DNA damage signaling triggers the cytoplasm-to-vacuole pathway of autophagy to regulate cell cycle progression

Budding yeast cells suffering a single unrepaired DNA double-strand break (DSB) trigger the ATR (Mec1)-dependent DNA damage checkpoint and arrest prior to anaphase for 12–15 h, following which they adapt and resume cell division. When the DNA lesion can be repaired, the checkpoint is extinguished and cells “recover” and resume mitosis. In this autophagic punctum, we report that hyperactivation of autophagy—specifically via the cytoplasm-to-vacuole targeting (Cvt) pathway—prevents both adaptation to, and recovery from, DNA damage, resulting in the permanent arrest of cells in G₂/M. We show that Saccharomyces cerevisiae deleted for genes encoding the Golgi-associated retrograde protein transport (GARP) complex are both adaptation- and recovery-defective. GARP mutants such as vps51Δ exhibit mislocalization of the key mitotic regulator, securin (Pds1), and its degradation by the vacuolar protease Prb1. In addition, separase (Esp1), is excluded from the nucleus, accounting for pre-anaphase arrest. Pds1 is degraded via the Cvt pathway. Many of the same defects seen by deleting GARP genes can be mimicked by hyperactivation of the Cvt pathway by overexpressing an unphosphorylatable form of ATG13 or by adding the TORC1 inhibitor rapamycin. These results suggest that nuclear events such as DNA damage can have profound effects on cytoplasmic processes and further expand the burgeoning connections between DNA damage and autophagy.     

Identifying potential entry inhibitors for emerging Nipah virus by molecular docking and chemical-protein interaction network

Abstract

Nipah Virus (NiV) is a newly emergent paramyxovirus that has caused various outbreaks in Asian countries. Despite its acute pathogenicity and lack of approved therapeutics for human use, there is an urgent need to determine inhibitors against NiV. Hence, this work includes prospection of potential entry inhibitors by implementing an integrative structure- and network-based drug discovery approach. FDA-approved drugs were screened against attachment glycoprotein (NiV-G, PDB: 2VSM), one of the prime targets to inhibit viral entry, using a molecular docking approach that was benchmarked both on CCDC/ASTEX and known NIV-G inhibitor set. The predicted small molecules were prioritized on the basis of topological analysis of the chemical-protein interaction network, which was inferred by integrating the drug-target network, NiV-human interaction network, and human protein-protein interaction network. A total of 17 drugs were predicted to be NiV-G inhibitors using molecular docking studies that were further prioritized to 3 novel leads???Nilotinib, Deslanoside and Acetyldigitoxin???on the basis of topological analysis of inferred chemical-protein interaction network. While Deslanoside and Acetyldigitoxin belong to an already known class of anti-NiV inhibitors, Nilotinib belongs to Benzenoids chemical class that has not been reported hitherto for developing anti-NiV inhibitors. These identified drugs are expected to be successful in further experimental evaluation and therefore could be used for anti-Nipah drug discovery. Apart, we also obtained various insights into the underlying chemical-protein interaction network, based on which several important network nodes were predicted. The applicability of our proposed approach was also demonstrated by prospecting for anti-NiV phytochemicals on an independent dataset.

Communicated by Ramaswamy H. Sarma     

Common variant in FUT2 gene is associated with levels of vitamin B12 in Indian population

Vitamin B₁₂ is an essential micronutrient synthesized by microorganisms. Mammals including humans have evolved ways for transport and absorption of this vitamin. Deficiency of vitamin B₁₂ (either due to low intake or polymorphism in genes involved in absorption and intracellular transport of this vitamin) has been associated with various complex diseases. Genome-wide association studies have recently identified several common single nucleotide polymorphisms (SNPs) in fucosyl transferase 2 gene (FUT2) to be associated with levels of vitamin B₁₂—the strongest association was with a non-synonymous SNP rs602662 in this gene. In the present study, we attempted to replicate the association of this SNP (rs602662) in an Indian population since a significant proportion has been reported to have low levels of vitamin B₁₂ in this population. A total of 1146 individuals were genotyped for this SNP using a single base extension method and association with levels of vitamin B₁₂ was assessed in these individuals. Regression analysis was performed to analyze the association considering various confounding factors like for age, sex, diet, hypertension, diabetes mellitus and coronary artery disease status. We found that the SNP rs602662 was significantly associated with the levels of vitamin B₁₂ (p value < 0.0001). We also found that individuals adhering to a vegetarian diet with GG (homozygous major genotype) have significantly lower levels of vitamin B₁₂ in these individuals. Thus, our study reveals that vegetarian diet along with polymorphism in the FUT2 gene may contribute significantly to the high prevalence of vitamin B₁₂ deficiency in India.     

