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Background Mounting evidence suggests that circulating microRNAs (miRNAs) are critical indicators of cardiovascular disease. However, prospective studies linking circulating miRNAs to incident acute coronary syndrome (ACS) are limited, and the underlying effect of associated miRNA on incident ACS remains unknown. Methods and Results Based on a 2-stage prospective nested case-control design within the Dongfeng-Tongji cohort, we profiled plasma miRNAs from 23 pairs of incident ACS cases and controls by microarray and validated the candidate miRNAs in 572 incident ACS case-control pairs using quantitative real-time polymerase chain reaction. We observed that plasma miR-4286 was associated with higher risk of ACS (adjusted odds ratio according to an interquartile range increase, 1.26 [95% CI, 1.07-1.48]). Further association analysis revealed that triglyceride was positively associated with plasma miR-4286, and an interquartile range increase in triglyceride was associated with an 11.04% (95% CI, 3.77%-18.83%) increase in plasma miR-4286. In addition, the Mendelian randomization analysis suggested a potential causal effect of triglyceride on plasma miR-4286 ( β coefficients: 0.27 [95% CI, 0.01-0.53] and 0.27 [95% CI, 0.07-0.47] separately by inverse variance-weighted and Mendelian randomization-pleiotropy residual sum and outlier tests). Moreover, the causal mediation analysis indicated that plasma miR-4286 explained 5.5% (95% CI, 0.7%-17.0%) of the association of triglyceride with incident ACS. Conclusions Higher level of plasma miR-4286 was associated with an increased risk of ACS. The upregulated miR-4286 in plasma can be attributed to higher triglyceride level and may mediate the effect of triglyceride on incident ACS.

The small heat shock proteins (sHsps) are a ubiquitous family of ATP-independent stress proteins found in all domains of life. Drosophila melanogaster Hsp27 (DmHsp27) is the only known nuclear sHsp in insect. Here analyzing sequences from HMMER, we identified 56 additional insect sHsps with conserved arginine-rich nuclear localization signal (NLS) in the N-terminal region. At this time, the exact role of nuclear sHsps remains unknown. DmHsp27 protein-protein interaction analysis from iRefIndex database suggests that this protein, in addition to a putative role of molecular chaperone, is likely involved in other nuclear processes (i.e., chromatin remodeling and transcription). Identification of DmHsp27 interactors should provide key insights on the cellular and molecular functions of this nuclear chaperone.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones found in all domains of life, possessing significant roles in protein quality control in cells and assisting the refolding of non-native proteins. They are efficient chaperones against many in vitro protein substrates. Nevertheless, the in vivo native substrates of sHsps are not known. To better understand the functions of sHsps and the mechanisms by which they enhance heat resistance, sHsp-interacting proteins were identified using affinity purification under heat shock conditions. This paper aims at providing some insights into the characteristics of natural substrate proteins of sHsps. It seems that sHsps of prokaryotes, as well as sHsps of some eukaryotes, can bind to a wide range of substrate proteins with a preference for certain functional classes of proteins. Using Drosophila melanogaster mitochondrial Hsp22 as a model system, we observed that this sHsp interacted with the members of ATP synthase machinery. Mechanistically, Hsp22 interacts with the multi-type substrate proteins under heat shock conditions as well as non-heat shock conditions.

Hereditary tyrosinemia type 1 (HT1), the most severe disease of the tyrosine catabolic pathway, is caused by a deficiency of fumarylacetoacetate hydrolase (FAH). More than 90 disease-causing variants have been identified in the fah gene. We investigated the molecular defect in a patient who presented atypical symptoms for the disease. No immunoreactive FAH was found in the liver and RNA analysis by RT-PCR suggested the presence of splicing mutations. Indeed, the patient was revealed to be a compound heterozygote for IVS6-1 g- > t and two new variants, namely p.V259L and p.G398E. Using splicing minigene constructs transfected in HeLa cells, the c.775G > C variant (p.V259L) was shown to affect partially exon 9 splicing thereby allowing the production of some full-length double-mutant FAH transcripts. The p.G398E variant had a major impact on enzyme activity, which was worsened by the p.V259L variant. Surprisingly, the double mutant protein was expressed to similar level as the wild-type protein upon transfection in HeLa cells but was absent in the patient liver extract, suggesting a higher propensity to be degraded in the hepatocellular context., (Copyright © 2019. Published by Elsevier Inc.)

Mitochondrial and nuclear genomes have to coevolve to ensure the proper functioning of the different mitochondrial complexes that are assembled from peptides encoded by both genomes. Mismatch between these genomes is believed to be strongly selected against due to the consequent impairments of mitochondrial functions and induction of oxidative stress. Here, we used a Drosophila model harboring an incompatibility between a mitochondrial tRNA tyr and its nuclear-encoded mitochondrial tyrosine synthetase to assess the cellular mechanisms affected by this incompatibility and to test the relative contribution of mitonuclear interactions and aging on the expression of impaired phenotypes. Our results show that the mitochondrial tRNA mutation caused a decrease in mitochondrial oxygen consumption in the incompatible nuclear background but no effect with the compatible nuclear background. Mitochondrial DNA copy number increased in the incompatible genotype but that increase failed to rescue mitochondrial functions. The flies harboring mismatch between nuclear and mitochondrial genomes had almost three times the relative mtDNA copy number and fifty percent higher rate of hydrogen peroxide production compared to other genome combinations at 25 days of age. We also found that aging exacerbated the mitochondrial dysfunctions. Our results reveal the tight interactions linking mitonuclear mismatch to mitochondrial dysfunction, mitochondrial DNA regulation, ROS production and aging.

