Novel Mutations that Enhance or Repress the Aggregation Potential of SOD1

Mutations in SOD1 cause FALS by a gain of function likely related to protein misfolding and aggregation. SOD1 mutations encompass virtually every domain of the molecule, making it difficult to identify motifs important in SOD1 aggregation. Zinc binding to SOD1 is important for structural integrity, and is hypothesized to play a role in mutant SOD1 aggregation. To address this question, we mutated the unique zinc binding sites of SOD1 and examined whether these changes would influence SOD1 aggregation. We generated single and multiple mutations in SOD1 zinc binding residues (H71, H80 and D83) either alone or in combination with an aggregate forming mutation (A4V) known to cause disease. These SOD1 mutants were assayed for their ability to form aggregates.Using an in vitro cellular SOD1 aggregation assay, we show that combining A4V with mutations in non-zinc binding domains (G37R or G85R) increases SOD1 aggregation potential. Mutations at two zinc binding residues (H71G and D83G) also increase SOD1 aggregation potential. However, an H80G mutation at the third zinc binding residue decreases SOD1 aggregation potential even in the context of other aggregate forming SOD1 mutations. These results demonstrate that various mutations have different effects on SOD1 aggregation potential and that the H80G mutation appears to uniquely act as a dominant inhibitor of SOD1 aggregation.     

Some novel approaches for radioprotection and the beneficial effect of natural products

Due to the increased use of ionizing radiation in various aspects of human life especially in areas pertaining to radiotherapy of cancer, food preservation, agriculture, industry and power generation, there is a need to develop an effective and non-toxic radioprotector. The currently available ones have many drawbacks including high cost, side effects and toxicity. Several novel approaches are on to locate a potent radioprotector. These include mimics of antioxidant enzymes, nitroxides, melatonin, growth factors, gene therapy, hyperthermia apart from natural products. The latter has several advantages since they are non-toxic with proven therapeutic benefits. These can be classified as natural compounds and plant extracts; polyherbal formulations; besides natural and semi-natural compounds of plant origin. A review of the above agents, their efficacy in radioprotection and possible mechanisms responsible has been carried out. As India and many Eastern countries have an enormous heritage of vast natural dietary and time tested medicinal resources it is worth exploring the possibility of developing efficient, economically viable and clinically acceptable radioprotectors for human application from these resources.     

Rapid cold-hardening increases membrane fluidity and cold tolerance of insect cells

The rapid cold-hardening (RCH) response not only confers dramatic protection against cold-shock (non-freezing) injury, but also "instantaneously" enhances organismal performance. Since cold-shock injury is associated with damage to the cell membrane, we investigated the relationship between RCH and changes in cold tolerance and membrane fluidity at the cellular level. None of the adult flies (Sarcophaga bullata) in the cold-shocked treatment group survived direct transfer to -8 degrees C for 2 h; in contrast, 64.5% of flies in the RCH group survived exposure to -8 degrees C. Differences between the treatment groups also were reflected at the cellular level; only 21.3% of fat body cells in the cold-shocked group survived compared to 68.5% in the RCH group. Using 31P solid-state NMR spectroscopy, we determined that membrane fluidity increased concurrently with rapid cold-hardening of fat body cells. This result suggests that membrane characteristics may be modified very rapidly to protect cells against cold-shock injury.     

Geometries and electronic structures of cyanide adducts of the non-heme iron active site of superoxide reductases: vibrational and ENDOR studies

We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide and have used vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Addition of cyanide changes the active site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13CN, C15N, and 13C15N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle approximately 175 degrees) and two distinct bent conformations (Fe-C-N angles <140 degrees). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state, indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13C-ENDOR spectra for the 13CN-bound ferric active sites in both 1Fe- and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123 degrees (C3) and 133 degrees (C2), taking a reference value from vibrational studies of 175 degrees (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.     

