Small heat-shock proteins: important players in regulating cellular proteostasis

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease.SM was supported by a Royal Society Dorothy Hodgkin Fellowship, HE is supported by an Australian Research Council Future Fellowship (FT110100586) and JC is supported by a National Health and Medical Research Council Project Grant (#1068087)

Small heat-shock proteins: important players in regulating cellular proteostasis

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease

Clusterin is an extracellular chaperone that specifically interacts with slowly aggregating proteins on their off-folding pathway

Clusterin is an extracellular mammalian chaperone protein which inhibits stress-induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off-folding (precipitation-bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of α-lactalbumin, γ-crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of α-lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced α-lactalbumin. Reduction-induced changes in the conformation of α-lactalbumin, in the absence and presence of clusterin, were monitored by real-time 1H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of α-lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of α-lactalbumin was stabilised in a non-aggregated state, possibly via transient interactions with clusterin prior to complexation. Additional experiments demonstrated that clusterin potently inhibited the slow precipitation, but did not inhibit the rapid precipitation, of lysozyme and γ-crystallin induced by different stresses. These results suggest that clusterin interacts with and stabilises slowly aggregating proteins but is unable to stabilise rapidly aggregating proteins. Collectively, our results suggest that during its chaperone action, clusterin preferentially recognises partly folded protein intermediates that are slowly aggregating whilst venturing along their irreversible off-folding pathway towards a precipitated protein

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

Evaluating women\u27s knowledge of the combined oral contraceptive pill in an Australian rural general practice setting

Background: In addition to the contraceptive action of the combined oral contraceptive pill (COCP), there are a number of other benefits to its use such as menstrual cycle regulation. However, COCP use is also associated with a higher risk of thromboembolism. Despite the prevalence of COCP use, studies have indicated that overall women have poor knowledge of the COCP. Aim: To evaluate women\u27s knowledge of the COCP in a rural general practice setting. The extent of knowledge was assessed in several domains including: COCP use and effectiveness, mechanism of action, and the risks and benefits of COCP use. Methods: An observational study design was utilised. Women aged 18-50 years self-selected to complete an anonymous survey at a general practice in rural NSW. Women who were currently using, had previously used, or had never used the COCP were invited to participate. Women using a progesterone-only contraceptive were excluded. A total knowledge score on the usage and effects of the COCP was calculated for each participant by assessing responses to 34 questions for an overall score out of 34. Results: A total of 80 surveys were completed revealing that 98% of respondents used the COCP at some time in their lives with almost 29% being current users. The mean total knowledge score for all participants was 14.4 (SD = 4.9) out of a possible 34 (range: 5 - 26). There was no significant difference in total knowledge score between current and previous users (p = 0.56). Conclusion: The women surveyed in this study appear to have substantial gaps in their knowledge of the COCP. This study provides insight into specific knowledge areas that require further education and clarification during COCP counselling sessions (especially those conducted by a GP) to encourage improved knowledge of the COCP by women in this particular setting

Docosahexaenoic acid varies in rat skeletal muscle membranes according to fibre type and provision of dietary fish oil

Background Dietary fish oil provides polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) and is associated with modified oxygen consumption, contractile fatigue and physiological responses to ischaemia or hypoxia in striated muscle. This study systematically investigated the membrane incorporation of fatty acids, with a focus on DHA, into skeletal muscle in relation to functional/metabolic differences and their responsiveness to fish oil doses. Methods Male Sprague-Dawley rats were randomised to isoenergetic diets (10% fat by weight). Human Western-style diets were simulated with 5.5% tallow, 2.5% n-6 PUFA sunflower seed oil and 2% olive oil (Control). High-DHA tuna oil exchanged for olive oil provided a Low (0.32%) or moderate (Mod) (1.25%) fish oil diet. Membrane phospholipid fatty acid composition was analysed in samples of five skeletal muscles selected for maximum variation in muscle fibre-type. Results Concentrations of DHA varied according to muscle fibre type, very strongly associated with fast oxidative glycolytic fibre population (r2 = 0.93; P \u3c 0.01). No relationship was evident between DHA and fast glycolytic or slow oxidative fibre populations. Fish oil diets increased membrane incorporation of DHA in all muscles, mainly at the expense of n-6 PUFA linoleic and arachidonic acid. Conclusion The exquisite responsiveness of all skeletal muscles to as little fish oil as the equivalent of 1-2 fish meals per week in a human diet and the selective relationship to fatigable muscle fibre-types supports an integral role for DHA in muscle physiology, and particularly in fatigue resistance of fast-twitch muscles

Studies on the molecular chaperone abilities of alphaS1- and alphaS2-casein from bovine milk

αs-Casein is a predominant milk protein with important nutritional properties. In addition it has been shown to have molecular chaperone properties similar to those of the sHsps (small heat-shock proteins) and cIusterin. In order to study their relative contributions to the chaperone ability of total αs-casein, the individual subunits, αs1- and αs2-casein, which are present in a ratio of approximately 4:1 (αs1:αs2) in bovine milk were separated and purified from commercially obtained αs-casein. Anion-exchange chromatography was performed in a denaturing and reducing imidazole-Hel buffer at pH 7 as per Thompson et al. (1966) with minor changes, dialyzed against a urea gradient and lyophilized in ammonium acetate buffer