The small heat shock protein (sHsp) Hsp22 from Drosophila melanogaster (DmHsp22) is part of the family of sHsps in this diptera. This sHsp is characterized by its presence in the mitochondrial matrix as well as by its preferential expression during ageing. Although DmHsp22 has been demonstrated to be an efficient in vitro chaperone, its function within mitochondria in vivo remains largely unknown. Thus, determining its protein-interaction network (interactome) in the mitochondrial matrix would help to shed light on its function(s). In the present study we combined immunoaffinity conjugation (IAC) with mass spectroscopy analysis of mitochondria from HeLa cells transfected with DmHsp22 in non-heat shock condition and after heat shock (HS). 60 common DmHsp22-binding mitochondrial partners were detected in two independent IACs. Immunoblotting was used to validate interaction between DmHsp22 and two members of the mitochondrial chaperone machinery; Hsp60 and Hsp70. Among the partners of DmHsp22, several ATP synthase subunits were found. Moreover, we showed that expression of DmHsp22 in transiently transfected HeLa cells increased maximal mitochondrial oxygen consumption capacity and ATP contents, providing a mechanistic link between DmHsp22 and mitochondrial functions.

The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).

The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.

The importance of the N-terminal region (NTR) in the oligomerization and chaperone-like activity of the Drosophila melanogaster small nuclear heat shock protein DmHsp27 was investigated by mutagenesis using size exclusion chromatography and native gel electrophoresis. Mutation of two sites of phosphorylation in the N-terminal region, S58 and S75, did not affect the oligomerization equilibrium or the intracellular localization of DmHsp27 when transfected into mammalian cells. Deletion or mutation of specific residues within the NTR region delineated a motif (FGFG) important for the oligomeric structure and chaperone-like activity of this sHsp. While deletion of the full N-terminal region, resulted in total loss of chaperone-like activity, removal of the (FGFG) at position 29 to 32 or single mutation of F29A/Y, G30R and G32R enhanced oligomerization and chaperoning capacity under non-heat shock conditions in the insulin assay suggesting the importance of this site for chaperone activity. Unlike mammalian sHsps DmHsp27 heat activation leads to enhanced association of oligomers to form large structures of approximately 1100 kDa. A new mechanism of thermal activation for DmHsp27 is presented.

Hereditary tyrosinemia type 1 (HT1) is caused by the lack of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine catabolic pathway. Up to now, around 100 mutations in the FAH gene have been associated with HT1, and despite many efforts, no clear correlation between genotype and clinical phenotype has been reported. At first, it seems that any mutation in the gene results in HT1. However, placing these mutations in their molecular context allows a better understanding of their possible effects. This chapter presents a closer look at the FAH gene and its corresponding protein in addition to provide a complete record of all the reported mutations causing HT1.

Inborn errors of metabolism (IEMs) are a group of diseases involving a genetic defect that alters a metabolic pathway and that presents usually during infancy. The tyrosine degradation pathway contains five enzymes, four of which being associated with IEMs. The most severe metabolic disorder associated with this catabolic pathway is hereditary tyrosinemia type 1 (HT1; OMIM 276700). HT1 is an autosomal recessive disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine catabolic pathway. Although a rare disease worldwide, HT1 shows higher incidence in certain populations due to founder effects. The acute form of the disease is characterized by an early onset and severe liver failure while the chronic form appears later and also involves renal dysfunctions. Until 1992 the only treatment for this disease was liver transplantation. Since then, NTBC/Nitisone (a drug blocking the pathway upstream of FAH) is successfully used in combination with a diet low in tyrosine and phenylalanine, but patients are still at risk of developing hepatocellular carcinoma. This chapter summarizes the biochemical and clinical features of HT1.

Untreated HT1 rapidly degenerates into very severe liver complications often resulting in liver cancer. The molecular basis of the pathogenic process in HT1 is still unclear. The murine model of FAH-deficiency is a suitable animal model, which represents all phenotypic and biochemical manifestations of the human disease on an accelerated time scale. After removal of the drug 2-(2-N-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), numerous signaling pathways involved in cell proliferation, differentiation and cancer are rapidly deregulated in FAH deficient mice. Among these, the Endoplasmic reticulum (ER) pathway, the heat stress response (HSR), the Nrf2, MEK and ERK pathways, are highly represented. The p21 and mTOR pathways critical regulators of proliferation and tumorigenesis have also been found to be dysregulated. The changes in these pathways are described and related to the development of liver cancer.

