Comparative analysis of differential gene expression of HSP40 and HSP70 family isoforms during heat stress and HIV-1 infection in T-cells

Heat shock proteins (HSPs) are a family of cellular proteins involved in a variety of biological functions including chaperone activity. HSPs are classified based on their molecular weight and each family has several isoforms in eukaryotes. HSP40 is the most diverse family acting as a co-chaperone for the highly conserved HSP70 family. Some of the isoforms are reported to be induced during heat stress. Few studies have also highlighted the diverse role of some isoforms in different stress conditions including viral infections. But till date, no study has comprehensively examined the expression profile of different HSP40 and 70 isoforms in either heat stress or HIV-1 infection, a virus that is responsible for the pandemic of AIDS. In the present study, we have compared the mRNA expression profile of HSP40 and HSP70 isoforms during heat stress and HIV-1 infection in a T-cell line and also validated the HIV-1 stress results in peripheral blood mononuclear cells. In case of HSP70, we observed that three isoforms (HSPA1A, HSPA1B, and HSPA6) are highly upregulated during heat stress, but these isoforms were found to be downregulated during the peak of HIV-1 infection. While in case of HSP40, we found that only DNAJA4, DNAJB1, and DNAJB4 showed significant upregulation during heat stress, whereas in HIV-1 infection, majority of the isoforms were induced significantly. Stress-dependent differential expression observed here indicates that different HSP40 and HSP70 isoforms may have specific roles during HIV-1 infection and thus could be important for future studies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-020-01185-y.     

Structure of the mammalian 80S ribosome at 8.7 A resolution

In this paper, we present a structure of the mammalian ribosome determined at approximately 8.7 A resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for approximately 20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.     

Characterization of an exopolysaccharide produced by a marine Enterobacter cloacae

An exopolysaccharide producing marine bacterium, Enterobacter cloacae, was isolated from marine sediment collected from Gujarat coast, India. Chemical investigation of exopolysaccharide (EPS 71 a) revealed that this exopolysaccharide was an acidic polysaccliaride containing high amount of uronic acid, fucose and sulfate which is rare for bacterial exopolysaccharides. EPS 71a was found to have fucose, galactose, glucose and glucuronic acid in a molar ratio of 2: 1: 1: 1.     

Proton Pump Inhibitor Usage and the Risk of Myocardial Infarction in the General Population

Background and Aims

Proton pump inhibitors (PPIs) have been associated with adverse clinical outcomes amongst clopidogrel users after an acute coronary syndrome. Recent pre-clinical results suggest that this risk might extend to subjects without any prior history of cardiovascular disease. We explore this potential risk in the general population via data-mining approaches.

Methods

Using a novel approach for mining clinical data for pharmacovigilance, we queried over 16 million clinical documents on 2.9 million individuals to examine whether PPI usage was associated with cardiovascular risk in the general population.

Results

In multiple data sources, we found gastroesophageal reflux disease (GERD) patients exposed to PPIs to have a 1.16 fold increased association (95% CI 1.09–1.24) with myocardial infarction (MI). Survival analysis in a prospective cohort found a two-fold (HR = 2.00; 95% CI 1.07–3.78; P = 0.031) increase in association with cardiovascular mortality. We found that this association exists regardless of clopidogrel use. We also found that H₂ blockers, an alternate treatment for GERD, were not associated with increased cardiovascular risk; had they been in place, such pharmacovigilance algorithms could have flagged this risk as early as the year 2000.

Conclusions

Consistent with our pre-clinical findings that PPIs may adversely impact vascular function, our data-mining study supports the association of PPI exposure with risk for MI in the general population. These data provide an example of how a combination of experimental studies and data-mining approaches can be applied to prioritize drug safety signals for further investigation.     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

Design and activity of AP endonuclease-1 inhibitors

Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1) is a critical component of base excision repair that excises abasic lesions created enzymatically by the action of DNA glycosylases on modified bases and non-enzymatically by hydrolytic depurination/depyrimidination of nucleobases. Many anticancer drugs generate DNA adducts that are processed by base excision repair, and tumor resistance is frequently associated with enhanced APE-1 expression. Accordingly, APE-1 is a potential therapeutic target to treat cancer. Using computational approaches and the high resolution structure of APE-1, we developed a 5-point pharmacophore model for APE-1 small molecule inhibitors. One of the nM APE-1 inhibitors (AJAY-4) that was identified based on this model exhibited an overall median growth inhibition (GI₅₀) of 4.19 μM in the NCI-60 cell line panel. The mechanism of action is shown to be related to the buildup of abasic sites that cause PARP activation and PARP cleavage, and the activation of caspase-3 and caspase-7, which is consistent with cell death by apoptosis. In a drug combination growth inhibition screen conducted in 10 randomly selected NCI-60 cell lines and with 20 clinically used non-genotoxic anticancer drugs, a synergy was flagged in the SK-MEL-5 melanoma cell line exposed to combinations of vemurafenib, which targets melanoma cells with V600E mutated BRAF, and AJAY-4, our most potent APE-1 inhibitor. The synergy between AJAY-4 and vemurafenib was not observed in cell lines expressing wild-type B-Raf protein. This synergistic combination may provide a solution to the resistance that develops in tumors treated with B-Raf-targeting drugs.

Electronic supplementary material

The online version of this article (doi:10.1007/s12154-015-0131-7) contains supplementary material, which is available to authorized users.     

Recovery of Aging-Related Size Increase of Skin Epithelial Cells: In vivo Mouse and In vitro Human Study

The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment). An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8). A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20–40% for cells of older passage (6–8 passages) whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.