Molecular origin of polyglutamine aggregation in neurodegenerative diseases

Expansion of polyglutamine (polyQ) tracts in proteins results in protein aggregation and is associated with cell death in at least nine neurodegenerative diseases. Disease age of onset is correlated with the polyQ insert length above a critical value of 35-40 glutamines. The aggregation kinetics of isolated polyQ peptides in vitro also shows a similar critical-length dependence. While recent experimental work has provided considerable insights into polyQ aggregation, the molecular mechanism of aggregation is not well understood. Here, using computer simulations of isolated polyQ peptides, we show that a mechanism of aggregation is the conformational transition in a single polyQ peptide chain from random coil to a parallel beta-helix. This transition occurs selectively in peptides longer than 37 glutamines. In the beta-helices observed in simulations, all residues adopt beta-strand backbone dihedral angles, and the polypeptide chain coils around a central helical axis with 18.5 +/- 2 residues per turn. We also find that mutant polyQ peptides with proline-glycine inserts show formation of antiparallel beta-hairpins in their ground state, in agreement with experiments. The lower stability of mutant beta-helices explains their lower aggregation rates compared to wild type. Our results provide a molecular mechanism for polyQ-mediated aggregation.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Sequence and structural determinants of Cu, Zn superoxide dismutase aggregation

Diverse point mutations in the enzyme Cu, Zn superoxide dismutase (SOD1) are linked to its aggregation in the familial form of the disease amyotrophic lateral sclerosis. The disease-associated mutations are known to destabilize the protein, but the structural basis of the aggregation of the destabilized protein and the structure of aggregates are not well understood. Here, we investigate in silico the sequence and structural determinants of SOD1 aggregation: (1) We identify sequence fragments in SOD1 that have a high aggregation propensity, using only the sequence of SOD1, and (2) we perform molecular dynamics simulations of the SOD1 dimer folding and misfolding. In both cases, we identify identical regions of the protein as having high propensity to form intermolecular interactions. These regions correspond to the N- and C-termini, and two crossover loops and two beta-strands in the Greek-key native fold of SOD1. Our results suggest that the high aggregation propensity of mutant SOD1 may result from a synergy of two factors: the presence of highly amyloidogenic sequence fragments ("hot spots"), and the presence of these fragments in regions of the protein that are structurally most likely to form intermolecular contacts under destabilizing conditions. Therefore, we postulate that the balance between the self-association of aggregation-prone sequences and the specific structural context of these sequences in the native state determines the aggregation propensity of proteins.     

The 3' --> 5' exonuclease of T4 DNA polymerase removes premutagenic alkyl mispairs and contributes to futile cycling at O6-methylguanine lesions

We have studied the processing of O(6)-methylguanine (m6G)-containing oligonucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3' --> 5' exonuclease of T4 DNA polymerase. In vitro biochemical analyses demonstrate that the exonuclease can remove bases opposite a defined m6G lesion. The efficiency of excision of a terminal m6G.T was similar to that of m6G.C, and both were excised as efficiently as a G.T substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3' --> 5' exonuclease may contribute to DNA synthesis inhibition by alkylating agents. Genetic data obtained using an in vitro herpes simplex virus-thymidine kinase assay support the inefficiency of the exonuclease as a "proofreading" activity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G --> A transitions. Comparison of MNU dose-response curves for exonuclease-proficient and -deficient forms of T4 polymerase reveals that the exonuclease efficiently removes 50-86% of total premutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the biochemical explanation for cellular cytotoxicity of methylating agents.     

Glutathione reductase and catalase as potential biomarkers for synergistic intoxication of pesticides in fish

Abstract

Synergy occurs when chemicals give pronounced effect on combination in contrast to their individual effect. The objective of this study was to investigate the synergistic effect of pesticides carbaryl (C) and methyl parathion (MP) on oxidative stress biomarkers viz catalase (CAT), glutathione reductase (GSSG-R) including different enzymes like lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acetyl cholinesterase (AChE) in different tissues of carps Catla catla. Fishes were exposed to 6.25?mg/L of MP and 2.3?mg/L of C in mixture (one-third of LC₅₀ value). CAT and GSSG-R were studied in gills, brain, liver and muscle of carp were found to be elevated significantly (p?<?0.005). LDH activity increased significantly (p?<?0.005) in synergistic group, there was a seven-fold (748%) increase in LDH activity in muscle compared to individual studies with same pesticides. Contrary to LDH, sudden decrease in SDH activity was accounted. Significant (p?<?0.005) decrease in AChE activity after initial 24?h was remarkable addressing to the shift in neurotransmission pathway in organism. Significant increase was observed in activity of CAT and GSSG-R in all tissues compared to control fishes in individual as well as synergistic (MP?+?C) group suggesting that CAT and GSSG-R can be a potential biomarker of oxidative stress when studied in combination.     

Identification of inhibitors against Mycobacterium tuberculosis thiamin phosphate synthase, an important target for the development of anti-TB drugs

Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6-9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M. tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M. tuberculosis. In this study, a comparative homology model of M. tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC(50) values ranging from 20-100 μg/ml and two of these exhibited weak inhibition of M. tuberculosis growth with MIC(99) values being 125 μg/ml and 162.5 μg/ml while one compound was identified as a very potent inhibitor of M. tuberculosis growth with an MIC(99) value of 6 μg/ml. This study establishes MtTPS as a novel drug target against M. tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis.     

Combined effect of chemical fertilisers and rhizosphere-competent Bacillus subtilis BSK17 on yield of Cicer arietinum

A chemical fertiliser-adaptive variant, Bacillus subtilis BSK17, showed induction in growth at 0.32?M of Urea, 0.05?M of DAP, 0.04?M of MoP and 0.08 of gypsum. In addition, B. subtilis BSK17 produced various plant growth-promoting substances and showed higher colony growth inhibition of Fusarium oxysporum that increased with increase in incubation time and reached the maximum by 78% at 120?h. In field, antibiotic-resistant marker strains of B. subtilis BSK17^ery+ and B. subtilis BSK17^tet+ showed more improvement in seed yield (90% than the control and 24% than full dose of chemical fertilisers) of Cicer arietinum when applied with half dose of chemical fertilisers (N₅₊₅P₁₅₊₁₅K₁₅S_10+10+10). Root length, shoot length, fresh and dry weight of root and shoot of plants were enhanced after 120?days in comparison to control; all values were significant at 1% CD. The strain significantly colonised the rhizosphere of C. arietinum by 6.64 log cfu after 120?days.