges in evolution.

Conclusion

Our findings show that a noticeable fraction of functionally important sites undergo coevolution and also point towards compensatory substitutions as a probable coevolutionary mechanism within spatially proximal coevolved functional sites.     

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy

Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2 ^gib005Δ8/+) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.