Aeropin from the extremophile Pyrobaculum aerophilum bypasses the serpin misfolding trap

Serpins are metastable proteinase inhibitors. Serpin metastability drives both a large conformational change that is utilized during proteinase inhibition and confers an inherent structural flexibility that renders serpins susceptible to aggregation under certain conditions. These include point mutations (the basis of a number of important human genetic diseases), small changes in pH, and an increase in temperature. Many studies of serpins from mesophilic organisms have highlighted an inverse relationship: mutations that confer a marked increase in serpin stability compromise inhibitory activity. Here we present the first biophysical characterization of a metastable serpin from a hyperthermophilic organism. Aeropin, from the archaeon Pyrobaculum aerophilum, is both highly stable and an efficient proteinase inhibitor. We also demonstrate that because of high kinetic barriers, aeropin does not readily form the partially unfolded precursor to serpin aggregation. We conclude that stability and activity are not mutually exclusive properties in the context of the serpin fold, and propose that the increased stability of aeropin is caused by an unfolding pathway that minimizes the formation of an aggregation-prone intermediate ensemble, thereby enabling aeropin to bypass the misfolding fate observed with other serpins.     

DNA accelerates the inhibition of human cathepsin V by serpins

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.     

Mechanisms of ataxin-3 misfolding and fibril formation: kinetic analysis of a disease-associated polyglutamine protein

The polyglutamine diseases are a family of nine proteins where intracellular protein misfolding and amyloid-like fibril formation are intrinsically coupled to disease. Previously, we identified a complex two-step mechanism of fibril formation of pathologically expanded ataxin-3, the causative protein of spinocerebellar ataxia type-3 (Machado-Joseph disease). Strikingly, ataxin-3 lacking a polyglutamine tract also formed fibrils, although this occurred only via a single-step that was homologous to the first step of expanded ataxin-3 fibril formation. Here, we present the first kinetic analysis of a disease-associated polyglutamine repeat protein. We show that ataxin-3 forms amyloid-like fibrils by a nucleation-dependent polymerization mechanism. We kinetically model the nucleating event in ataxin-3 fibrillogenesis to the formation of a monomeric thermodynamic nucleus. Fibril elongation then proceeds by a mechanism of monomer addition. The presence of an expanded polyglutamine tract leads subsequently to rapid inter-fibril association and formation of large, highly stable amyloid-like fibrils. These results enhance our general understanding of polyglutamine fibrillogenesis and highlights the role of non-poly(Q) domains in modulating the kinetics of misfolding in this family.     

Identification of Protor as a novel Rictor-binding component of mTOR complex-2

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.     

The biodiversity and ecology of Antarctic lakes: models for evolution

Antarctic lakes are characterised by simplified, truncated food webs. The lakes range from freshwater to hypersaline with a continuum of physical and chemical conditions that offer a natural laboratory in which to study evolution. Molecular studies on Antarctic lake communities are still in their infancy, but there is clear evidence from some taxonomic groups, for example the Cyanobacteria, that there is endemicity. Moreover, many of the bacteria have considerable potential as sources of novel biochemicals such as low temperature enzymes and anti-freeze proteins. Among the eukaryotic organisms survival strategies have evolved, among which dependence on mixotrophy in phytoflagellates and some ciliates is common. There is also some evidence of evolution of new species of flagellate in the marine derived saline lakes of the Vestfold Hills. Recent work on viruses in polar lakes demonstrates high abundance and high rates of infection, implying that they may play an important role in genetic exchange in these extreme environments.     

The global impact of income inequality on health by age: an observational study

Objectives To explore whether the apparent impact of income inequality on health, which has been shown for wealthier nations, is replicated worldwide, and whether the impact varies by age.Design Observational study. Setting 126 countries of the world for which complete data on income inequality and mortality by age and sex were available around the year 2002 (including 94.4% of world human population).Data sources Data on mortality were from the World Health Organization and income data were taken from the annual reports of the United Nations Development Programme.Main outcome measures Mortality in 5-year age bands for each sex by income inequality and income level.Results At ages 15-29 and 25-39 variations in income inequality seem more closely correlated with mortality worldwide than do variations in material wealth. This relation is especially strong among the poorest countries in Africa. Mortality is higher for a given level of overall income in more unequal nations.Conclusions Income inequality seems to have an influence worldwide, especially for younger adults. Social inequality seems to have a universal negative impact on health.     

Alpha-substituted N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues as potent and stereospecific TRPV1 antagonists

A series of alpha-substituted N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. alpha-Methyl substituted analogues showed potent and stereospecific antagonism to the action of capsaicin on rat TRPV1 heterologously expressed in Chinese hamster ovary cells. In particular, compounds 14 and 18, which possess the R-configuration, exhibited excellent potencies (respectively, K(i)=41 and 39.2 nM and K(i(ant))=4.5 and 37 nM).     

Localized temperature adaptation of cottonwoods from elevational ecoregions in the Rocky Mountains

To investigate temperature adaptation of cool-climate trees, we studied 14 cottonwood genotypes from riparian (streamside) zones in three ecoregions differing in elevation in the Rocky Mountains of Alberta, Canada. Black cottonwoods (Populus trichocarpa) were collected from the higher, cooler montane and intermediate (aspen) parkland, and narrowleaf cottonwoods (P. angustifolia) were collected from the lower, warmer fescue prairie. The genotypes were grown in growth chambers under three temperature regimes reflecting the ecoregion variation. Sapling growth (dry weight) varied significantly across temperatures and for the genotypes from within and particularly across the ecoregions. Significant temperature × genotype interactions further indicated differentiation of the temperature response. Growth of the montane clones increased by 209% between 15/10 and 20/15°C and was 37% lower at 25/20°C. In contrast, genotypes from the lower ecoregions grew more slowly at the cool and intermediate temperatures (59 and 58% of montane) and then demonstrated constant (−3% parkland) or slightly increased (+16% prairie) growth at 25/20°C. This suggests the existence of P. trichocarpa ecotypes, localized populations with different temperature responses. This differentiation may explain our previous observation of comparable growth across these ecoregions despite substantial temperature variation, and the existence of ecotypes would produce a range of responses to climate warming that should produce an upward shift of the mountain ecoregions.     

The N terminus of the serpin, tengpin, functions to trap the metastable native state

Serpins fold to a metastable native state and are susceptible to undergoing spontaneous conformational change to more stable conformers, such as the latent form. We investigated conformational change in tengpin, an unusual prokaryotic serpin from the extremophile Thermoanaerobacter tengcongensis. In addition to the serpin domain, tengpin contains a functionally uncharacterized 56-amino-acid amino-terminal region. Deletion of this domain creates a variant--tengpinDelta51--which folds past the native state and readily adopts the latent conformation. Analysis of crystal structures together with mutagenesis studies show that the N terminus of tengpin protects a hydrophobic patch in the serpin domain and functions to trap tengpin in its native metastable state. A 13-amino-acid peptide derived from the N terminus is able to mimick the role of the N terminus in stabilizing the native state of tengpinDelta51. Therefore, the function of the N terminus in tengpin resembles protein cofactors that prevent mammalian serpins from spontaneously adopting the latent conformation.