We previously reported the in silico characterization of Synechococcus sp. phage 18 kDa small heat shock protein (HspSP-ShM2). This small heat shock protein (sHSP) contains a highly conserved core alpha crystalline domain of 92 amino acids and relatively short N- and C-terminal arms, the later containing the classical C-terminal anchoring module motif (L-X-I/L/V). Here we establish the oligomeric profile of HspSP-ShM2 and its structural dynamics under in vitro experimental conditions using size exclusion chromatography (SEC/FPLC), gradient native gels electrophoresis and dynamic light scattering (DLS). Under native conditions, HspSP-ShM2 displays the ability to form large oligomers and shows a polydisperse profile. At higher temperatures, it shows extensive structural dynamics and undergoes conformational changes through an increased of subunit rearrangement and formation of sub-oligomeric species. We also demonstrate its capacity to prevent the aggregation of citrate synthase, malate dehydrogenase and luciferase under heat shock conditions through the formation of stable and soluble hetero-oligomeric complexes (sHSP:substrate). In contrast, the host cyanobacteria Synechococcus sp. WH7803 15 kDa sHSP (HspS-WH7803) aggregates when in the same conditions as HspSP-ShM2. However, its solubility can be maintained in the presence of non-ionic detergent Triton™X-100 and forms an oligomeric structure estimated to be between dimer and tetramer but exhibits no apparent inducible structural dynamics neither chaperon-like activity in all the assays and molar ratios tested. SEC/FPLC and thermal aggregation prevention assays results indicate no formation of hetero-oligomeric complex or functional interactions between both sHSPs. Taken together these in vitro results portray the phage HspSP-ShM2 as a classical sHSP and suggest that it may be functional at the in vivo level while behaving differently than its host amphitropic sHSP., Competing Interests: The authors have declared that no competing interests exist.

Hereditary tyrosinemia type 1 (HT1) is a severe inborn error of metabolism, impacting the tyrosine catabolic pathway with a high incidence of hepatocellular carcinoma (HCC). Using a HT1 murine model, we investigated the changes in profiles of circulating and hepatic miRNAs. The aim was to determine if plasma miRNAs could be used as non-invasive markers of liver damage in HT1 progression. Plasma and liver miRNAome was determined by deep sequencing after HT1 phenotype was induced. Sequencing analysis revealed deregulation of several miRNAs including let-7/miR-98 family, miR-21 and miR-148a, during manifestation of liver pathology. Three miRNAs (miR-98, miR-200b, miR-409) presenting the highest plasmatic variations among miRNAs found in both plasma and liver and with >1000 reads in at least one plasma sample, were further validated by RT-qPCR. Two of these miRNAs have protein targets involved in HT1 and significant changes in their circulating levels are detectable prior an increase in protein expression of alpha-fetoprotein, the current biomarker for HCC diagnosis. Future assessment of these miRNAs in HT1 patients and their association with liver neoplastic lesions might designate these molecules as potential biomarkers for monitoring HT1 damage progression, improving diagnosis for early HCC detection and the design of novel therapeutic targets.

The Seventh International Congress of the Cell Stress Society International (CSSI) was held as a joint meeting with the newly organized committee of Stress Physiology, the Chinese Association for Physiological Sciences (CAPS). There were over 200 colleagues and their students in attendance from 22 different countries. The topics of the congress were core scientific areas in the field of stress and health. The keynote speakers were Fu-Chu He (China), E.R. (Ron) de Kloet (The Netherlands), and Kazuhiro Nagata (Japan). The CSSI Medallion for Career Achievement in the cell stress and chaperones field was awarded to Kazutoshi Mori (Japan). Twelve student post awards were given in recognition of a very high quality poster session. In the tradition of this series of congresses, cultural events were an important part of the program. In addition, participants became better acquainted during trips to the ancient shopping street, an evening at the Chinese opera, and a lesson in Tai Chi from a master. The first groups of CSSI Fellows and Senior Fellows were presented their rosettes and certificates during the congress.

