The trajectory taken by dimeric Cu/Zn superoxide dismutase through the protein unfolding and dissociation landscape is modulated by salt-bridge formation

Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel are explored by activation of protein ions in vacuum. Here we examine the trajectory that Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in vacuum. We examined wild-type SOD1 and six disease-related point-mutants by using tandem MS and ion-mobility MS as a function of collisional activation. For six of the seven SOD1 variants, increasing activation prompted dimers to transition through two unfolding events and dissociate symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition, and displayed a much higher abundance of asymmetric products. Supported by the observation that ejected asymmetric G37R monomers were more compact than symmetric G37R ones, we localised this effect to the formation of a gas-phase salt-bridge in the first activated conformation. To examine the data quantitatively, we applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals a heightening of the barrier to unfolding in G37R >5 kJmol-1 over the other variants, consistent with expectations for the strength of a salt-bridge. Our work demonstrates weaknesses in the simple general framework for understanding protein complex dissociation in vacuum, and highlights the importance of individual residues, their local environment, and specific interactions in governing product formation

Site-directed mutations in the C-terminal extension of human aB-Crystalline affect chaperone function and block amyloid fibril formation

Copyright: 2007 Treweek et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background. Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including aB-crystallin, play a role in the prevention of protein deposition. Methodology/Principal Findings. A series of site-directed mutants of the human molecular chaperone, aBcrystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of aB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of aB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein. Conclusions/Significance. Together, our results highlight the important role of the C-terminal region of aB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify aB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation

Hydrothermal activity, functional diversity and chemoautotrophy are major drivers of seafloor carbon cycling

Hydrothermal vents are highly dynamic ecosystems and are unusually energy rich in the deep-sea. In situ hydrothermal-based productivity combined with sinking photosynthetic organic matter in a soft-sediment setting creates geochemically diverse environments, which remain poorly studied. Here, we use comprehensive set of new and existing field observations to develop a quantitative ecosystem model of a deep-sea chemosynthetic ecosystem from the most southerly hydrothermal vent system known. We find evidence of chemosynthetic production supplementing the metazoan food web both at vent sites and elsewhere in the Bransfield Strait. Endosymbiont-bearing fauna were very important in supporting the transfer of chemosynthetic carbon into the food web, particularly to higher trophic levels. Chemosynthetic production occurred at all sites to varying degrees but was generally only a small component of the total organic matter inputs to the food web, even in the most hydrothermally active areas, owing in part to a low and patchy density of vent-endemic fauna. Differences between relative abundance of faunal functional groups, resulting from environmental variability, were clear drivers of differences in biogeochemical cycling and resulted in substantially different carbon processing patterns between habitats

MLH1 mediates PARP-dependent cell death in response to the methylating agent N-methyl-N-nitrosourea

Background:Methylating agents such as N-methyl-N-nitrosourea (MNU) can cause cell cycle arrest and death either via caspase-dependent apoptosis or via a poly(ADP-ribose) polymerase (PARP)-dependent form of apoptosis. We wished to investigate the possible role of MLH1 in signalling cell death through PARP.Methods:Fibroblasts are particularly dependent on a PARP-mediated cell death response to methylating agents. We used hTERT-immortalised normal human fibroblasts (WT) to generate isogenic MLH1-depleted cells, confirmed by quantitative PCR and western blotting. Drug resistance was measured by clonogenic and cell viability assays and effects on the cell cycle by cell sorting. Damage signalling was additionally investigated using immunostaining.Results:MLH1-depleted cells were more resistant to MNU, as expected. Despite having an intact G2/M checkpoint, the WT cells did not initially undergo cell cycle arrest but instead triggered cell death directly by PARP overactivation and nuclear translocation of apoptosis-inducing factor (AIF). The MLH1-depleted cells showed defects in this pathway, with decreased staining for phosphorylated H2AX, altered PARP activity and reduced AIF translocation. Inhibitors of PARP, but not of caspases, blocked AIF translocation and greatly decreased short-term cell death in both WT and MLH1-depleted cells. This MLH1-dependent response to MNU was not blocked by inhibitors of ATM/ATR or p53.Conclusion:These novel data indicate an important role for MLH1 in signalling PARP-dependent cell death in response to the methylating agent MNU