DFT study of structure and vibrational spectra of ceramide 3: comparison to experimental data

The Fourier transform Raman and infrared (IR) spectra of the Ceramide 3 (CER3) have been recorded in the regions 200–3500 cm^? 1 and 680–4000 cm^? 1, respectively. We have calculated the equilibrium geometry, harmonic vibrational wavenumbers, electrostatic potential surfaces, absolute Raman scattering activities and IR absorption intensities by the density functional theory with B3LYP functionals having extended basis set 6-311G. This work is undertaken to study the vibrational spectra of CER3 completely and to identify the various normal modes with better wavenumber accuracy. Good consistency is found between the calculated results and experimental data for the IR and Raman spectra.     

Tryptophan to Glycine mutation in the position 116 leads to protein aggregation and decreases the stability of the LITAF protein

Mutations in the gene-encoding vesicle lipopolysaccharide-induced tumor necrosis factor (LITAF) protein cause Charcot–Marie–Tooth type 1C (CMT1C) disease, a neurological disorder. The LITAF gene is mapped to chromosome number 16 and can be found at cytogenetic location 16p13 of the chromosome. CMT1C-linked small integral membrane protein of lysosome/late endosome mutants are loss-of-function mutants that act in a dominant negative manner to impair endosomal trafficking, leading to prolonged extracellular signal-regulated kinases 1/2 signaling downstream of ErbB activation. Mutation W116G in the LITAF decreases the stability of the protein and also interrupts the functioning of gene. We have analyzed the single nucleotide polymorphism (SNP) results of 28 nsSNPs obtained from dbSNP. We also carried out multiple molecular dynamics simulations of 200 ns and obtained results of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent-accessible surface area, H-bond, and principal component analysis to check and prove the stability of both the wild type and the mutant. The protein was then checked for its aggregation and the results showed loss of helix. The loss of helix leads to the instability of the protein.     

The Ebola Virus Matrix Protein Penetrates into the Plasma Membrane: A KEY STEP IN VIRAL PROTEIN 40 (VP40) OLIGOMERIZATION AND VIRAL EGRESS*

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu²¹³, Ile²⁹³, Leu²⁹⁵, and Val²⁹⁸ demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.     

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.     

Interaction between mosquito-larvicidal Lysinibacillus sphaericus binary toxin components: Analysis of complex formation

The two components (BinA and BinB) of Lysinibacillus sphaericus binary toxin together are highly toxic to Culex and Anopheles mosquito larvae, and have been employed world-wide to control mosquito borne diseases. Upon binding to the membrane receptor an oligomeric form (BinA2.BinB2) of the binary toxin is expected to play role in pore formation. It is not clear if these two proteins interact in solution as well, in the absence of receptor. The interactions between active forms of BinA and BinB polypeptides were probed in solution using size-exclusion chromatography, pull-down assay, surface plasmon resonance, circular dichroism, and by chemically crosslinking BinA and BinB components. We demonstrate that the two proteins interact weakly with first association and dissociation rate constants of 4.5 × 10³ M^?1 s^?1 and 0.8 s^?1, resulting in conformational change, most likely, in toxic BinA protein that could kinetically favor membrane translocation of the active oligomer. The weak interactions between the two toxin components could be stabilized by glutaraldehyde crosslinking. The cross-linked complex, interestingly, showed maximal Culex larvicidal activity (LC₅₀ value of 1.59 ng mL^?1) reported so far for combination of BinA/BinB components, and thus is an attractive option for development of new bio-pesticides for control of mosquito borne vector diseases.     

Identification of Penicillin-binding proteins employing support vector machines and random forest

Penicillin-Binding Proteins are peptidases that play an important role in cell-wall biogenesis in bacteria and thus maintaining bacterial infections. A wide class of β-lactam drugs are known to act on these proteins and inhibit bacterial infections by disrupting the cell-wall biogenesis pathway. Penicillin-Binding proteins have recently gained importance with the increase in the number of multi-drug resistant bacteria. In this work, we have collected a dataset of over 700 Penicillin-Binding and non-Penicillin Binding Proteins and extracted various sequence-related features. We then created models to classify the proteins into Penicillin-Binding and non-binding using supervised machine learning algorithms such as Support Vector Machines and Random Forest. We obtain a good classification performance for both the models using both the methods.