Hsp22 is a small mitochondrial heat shock protein (sHSP) preferentially up-regulated during aging in Drosophila melanogaster. Its developmental expression is strictly regulated and it is rapidly induced in conditions of stress. Hsp22 is one of the few sHSP to be localized inside mitochondria, and is the first sHSP to be involved in the mitochondrial unfolding protein response (UPR(MT)) together with Hsp60, mitochondrial Hsp70 and TRAP1. The UPR(MT) is a pro-longevity mechanism, and interestingly Hsp22 over-expression by-itself increases lifespan and resistance to stress. To unveil the effect of Hsp22 on the mitochondrial proteome, comparative IEF/SDS polyacrylamide 2D gels were done on mitochondria from Hsp22+ flies and controls. Among the proteins influenced by Hsp22 expression were proteins from the electron transport chain (ETC), the TCA cycle and mitochondrial Hsp70. Hsp22 co-migrates with ETC components and its over-expression is associated with an increase in mitochondrial protease activity. Interestingly, the only protease that showed significant changes upon Hsp22 over-expression in the comparative IEF/SDS-PAGE analysis was cathepsin D, which is localized in mitochondria in addition to lysosome in D. melanogaster as evidenced by cellular fractionation. Together the results are consistent with a role of Hsp22 in the UPR(MT) and in mitochondrial proteostasis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Aging is characterized by the accumulation of dysfunctional mitochondria. Since these organelles are involved in many important cellular processes, different mechanisms exist to maintain their integrity. Among them is the mitochondrial unfolding protein response, which triggers the expression of a set of proteins aimed at re-establishing mitochondrial homeostasis. The induction of mitochondrial chaperones expression, particularly of Hsp60 and Hsp70, is a hallmark of this pathway. In Drosophila melanogaster, Hsp22 is also up-regulated by mitochondrial stress. This small heat shock protein is one of the members of the family to be localized inside mitochondria. One characteristic of Drosophila Hsp22 is its preferential up-regulation during aging and in oxidative stress conditions. It is a beneficial protein since its over-expression increases lifespan and resistance to stress while its down-regulation is detrimental. This review focuses on Drosophila Hsp22 and its links with the mitochondrial unfolding protein response and the aging process, in addition to highlight the important role of this sHSP in mitochondrial homeostasis.

Hereditary tyrosinemia type 1 (HT1) is the most severe inherited metabolic disease of the tyrosine catabolic pathway, with a progressive hepatic and renal injury and a fatal outcome if untreated. Toxic metabolites accumulating in HT1 have been shown to elicit endoplasmic reticulum (ER) stress response, and to induce chromosomal instability, cell cycle arrest and apoptosis perturbation. Although many studies have concentrated on elucidating these events, the molecular pathways responsible for development of hepatocellular carcinoma (HCC) still remain unclear. In this study the fah knockout murine model (fah(-/-)) was used to investigate the cellular signaling implicated in the pathogenesis of HT1. Fah(-/-) mice were subjected to drug therapy discontinuation (Nitisinone withdrawal), and livers were analyzed at different stages of the disease. Monitoring of mice revealed an increasing degeneration of the overall physiological conditions following drug withdrawal. Histological analysis unveiled diffuse hepatocellular damage, steatosis, oval-like cells proliferation and development of liver cell adenomas. Immunoblotting results revealed a progressive and chronic activation of stress pathways related to cell survival and proliferation, including several stress regulators such as Nrf2, eIF2α, CHOP, HO-1, and some members of the MAPK signaling cascade. Impairment of stress defensive mechanisms was also shown by microarray analysis in fah(-/-) mice following prolonged therapy interruption. These results suggest that a sustained activation of stress pathways in the chronic HT1 progression might play a central role in exacerbating liver degeneration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Dystonia1 (DYT1) dystonia is caused by a glutamic acid deletion (ΔE) mutation in the gene encoding Torsin A in humans (HTorA). To investigate the unknown molecular and cellular mechanisms underlying DYT1 dystonia, we performed an unbiased proteomic analysis., Results: We found that the amount of proteins and transcripts of an Endoplasmic reticulum (ER) resident chaperone Heat shock protein cognate 3 (HSC3) and a mitochondria chaperone Heat Shock Protein 22 (HSP22) were significantly increased in the HTorA(ΔE)- expressing brains compared to the normal HTorA (HTorA(WT)) expressing brains. The physiological consequences included an increased susceptibility to oxidative and ER stress compared to normal HTorA(WT) flies. The alteration of transcripts of Inositol-requiring enzyme-1 (IRE1)-dependent spliced X box binding protein 1(Xbp1), several ER chaperones, a nucleotide exchange factor, Autophagy related protein 8b (ATG8b) and components of the ER associated degradation (ERAD) pathway and increased expression of the Xbp1-enhanced Green Fluorescence Protein (eGFP) in HTorA(ΔE) brains strongly indicated the activation of the unfolded protein response (UPR). In addition, perturbed expression of the UPR sensors and inducers in the HTorA(ΔE) Drosophila brains resulted in a significantly reduced life span of the flies. Furthermore, the types and quantities of proteins present in the anti-HSC3 positive microsomes in the HTorA(ΔE) brains were different from those of the HTorA(WT) brains., Conclusion: Taken together, these data show that HTorA(ΔE) in Drosophila brains may activate the UPR and increase the expression of HSP22 to compensate for the toxic effects caused by HTorA(ΔE) in the brains.

