Search

Defining the molecular mechanisms underlying cardiac resilience is crucial to find effective approaches to protect the heart. A physiologic level of ROS is produced in the heart by fatty acid oxidation, but stressful events can boost ROS and cause mitochondrial dysfunction and cardiac functional impairment. Melusin is a muscle specific chaperone required for myocardial compensatory remodeling during stress. Here we report that Melusin localizes in mitochondria where it binds the mitochondrial trifunctional protein, a key enzyme in fatty acid oxidation, and decreases it activity. Studying both mice and human induced pluripotent stem cell-derived cardiomyocytes, we found that Melusin reduces lipid oxidation in the myocardium and limits ROS generation in steady state and during pressure overload and doxorubicin treatment, preventing mitochondrial dysfunction. Accordingly, the treatment with the lipid oxidation inhibitor Trimetazidine concomitantly with stressful stimuli limits ROS accumulation and prevents long-term heart dysfunction. These findings disclose a physiologic mechanism of metabolic regulation in the heart and demonstrate that a timely restriction of lipid metabolism represents a potential therapeutic strategy to improve cardiac resilience to stress. Synopsis: FAO is catalyzed by the mitochondrial trifunctional protein (MTP) and, if overloaded, generates ROS. ROS are boosted during stressful events, favoring cardiac maladaptive remodeling. Melusin is a muscle-specific chaperone able to sustain heart resilience during a wide variety of stressful insults. Melusin interacts and inhibits the MTP, restraining FAO and ROS generation in the myocardium. Heart resilience depends on the rate of FAO during stressful events occurrence. Inhibiting lipid metabolism in a timely manner during stress protects the heart from dysfunction and prolongs the survival in preclinical models. FAO is catalyzed by the mitochondrial trifunctional protein (MTP) and, if overloaded, generates ROS. ROS are boosted during stressful events, favoring cardiac maladaptive remodeling. Melusin is a muscle-specific chaperone able to sustain heart resilience during a wide variety of stressful insults. [ABSTRACT FROM AUTHOR]

Abstract Defining the molecular mechanisms underlying cardiac resilience is crucial to find effective approaches to protect the heart. A physiologic level of ROS is produced in the heart by fatty acid oxidation, but stressful events can boost ROS and cause mitochondrial dysfunction and cardiac functional impairment. Melusin is a muscle specific chaperone required for myocardial compensatory remodeling during stress. Here we report that Melusin localizes in mitochondria where it binds the mitochondrial trifunctional protein, a key enzyme in fatty acid oxidation, and decreases it activity. Studying both mice and human induced pluripotent stem cell-derived cardiomyocytes, we found that Melusin reduces lipid oxidation in the myocardium and limits ROS generation in steady state and during pressure overload and doxorubicin treatment, preventing mitochondrial dysfunction. Accordingly, the treatment with the lipid oxidation inhibitor Trimetazidine concomitantly with stressful stimuli limits ROS accumulation and prevents long-term heart dysfunction. These findings disclose a physiologic mechanism of metabolic regulation in the heart and demonstrate that a timely restriction of lipid metabolism represents a potential therapeutic strategy to improve cardiac resilience to stress.

Intracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, this work reports on a cell‐permeable photosensitizer (P8, Rose Bengal derivative) for selective photo induced proximity labeling and crosslinking of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect. Further acetylation permeabilizes Rose Bengal to selectively image, label, and crosslink aggregated proteome in live stressed cells. A combination of photo‐chemical, tandem mass spectrometry, and protein biochemistry characterizations reveals the complexity in photosensitizing pathways (both Type I & II), modification sites and labeling mechanisms. The diverse labeling sites and reaction types result in highly effective enrichment and identification of aggregated proteome. Finally, aggregated proteomics and interaction analyses thereby reveal extensive entangling of proteostasis network components mediated by HSP70 chaperone (HSPA1B) and active participation of autophagy pathway in combating proteasome inhibition. Overall, this work exemplifies the first photo induced proximity labeling and crosslinking method (namely AggID) to profile intracellular aggregated proteome and analyze its interactions. [ABSTRACT FROM AUTHOR]

Carbon monoxide dehydrogenase derived from Carboxydothermus hydrogenoformans (ChCODH) is a representative anaerobic enzyme that contains [4Fe–4S] clusters. To functionally express ChCODH under aerobic conditions in genetically engineered E. coli, co-expression with the SUF system that facilitates the assembly of iron–sulfur clusters under oxidative stress and iron limitation was performed. Results showed that compared to the expression of ChCODH alone under aerobic conditions, a 10-fold and 2-fold increase in the specific activity and protein yield, respectively, resulting in a total activity of up to 29,185 U/L was observed in E. coli co-expressed with ChCODH and the SUF system. Co-expression of Tig, a chaperone protein, and the addition of 2 mM FeSO4, which constitutes the iron-sulfur cluster, further increased the total activity up to 97,758 U/L. Moreover, the amount of Fe incorporated into ChCODH was proportional to the specific activity, while the amount of Ni was not correlated with the specific activity. Finally, high cell density cultivation under optimized aerobic conditions in the SUF system and the chaperone protein Tig co-expressed in recombinant E. coli resulted in a total activity of up to 235,689 U/L. [ABSTRACT FROM AUTHOR]