Optical effects of exposing intact human lenses to ultraviolet radiation and visible light

<p>Abstract</p> <p>Background</p> <p>The human lens is continuously exposed to high levels of light. Ultraviolet radiation is believed to play a causative role in the development of cataract. In vivo, however, the lens is mainly exposed to visible light and the ageing lens absorbs a great part of the short wavelength region of incoming visible light. The aim of the present study was to examine the optical effects on human lenses of short wavelength visible light and ultraviolet radiation.</p> <p>Methods</p> <p>Naturally aged human donor lenses were irradiated with UVA (355 nm), violet (400 and 405 nm) and green (532 nm) lasers. The effect of irradiation was evaluated qualitatively by photography and quantitatively by measuring the direct transmission before and after irradiation. Furthermore, the effect of pulsed and continuous laser systems was compared as was the effect of short, intermediate and prolonged exposures.</p> <p>Results</p> <p>Irradiation with high intensity lasers caused scattering lesions in the human lenses. These effects were more likely to be seen when using pulsed lasers because of the high pulse intensity. Prolonged irradiation with UVA led to photodarkening whereas no detrimental effects were observed after irradiation with visible light.</p> <p>Conclusions</p> <p>Irradiation with visible light does not seem to be harmful to the human lens except if the lens is exposed to laser irradiances that are high enough to warrant thermal protein denaturation that is more readily seen using pulsed laser systems.</p

Human MLH1 Protein Participates in Genomic Damage Checkpoint Signaling in Response to DNA Interstrand Crosslinks, while MSH2 Functions in DNA Repair

DNA interstrand crosslinks (ICLs) are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA) is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSα and MutSβ mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents

Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans

Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50–70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50–80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70–180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage brought about by an environmental stressor, demonstrate important differences in aging- versus stress-induced protein damage, and challenge the widely held view that chemical chaperones are accumulated during hypertonic stress to protect protein structure/function

αA-crystallin R49Cneo mutation influences the architecture of lens fiber cell membranes and causes posterior and nuclear cataracts in mice

<p>Abstract</p> <p>Background</p> <p>αA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency. The R49C mutation in the αA-crystallin protein is linked with non-syndromic, hereditary human cataracts in a four-generation Caucasian family.</p> <p>Methods</p> <p>This study describes a mouse cataract model generated by insertion of a neomycin-resistant (neo^r) gene into an intron of the gene encoding mutant R49C αA-crystallin. Mice carrying the neo^rgene and wild-type <it>Cryaa </it>were also generated as controls. Heterozygous knock-in mice containing one wild type gene and one mutated gene for αA-crystallin (WT/R49C^neo) and homozygous knock-in mice containing two mutated genes (R49C^neo/R49C^neo) were compared.</p> <p>Results</p> <p>By 3 weeks, WT/R49C^neomice exhibited large vacuoles in the cortical region 100 μm from the lens surface, and by 3 months posterior and nuclear cataracts had developed. WT/R49C^neomice demonstrated severe posterior cataracts at 9 months of age, with considerable posterior nuclear migration evident in histological sections. R49C^neo/R49C^neomice demonstrated nearly complete lens opacities by 5 months of age. In contrast, R49C mice in which the neo^rgene was deleted by breeding with CreEIIa mice developed lens abnormalities at birth, suggesting that the neo^rgene may suppress expression of mutant R49C αA-crystallin protein.</p> <p>Conclusion</p> <p>It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the αA-crystallin mutation and rapidly leads to lens cell pathology <it>in vivo</it>.</p