Insulin receptor (IR) endocytosis requires a remodelling of the actin cytoskeleton. We show here that ANXA2 is SUMOylated at the K10 located in a non-consensus SUMOylation motif in the N-terminal domain. The Y24F mutation decreased the SUMOylation signal, whereas insulin stimulation increased ANXA2 SUMOylation. A survey of protein SUMOylation in hepatic Golgi/endosome (G/E) fractions after insulin injections revealed the presence of a SUMOylation pattern and confirmed the SUMOylation of ANXA2. The construction of an IR/ANXA2/SUMO network (IRASGEN) in the G/E context reveals the presence of interacting nodes whereby SUMO1 connects ANXA2 to actin and microtubule-mediated changes in membrane topology. Heritable variants associated with type 2 diabetes represent 41% of the IRASGEN thus pointing out the physio-pathological importance of this subnetwork., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Mitochondria are involved in many key cellular processes and therefore need to rely on good protein quality control (PQC). Three types of mechanisms are in place to insure mitochondrial protein integrity: reactive oxygen species scavenging by anti-oxidant enzymes, protein folding/degradation by molecular chaperones and proteases and clearance of defective mitochondria by mitophagy. Drosophila melanogaster Hsp22 is part of the molecular chaperone axis of the PQC and is characterized by its intra-mitochondrial localization and preferential expression during aging. As a stress biomarker, the level of its expression during aging has been shown to partially predict the remaining lifespan of flies. Since over-expression of this small heat shock protein increases lifespan and resistance to stress, Hsp22 most likely has a positive effect on mitochondrial integrity. Accordingly, Hsp22 has recently been implicated in the mitochondrial unfolding protein response of flies. This review will summarize the key findings on D. melanogaster Hsp22 and emphasis on its links with the aging process.

The workshop was entitled "The Small HSP World" and had the mission to bring together investigators studying small heat shock proteins (sHSPs). It was held at Le Bonne Entente in Quebec City (Quebec, Canada) from October 2 to October 5 2014. Forty-four scientists from 14 different countries attended this workshop of the Cell Stress Society International (CSSI). The small number of participants stimulated interesting discussions, and the resulting informal atmosphere was appreciated by everybody. This article provides highlights from talks and discussions of the workshop, giving an overview of the latest work on sHSPs.

Hereditary tyrosinemia type 1 (HT1) (OMIM 276700) is a severe inherited metabolic disease affecting mainly hepatic and renal functions that leads to a fatal outcome if untreated. HT1 results from a deficiency of the last enzyme of tyrosine catabolism, fumarylacetoacetate hydrolase (FAH). Biochemical findings include elevated succinylacetone in blood and urine; elevated plasma concentrations of tyrosine, methionine and phenylalanine; and elevated tyrosine metabolites in urine. The HT1 frequency worldwide is about 1 in 100,000 individuals. In some areas, where the incidence of HT1 is noticeably higher, prevalence of characteristic mutations has been reported, and the estimated incidence of carriers of a specific mutation can be as high as 1 out of 14 adults. Because the global occurrence of HT1 is relatively low, a considerable number of cases may go unrecognized, underlining the importance to establish efficient prenatal and carrier testing to facilitate an early detection of the disease. Here we describe the 95 mutations reported so far in HT1 with special emphasis on their geographical and ethnic distributions. Such information should enable the establishment of a preferential screening process for mutations most predominant in a given region or ethnic group.

Stress granules (SGs) are well characterized cytoplasmic RNA bodies that form under various stress conditions. We have observed that exposure of mammalian cells in culture to low doses of UVC induces the formation of discrete cytoplasmic RNA granules that were detected by immunofluorescence staining using antibodies to RNA-binding proteins. UVC-induced cytoplasmic granules are not Processing Bodies (P-bodies) and are bone fide SGs as they contain TIA-1, TIA-1/R, Caprin1, FMRP, G3BP1, PABP1, well known markers, and mRNA. Concomitant with the accumulation of the granules in the cytoplasm, cells enter a quiescent state, as they are arrested in G1 phase of the cell cycle in order to repair DNA damages induced by UVC irradiation. This blockage persists as long as the granules are present. A tight correlation between their decay and re-entry into S-phase was observed. However the kinetics of their formation, their low number per cell, their absence of fusion into larger granules, their persistence over 48 hours and their slow decay, all differ from classical SGs induced by arsenite or heat treatment. The induction of these SGs does not correlate with major translation inhibition nor with phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). We propose that a restricted subset of mRNAs coding for proteins implicated in cell cycling are removed from the translational apparatus and are sequestered in a repressed form in SGs.

Hereditary Tyrosinemia type 1 (HT1) is a metabolic liver disease caused by genetic defects of fumarylacetoacetate hydrolase (FAH), an enzyme necessary to complete the breakdown of tyrosine. The severe hepatic dysfunction caused by the lack of this enzyme is prevented by the therapeutic use of NTBC (2-[2-nitro-4-(trifluoromethyl)benzoyl] cyclohexane-1,3-dione). However despite the treatment, chronic hepatopathy and development of hepatocellular carcinoma (HCC) are still observed in some HT1 patients. Growing evidence show the important role of heat shock proteins (HSPs) in many cellular processes and their involvement in pathological diseases including cancer. Their survival-promoting effect by modulation of the apoptotic machinery is often correlated with poor prognosis and resistance to therapy in a number of cancers. Here, we sought to gain insight into the pathophysiological mechanisms associated with liver dysfunction and tumor development in a murine model of HT1. Differential gene expression patterns in livers of mice under HT1 stress, induced by drug retrieval, have shown deregulation of stress and cell death resistance genes. Among them, genes coding for HSPB and HSPA members, and for anti-apoptotic BCL-2 related mitochondrial proteins were associated with the hepatocarcinogenetic process. Our data highlight the variation of stress pathways related to HT1 hepatocarcinogenesis suggesting the role of HSPs in rendering tyrosinemia-affected liver susceptible to the development of HCC.