Abstract Intracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, this work reports on a cell‐permeable photosensitizer (P8, Rose Bengal derivative) for selective photo induced proximity labeling and crosslinking of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect. Further acetylation permeabilizes Rose Bengal to selectively image, label, and crosslink aggregated proteome in live stressed cells. A combination of photo‐chemical, tandem mass spectrometry, and protein biochemistry characterizations reveals the complexity in photosensitizing pathways (both Type I & II), modification sites and labeling mechanisms. The diverse labeling sites and reaction types result in highly effective enrichment and identification of aggregated proteome. Finally, aggregated proteomics and interaction analyses thereby reveal extensive entangling of proteostasis network components mediated by HSP70 chaperone (HSPA1B) and active participation of autophagy pathway in combating proteasome inhibition. Overall, this work exemplifies the first photo induced proximity labeling and crosslinking method (namely AggID) to profile intracellular aggregated proteome and analyze its interactions.

This study aims to evaluate the effects of smoking on extracellular heat shock protein levels. Heat shock proteins have been associated with numerous diseases. Studies on the relationship between smoking exposure and heat shock proteins were mostly evaluated with intracellular heat shock proteins. This crosssectional case--control study was conducted on patients who applied to outpatient clinics for smoking cessation and routine control in our faculty hospital. Of the smoking men, 75 between the ages of 30 and 65 years and 82 nonsmoking men were included in the study. Heat shock protein levels in plasma samples were determined using ELX 800 plate reader and Human HSP ELISA kit. Plasma heat shock protein levels were found to be significantly higher in smokers. There is no significant relationship between all three heat shock proteins and age, the number of cigarettes smoked daily, and the number of cigarettes smoked so far (package/year) (p > .050). Smoking significantly increases plasma heat shock protein 90, heat shock protein 70, and heat shock protein 40 levels. [ABSTRACT FROM AUTHOR]

Heterologous expression systems (e.g., Xenopus laevis oocytes) are useful to study the biophysical properties and pharmacology of ionotropic receptors such as ionotropic glutamate (iGLuRs) and nicotinic acetylcholine (nAChRs) receptors. However, insect receptors often require the co‐expression of chaperone proteins to be functional. Only few iGluRs and nAChRs have been successfully expressed in such systems. Here, we compared the efficiency of chaperone proteins to promote the functional expression of one Apis mellifera iGluR and several nAChR subunit combinations (α1α8β1, α7, α2α8β1 and α2α7α8β1) in Xenopus oocytes. To this end, we cloned a new iGluR (GluR‐1) and potential chaperone proteins (e.g., SOL‐1, Neto, NACHO) and tested more than 40 combinations of human, nematode and honeybee proteins. We obtained robust expression of GluR‐1 and α1α8β1 when co‐expressed with honeybee chaperone proteins and found that nAChR expression critically depended on the α1 subunit N‐terminal sequence. We recorded small ACh‐gated currents in few oocytes when the α7 subunit was co‐expressed with Caenorhabditis elegans RIC‐3, but none of the chaperone proteins allowed efficient expression of α2α8β1 or α2α7α8β1. Our results show that only some protein combinations can reconstitute functional receptors in Xenopus oocytes and that protein combination efficient in one species is not always efficient in another species. [ABSTRACT FROM AUTHOR]

The heterologous overexpression states of prion proteins play a critical role in understanding the mechanisms of prion‐related diseases. We report herein the identification of soluble monomer and complex states for a bakers' yeast prion, Sup35, when expressed in Escherichia coli. Two peaks are apparent with the elution of His‐tagged Sup35 by imidazole from a Ni2+ affinity column. Peak I contains Sup35 in both monomer and aggregated states. Sup35 aggregate is abbreviated as C‐aggregate and includes a non‐fibril complex comprising Sup35 aggregate‐HSP90‐Dna K, ATP synthase β unit (chain D), 30S ribosome subunit, and Omp F. The purified monomer and C‐aggregate can remain stable for an extended period of time. Peak II contains Sup35 also in both monomer and aggregated (abbreviated as S‐aggregate) states, but the aggregated states are caused by the formation of inter‐Sup35 disulfide bonds. This study demonstrates that further assembly of Sup35 non‐fibril C‐aggregate can be interrupted by the chaperone repertoire system in E. coli. [ABSTRACT FROM AUTHOR]

