Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.     

6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death

Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44⁺CD24^-/^low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.     

Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle

A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.     

Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV–vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV–vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.     

Defective chromatin recruitment and retention of NHEJ core components in human tumor cells expressing a Cyclin E fragment

Exposure to genotoxic agents, such as ionizing radiation (IR), produces double-strand breaks, repaired predominantly in mammalian cells by non-homologous end-joining (NHEJ). Ku70 was identified as an interacting partner of a proteolytic Cyclin E (CycE) fragment, p18CycE. p18CycE endogenous generation during IR-induced apoptosis in leukemic cells and its stable expression in epithelial tumor cells sensitized to IR. γH2AX IR-induced foci (IRIFs) and comet assays indicated ineffective NHEJ DNA repair in p18CycE-expressing cells. DNA pull-down and chromatin recruitment assays revealed that retention of NHEJ factors to double-strand breaks, but not recruitment, was diminished. Similarly, IRIFs of phosphorylated T2609 and S2056-DNA-PKcs and its target S1778-53BP1 were greatly decreased in p18CycE-expressing cells. As a result, DNA-PKcs chromatin association was also increased. 53BP1 IRIFs were suppressed when p18CycE was generated in leukemic cells and in epithelial cells stably expressing p18CycE. Ataxia telangiectasia mutated was activated but not its 53BP1 and MDC1 targets. These data indicate a profound influence of p18CycE on NHEJ through its interference with DNA-PKcs conformation and/or dimerization, which is required for effective DNA repair, making the p18CycE-expressing cells more IR sensitive. These studies provide unique mechanistic insights into NHEJ misregulation in human tumor cells, in which defects in NHEJ core components are rare.     

Influence of ethnicity, geography and climate on the variation of stature among Indian populations

This paper analyzes the variation in the mean stature of adult males of a variety of population groups in India and examines the influence of geographical, climatic and ethnic factors on it. A considerable variation in mean stature has been found with respect to these three attributes. Variation "between" ethnic groups compared with "within" ethnic groups was found to be much more than that of geographical and climatic zones. Scheduled Castes (SC) and Scheduled Tribes (ST) populations have much low average height than that of General Castes (GC). Climatically dry and semiarid zones have a tendency to have higher stature than in the Monsoon areas. The mean height has been found to be the highest in north India. It is closely followed by west India. An interesting feature is that as one goes towards east and south the mean height gradually decreases. It is the lowest in islands. The mean heights have been regressed on geographical, climatic and ethnic factors, after converting these factors into binary variables. The regression analysis has strengthened the findings, that there is a highly significant relationship between height and geographical, climatic and ethnic factors.     

Live Cell Fluorescence Resonance Energy Transfer Predicts an Altered Molecular Association of Heterologous PrPSc with PrPC

Prion diseases result from the accumulation of a misfolded isoform (PrP^Sc) of the normal host prion protein (PrP^C). PrP^Sc propagates by templating its conformation onto resident PrP^C to generate new PrP^Sc. Although the nature of the PrP^Sc-PrP^C complex is unresolved, certain segments or specific residues are thought to feature critically in its formation. The polymorphic residue 129 is one such site under considerable study. We combined transmission studies with a novel live cell yeast-based fluorescence resonance energy transfer (FRET) system that models the molecular association of PrP in a PrP^Sc-like state, as a way to explore the role of residue 129 in this process. We show that a reduction in efficiency of prion transmission between donor PrP^Sc and recipient PrP^C that are mismatched at residue 129 correlates with a reduction in FRET between PrP-129M and PrP-129V in our yeast model. We further show that this effect depends on the different secondary structure propensities of Met and Val, rather than the specific amino acids. Finally, introduction of the disease-associated P101L mutation (mouse- equivalent) abolished FRET with wild-type mouse PrP, whereas mutant PrP-P101L displayed high FRET with homologous PrP-P101L, as long as residue 129 matched. These studies provide the first evidence for a physical alteration in the molecular association of PrP molecules differing in one or more residues, and they further predict that the different secondary structure propensities of Met and Val define the impaired association observed between PrP^Sc and PrP^C mismatched at residue 129.     

Socioeconomic differentials in nutritional status of children in the states of West Bengal and Assam, India

Malnutrition among children is prevalent in almost all the states in India. This study assesses the extent and causes of malnutrition in two eastern Indian states with similar climates, namely West Bengal and Assam, using data from the National Family Health Survey 1998-99 (NFHS-2). The three indices of malnutrition taken for analysis are weight-for-height (WHZ), height-for-age (HAZ) and weight-for-age (WAZ). These are assumed to depend on birth order, preceding birth interval, parent's educational status, working status of the mother, mother's age at delivery of the children, source of drinking water, toilet facilities and standard of living of the household. Logistic regression was carried out separately for each of the three indices on the explanatory variables for both the states. It was found that not all variables are equally important in determining whether a baby is underweight, or suffering from acute or chronic malnutrition. Also, the importance of variables is not the same in the two states. It was observed that the coefficients associated with the variables in determining weight-for-height are not significant compared with those for weight-for-age and height-for-age.     

Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome

During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states. Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation. Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome. Isolated L12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment. The overall structure can be described as three ellipsoids joined by flexible linkers. No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution. In the (1)H-(15)N HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies. The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12. These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors.     

Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G

The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.