Background: Circulating microRNAs ( miRNAs) are emerging as novel disease biomarkers. We aimed to explore the association between circulating miRNAs and the occurrence of acute myocardial infarction (AMI) in Chinese populations., Methods and Results: In the discovery stage, the plasma of 20 patients with AMI and 20 controls were pooled respectively and profiled by massively parallel sequencing. Seventy-seven miRNAs showed differential expression. Selected miRNAs were validated in 178 patients with AMI and 198 controls using quantitative reverse transcriptase polymerase chain reaction assays and further replicated in 150 patients with AMI and 150 controls. Results suggest that miR-320b and miR-125b levels were significantly lower in patients with AMI than in controls in both validation populations (P<0.0001). Lower levels of miR-320b and miR-125b were associated with increased occurrence of AMI (adjusted odds ratio, 4.71; 95% confidence interval, 2.96-7.48 and odds ratio, 4.27; 95% confidence interval, 2.84-6.41, respectively). Addition of the 2 miRNAs to traditional risk factors led to a significant improvement in the area under the curve from 0.822 (95% confidence interval, 0.787-0.856) to 0.871 (95% confidence interval, 0.842-0.900), with a net reclassification improvement of 20.45% (P<0.0001) and an integrated discrimination improvement of 0.16 (P<0.0001) for patients with AMI. A functional study showed that miR-320b and miR-125b could regulate the expression profiles of genes enriched in several signal transduction pathways critical for coronary heart disease in human vascular endothelial cells., Conclusions: The plasma levels of miR-320b and miR-125b were significantly lower in patients with AMI when compared with controls, and these miRNAs may be involved in the pathogenesis of coronary heart disease.

Small heat shock proteins (sHSPs) are oligomeric stress proteins characterized by an α-crystallin domain (ACD) surrounded by a N-terminal arm and C-terminal extension. Publications on sHSPs have reported that they exist in prokaryotes and eukaryotes but, to our knowledge, not in viruses. Here we show that sHSPs are present in some cyanophages that infect the marine unicellular cyanobacteria, Synechococcus and Prochlorococcus. These phage sHSPs contain a conserved ACD flanked by a relatively conserved N-terminal arm and a short C-terminal extension with or without the conserved C-terminal anchoring module (CAM) L-X-I/V, suggested to be implicated in the oligomerization. In addition, cyanophage sHSPs have the signature pattern, P-P-[YF]-N-[ILV]-[IV]-x(9)-[EQ], in the predicted β2 and β3 strands of the ACD. Phylogenetically, cyanophage sHSPs form a monophyletic clade closer to bacterial class A sHSPs than to cyanobacterial sHSPs. Furthermore, three sHSPs from their cellular host, Synechococcus, are phylogenetically close to plants sHSPs. Implications of evolutionary relationships between the sHSPs of cyanophages, bacterial class A, cyanobacteria, and plants are discussed.

Mouse prostate membrane-associated proteins of the annexin family showed changes in SUMOylation during androgen treatment. Among these the calcium-binding annexin A1 protein (ANXA1) was chosen for further characterization given its role in protein secretion and cancer. SUMOylation of ANXA1 was confirmed by overexpressing SUMO-1 in LNCaP cells. Site-directed mutagenesis indicated that K257 located in a SUMOylation consensus motif in the C-terminal calcium-binding DA3 repeat domain is SUMOylated. Mutation of the N-terminal Y21 decreased markedly the SUMOylation signal while EGF stimulation increased ANXA1 SUMOylation. A structural analysis of ANXA1 revealed that K257 is located in a hot spot where Ca(2+) and SUMO-1 bind and where a nuclear export signal and a polyubiquitination site are also present. Also, Y21 is buried inside an α-helix structure in the Ca(2+)-free conformation implying that Ca(2+) binding, and the subsequent expelling of the N-terminal α-helix in a disordered conformation, is permissive for its phosphorylation. These results show for the first time that SUMOylation can be regulated by an external signal (EGF) and indicate the presence of a cross-talk between the N-terminal and C-terminal domains of ANXA1 through post-translational modifications., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the effect of temperature changes on the functional properties of mitochondria from two sets of D. simulans fly lines harboring the siII and siIII haplotypes in a common nuclear genetic background. We studied four introgressed isofemale lines possessing the mtDNA of siII and the nuclear background of siIII (siII-introgressed) and four lines possessing siIII mitochondria with its native nuclear genome (siIII-controls). We assessed the catalytic capacities of electron transport system (ETS) at four different temperatures (12, 18, 24 and 28 ºC). The impact of temperature on the pyruvate dehydrogenase (PDH) activity, the mitochondrial respiration (coupled and uncoupled respiration), cytochrome c oxidase activity, as well as the excess capacity of complex IV (COX) were evaluated in these two sets of flies. Our results showed that the temperature coefficient values (Q(10)) measured for mitochondrial respiration in the lower range of temperatures (12 to 18 ºC) showed a 2 to 3 fold increase in siII-introgressed when compared to siIII-controls. This result shows that the impact of temperature on mitochondrial function is different between the two mitotypes studied. The Q(10) results seem to be linked to the apparent COX excess capacity of 193% for siIII-controls that is inexistent for siII-introgressed at 12 ºC. One explanation for these results is that the mitochondria can compensate for the disruption of mito-nuclear interactions at 24 ºC but not at lower temperatures. An alternate explanation would be that siII haplotype confer divergent kinetic properties to the ETS that translate to different temperature sensitivities.