High-avidity, high-IFNγ-producing CD8 T-cell responses following immune selection during HIV-1 infection

HIV-1 mutations, which reduce or abolish CTL responses against virus-infected cells, are frequently selected in acute and chronic HIV infection. Among population HIV-1 sequences, immune selection is evident as human leukocyte antigen (HLA) allele-associated substitutions of amino acids within or near CD8 T-cell epitopes. In these cases, the non-adapted epitope is susceptible to immune recognition until an escape mutation renders the epitope less immunogenic. However, several population-based studies have independently identified HLA-associated viral changes, which lead to the formation of a new T-cell epitope, suggesting that the immune responses that these variants or 'neo-epitopes' elicit provide an evolutionary advantage to the virus rather than the host. Here, we examined the functional characteristics of eight CD8 T-cell responses that result from viral adaptation in 125 HLA-genotyped individuals with chronic HIV-1 infection. Neo-epitopes included well-characterized immunodominant epitopes restricted by common HLA alleles, and in most cases the T-cell responses against the neo-epitope showed significantly greater functional avidity and higher IFNγ production than T cells for non-adapted epitopes, but were not more cytotoxic. Neo-epitope formation and emergence of cognate T-cell response coincident with a rise in viral load was then observed in vivo in an acutely infected individual. These findings show that HIV-1 adaptation not only abrogates the immune recognition of early targeted epitopes, but may also increase immune recognition to other epitopes, which elicit immunodominant but non-protective T-cell responses. These data have implications for immunodominance associated with polyvalent vaccines based on the diversity of chronic HIV-1 sequences.     

Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy

Numerous immunotherapy treatments for cancer are undergoing clinical trials or are already approved for use. One particular area of interest is targeting mechanisms of immune tolerance. Using a murine model of mesothelioma, we investigated the roles of regulatory T-cells, intratumoural transforming growth factor (TGF)-β and the negative regulator molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in immune tolerance to tumours. It was found that treatments targeting a single negative regulator molecule mechanism were not as effective against tumours as targeting multiple mechanisms simultaneously. Most importantly, it was found that a combined triple treatment of anti-CD25 monoclonal antibody (mAb), anti-CTLA-4 mAb and TGF-β soluble receptor resulted in long-term clearance of tumours and memory against tumour rechallenge. These data suggest that clinical application of immunotherapies against tumours may be improved by simultaneously targeting multiple mechanisms of immune suppression.     

Purification and assay protocols for obtaining highly active Jumonji C demethylases

Jumonji C (JmjC) lysine demethylases (KDMs) are Fe(II)-dependent hydroxylases that catalyze the oxidative demethylation of methyllysine residues in histones and nonhistone proteins. These enzymes play vital roles in regulating cellular processes such as gene expression, cell cycle progression, and stem cell self-renewal and differentiation. Despite their biological importance, recombinant forms of JmjC KDMs generally display low enzymatic activity and have remained challenging to isolate in a highly active form. Here we present a simple affinity purification scheme for Strep(II)-tagged JmjC KDMs that minimizes contamination by transition state metal ions, yielding highly active and pure enzyme. We also describe an optimized continuous fluorescent assay for KDMs that detects formaldehyde production during demethylation via a coupled reaction using formaldehyde dehydrogenase. Purification and kinetic analysis of the human KDMs JMJD2A and JMJD2D using these methods yielded activities substantially higher than those previously reported for these enzymes, which are comparable to that of the flavin-dependent KDM LSD1. In addition, we show that JMJD2A exhibited a lower catalytic efficiency toward a histone peptide bearing a chemically installed trimethyllysine analog compared with a bona fide trimethylated substrate. The methodology described here is broadly applicable to other JmjC KDMs, facilitating their biochemical characterization and high-throughput screening applications.     

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9?? resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.     

Ecology and Impacts of the Invasive Species, Lantana camara, in a Social-Ecological System in South India: Perspectives from Local Knowledge

We explored how the forest-dwelling Soliga community of South India views and explains biological invasions, and how local knowledge can inform scientific knowledge on biological invasions. We used an interview schedule with open-ended questions to solicit Soliga opinion on Lantana camara (lantana) invasion. The Soliga cited three reasons for lantana spread: its prolific fruit output and wide seed dispersal, change in fire management, and historical extraction of grass and bamboo. The Soliga believe that lantana invasion has had negative effects on the ecosystem and their livelihoods. Tabling scientific knowledge with local knowledge has improved our understanding of lantana invasion. The role of existing lantana in colonizing neighboring areas, and the response of native tree communities to lantana were common to both local and scientific sources. However, the Soliga view provides a more nuanced perspective of the lantana-fire relationship (contextually based on lantana density) with fires suppressing lantana when lantana density was low. This is contrary to views held by foresters and biologists, that fires are uniformly detrimental and promote lantana. Our study shows that examining Soliga observations has improved understanding of the invasion process and presents avenues for future lantana management.