Linking the mitochondrial genotype and the organismal phenotype is of paramount importance in evolution of mitochondria. In this study, we determined the differences in catalytic properties of mitochondria dictated by divergences in the siII and siIII haplogroups of Drosophila simulans using introgressions of siII mtDNA type into the siIII nuclear background. We used a novel in situ method (permeabilized fibers) that allowed us to accurately measure the consumption of oxygen by mitochondria in constructed siII-introgressed flies and in siIII-control flies. Our results showed that the catalytic capacity of the electron transport system is not impaired by introgressions, suggesting that the functional properties of mitochondria are tightly related to the mtDNA haplogroup and not to the nuclear DNA or to the mito-nuclear interactions. This is the first study, to our knowledge, that demonstrates a naturally occurring haplogroup can confer specific functional differences in aspects of mitochondrial metabolism. This study illustrates the importance of mtDNA changes on organelle evolution and highlights the potential bioenergetic and metabolic impacts that divergent mitochondrial haplogroups may have upon a wide variety of species including humans., (© 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.)

The expression of small heat shock proteins is tightly regulated during development in multiple organisms. As housekeeping proteins, small heat shock proteins help protect cells from apoptosis, stabilize the cytoskeleton and contribute to proteostasis. Consistently, depletion of one small heat shock protein is usually not detrimental due to a certain level of redundancy between the functions of each small heat shock protein. However, while their stress-induced expression is regulated by heat shock factors, their constitutive expression is under the control of other specific transcription factors, suggesting the existence of very specialized functions. This review focuses on the expression patterns and functions of small heat shock proteins in various organisms during development. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The human fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg(2+) was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.

The most common clinical tachycardia, Atrial Fibrillation (AF), is a progressive disease, caused by cardiomyocyte remodeling, which finally results in contractile dysfunction and AF persistence. Recently, we identified a protective role of heat shock proteins (HSPs), especially the small HSPB1 member, against tachycardia remodeling in experimental AF models. Our understanding of tachycardia remodeling and anti-remodeling drugs is currently hampered by the lack of suitable (genetic) manipulatable in vivo models for rapid screening of key targets in remodeling. We hypothesized that Drosophila melanogaster can be exploited to study tachycardia remodeling and protective effects of HSPs by drug treatments or by utilizing genetically manipulated small HSP-overexpressing strains. Tachypacing of Drosophila pupae resulted in gradual and significant cardiomyocyte remodeling, demonstrated by reduced contraction rate, increase in arrhythmic episodes and reduction in heart wall shortening, compared to normal paced pupae. Heat shock, or pre-treatment with HSP-inducers GGA and BGP-15, resulted in endogenous HSP overexpression and protection against tachycardia remodeling. DmHSP23 overexpressing Drosophilas were protected against tachycardia remodeling, in contrast to overexpression of other small HSPs (DmHSP27, DmHSP67Bc, DmCG4461, DmCG7409, and DmCG14207). (Ultra)structural evaluation of the tachypaced heart wall revealed loss of sarcomeres and mitochondrial damage which were absent in tachypaced DmHSP23 overexpressing Drosophila. In addition, tachypacing induced a significant increase in calpain activity, which was prevented in tachypaced Drosophila overexpressing DmHSP23. Tachypacing of Drosophila resulted in cardiomyocyte remodeling, which was prevented by general HSP-inducing treatments and overexpression of a single small HSP, DmHSP23. Thus, tachypaced D. melanogaster can be used as an in vivo model system for rapid identification of novel targets to combat AF associated cardiomyocyte remodeling., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In ectotherms, the external temperature is experienced by the mitochondria, and the mitochondrial respiration of different genotypes is likely to change as a result. Using high-resolution respirometry with permeabilized fibers (an in situ approach), we tried to identify differences in mitochondrial performance and thermal sensitivity of two Drosophila simulans populations with two different mitochondrial types (siII and siIII) and geographical distributions. Maximal state 3 respiration rates obtained with electrons converging at the Q junction of the electron transport system (ETS) differed between the mitotypes at 24°C. Catalytic capacities were higher in flies harboring siII than in those harboring siIII mitochondrial DNA (2,129 vs. 1,390 pmol O(2)·s(-1)·mg protein(-1)). The cytochrome c oxidase activity was also higher in siII than siIII flies (3,712 vs. 2,688 pmol O(2)·s(-1)·mg protein(-1)). The higher catalytic capacity detected in the siII mitotype could provide an advantage in terms of intensity of aerobic activity, endurance, or both, if the intensity of exercise that can be aerobically performed is partly dictated by the aerobic capacity of the tissue. Moreover, thermal sensitivity results showed that even if temperature affects the catalytic capacity of the different enzymes of the ETS, both mitotypes revealed high tolerance to temperature variation. Previous in vitro study failed to detect any consistent functional mitochondrial differences between the same mitotypes. We conclude that the in situ approach is more sensitive and that the ETS is a robust system in terms of functional and regulatory properties across a wide range of temperatures.

Drosophila simulans possesses three different mitochondrial haplotypes (siI, II and III) that are nonrandomly geographically subdivided with a 3% interhaplogroup variation. The aim of this study was to determine whether perturbation of mitochondrial metabolism and ROS management by temperature variation and mtDNA introgression would influence the development of aerobic capacity and the intensity of oxidative stress in D. simulans at different ages. Environmental temperature divergences during development had few impacts on metabolic capacities. Our data suggested strong functional conservatism of mitochondrial haplotypes between the D. simulans lines studied. This conservatism was expressed by the low divergences in either mitochondrial or ROS buffering enzyme activities, or even markers of ROS damage even after disruption of coevolved genomes. Disruption of coevolved mitochondrial and nuclear genomes through mtDNA introgression induced no clear divergence on metabolic phenotype at any state of development. Reduction of cytochrome c oxidase activity that was observed after introgression of one mitochondrial haplotype will require further investigation to delineate whether it is associated with any modification of mito-nuclear interactions., (Copyright © 2010 Wiley-Liss, Inc., A Wiley Company.)

Objectives: It was the aim of our study to investigate whether polymorphisms of HSP70 have an affect on antigen and antibody levels in acute coronary syndrome (ACS) patients and normal controls, and the possible joint effect of variants and antigen and antibody levels on the risk of ACS., Methods: Three single nucleotide polymorphisms of HSPA1A and HSPA1L were evaluated in 520 ACS patients and 520 age- and sex-matched controls. Plasma extracellular Hsp70 (eHsp70) and anti-Hsp70 antibody levels were determined using ELISA., Results: Individuals with +190G/C (rs1043618) CC genotype in HSPA1A had higher levels of eHsp70 in controls and lower levels of anti-Hsp70 body in ACS, compared with +190G/C GG carriers. Significantly increased ACS risks of 2.93 and 3.53 fold were found in subjects with the +190G/C CC genotype and high eHsp70 levels or low anti-Hsp70 antibody levels, respectively. The highest risk of ACS was found in subjects with +190G/C CC genotypes, high eHsp70 and low anti-Hsp70 antibody levels compared with those in the reference group (OR = 7.57, 95% CI 3.04-18.87)., Conclusions: The +190G/C polymorphism of HSPA1A may contribute to influence eHsp70 levels in controls and anti-Hsp70 antibody levels in ACS, and the +190G/C genotypes, eHsp70 and anti-Hsp70 antibody levels may have a joint effect on the risk of ACS., (Copyright © 2011 S. Karger AG, Basel.)

Although immune reactions against heat shock proteins have been implicated in the pathogenesis of atherosclerosis, conflicting associations between Hsp70, anti-Hsp70 antibody and coronary heart disease (CHD) have been reported. This study assessed whether there is a significant association between extracellular human Hsp70, anti-Hsp70 antibody and acute coronary syndrome (ACS) and stable angina (SA), and examined dynamic changes in Hsp70 and anti-Hsp70 antibody levels induced by acute myocardial infarction (AMI). Plasma Hsp70 and anti-Hsp70 antibody levels in 291 patients with ACS (179 AMI, 112 unstable angina), 126 patients with SA and 417 age and sex-matched healthy subjects, and in 40 patients after admission for AMI, and on day 2, 3, and 7 after the onset of AMI were determined using enzyme-linked immunosorbent assays. Hsp70 levels were significantly higher in ACS and SA and anti-Hsp70 antibody levels were only markedly lower in ACS than controls. After adjustment for traditional CHD risk factors, increasing levels of Hsp70 were significantly associated with an increased risk and severity of ACS (P for trend < 0.001), whereas increasing levels of anti-Hsp70 antibody were associated with a decreased risk of ACS (P for trend = 0.0003). High levels of Hsp70 combined with low levels of anti-Hsp70 antibody had a joint effect on the risk of ACS (OR, 5.14, 95% CI, 3.00-8.79; P < 0.0001). In patients with AMI, Hsp70 levels decreased rapidly from days 1-7 after onset, whereas anti-Hsp70 antibody levels increased in patients with AMI. These findings suggest that higher Hsp70 levels or lower anti-Hsp70 antibody levels are independently associated with a higher risk of ACS. Higher Hsp70 levels and lower anti-Hsp70 antibody levels combine to further increase this risk.