The stress-inducible mammalian heat shock protein Hsp70 and its bacterial orthologue DnaK are highly conserved molecular chaperones and a crucial part of the machinery responsible for protein folding and homeostasis. Hsp70 is a three-domain, 70 kDa protein that cycles between an ATP-bound state in which all three domains are securely coupled into one unit and an ADP-bound state in which they are loosely attached via a flexible interdomain linker. The Hsp70 presents an alluring novel therapeutic target since it is crucial for maintaining cellular proteostasis and is particularly crucial to cancer cells. We have performed molecular dynamics simulations of the SBD (substrate binding domain) along with the Lid domain in response to experimental efforts to identify small molecule inhibitors that impair the functioning of Hsp70. Our intent has been to characterize the motion of the SBD/Lid allosteric machinery and in, addition, to identify the effect of the PET16 molecule on this motion. Interestingly, we noticed the opening of the entire Lid domain in the apo-form of the dimer. The configuration of the open structure was very different from previously published structures (PDB 4JN4) of the open and docked conformation of the ATP bound form. MD simulations revealed the Lid to be capable of far greater dynamical excursions than has been anticipated by experimental structural biology. This is of value in future drug discovery efforts targeted to modulating Hsp70 activity. The PET16 molecule appears to be weakly bound and its effect on the dynamics of the complex is yet to be elucidated. [Display omitted] • The heat shock protein Hsp70 is responsible for protein folding and homeostasis. • Molecular dynamics simulations of the SBD and the Lid in response to experimental efforts to identify small molecule inhibitors. • MD simulations revealed the Lid to be capable of far greater dynamical excursions than has been anticipated by experimental structural biology. • MD trajectories reveal opening of the entire Lid domain by itself in the apo form of the SBD-Lid structure in one chain [ 73 ]. [ABSTRACT FROM AUTHOR]

Protein misassembly leads to the formation of dysfunctional and toxic molecular species relating to neurodegeneration in Parkinson's disease and Alzheimer's disease. Here, we tailored a nanochaperone (αS‐nChap) for α‐synuclein to regulate its assembly. The αS‐nChap is capable of i) specifically recognizing α‐synuclein; ii) dynamically capturing and stabilizing monomeric α‐synuclein and retarding oligomerization; iii) tightly capturing oligomeric α‐synuclein to prevent fibrillization; and iv) transporting α‐synuclein oligomers to the lysosomal degradation system. The regulation of α‐synuclein assembly by αS‐nChap was studied in vitro. Moreover, the role of αS‐nChap preventing α‐synuclein pathology in cells and protecting neurons from apoptosis was investigated. The strategy of tailoring a nanochaperone to regulate aberrant assembly of pathogenic proteins provides important insights into protein misfolding diseases. We foresee that αS‐nChap has therapeutic value for Parkinson's disease. [ABSTRACT FROM AUTHOR]

Copyright of Advances in Biochemistry / Postepy Biochemii is the property of Polish Biochemical Society / Acta Biochimica Polonica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Intracerebral hemorrhage (ICH) is a life-threatening type of stroke that disrupts the normal neurological function of the brain. Clinical studies have reported a non-linear J-shaped association between alcohol consumption levels and the occurrence of cerebral stroke. Specifically, alcohol intoxication increases stroke incidence, while moderate alcohol pre-conditioning decreases stroke frequency and improves outcomes. Although alcohol pre-consumption is likely a crucial player in ICH, the underlying mechanism remains unclear. We performed 1-h alcohol pre-conditioning followed by ICH induction in Sprague-Dawley (SD) rats to investigate the role of alcohol pre-conditioning in ICH. Interestingly, behavioral test analysis found that ethanol intoxication (3 g/kg) aggravated ICH-induced neurological deficits, but moderate ethanol pre-conditioning (0.75 g/kg) ameliorated ICH-induced neurological deficits by reducing the oxidative stress and proinflammatory cytokines release. Moreover, we found that moderate ethanol pretreatment improved the striatal endoplasmic reticulum (ER) homeostasis by increasing the chaperone protein expression and reducing oxidative stress and apoptosis caused by ICH. Our findings show that the mechanism regulated by moderate ethanol pre-conditioning might be beneficial for ICH, indicating the importance of ER homeostasis, oxidative stress, and differential cytokines release in ICH.

Intracerebral hemorrhage (ICH) is a life-threatening type of stroke that disrupts the normal neurological function of the brain. Clinical studies have reported a non-linear J-shaped association between alcohol consumption levels and the occurrence of cerebral stroke. Specifically, alcohol intoxication increases stroke incidence, while moderate alcohol pre-conditioning decreases stroke frequency and improves outcomes. Although alcohol pre-consumption is likely a crucial player in ICH, the underlying mechanism remains unclear. We performed 1-h alcohol pre-conditioning followed by ICH induction in Sprague-Dawley (SD) rats to investigate the role of alcohol pre-conditioning in ICH. Interestingly, behavioral test analysis found that ethanol intoxication (3 g/kg) aggravated ICH-induced neurological deficits, but moderate ethanol pre-conditioning (0.75 g/kg) ameliorated ICH-induced neurological deficits by reducing the oxidative stress and proinflammatory cytokines release. Moreover, we found that moderate ethanol pretreatment improved the striatal endoplasmic reticulum (ER) homeostasis by increasing the chaperone protein expression and reducing oxidative stress and apoptosis caused by ICH. Our findings show that the mechanism regulated by moderate ethanol pre-conditioning might be beneficial for ICH, indicating the importance of ER homeostasis, oxidative stress, and differential cytokines release in ICH. [ABSTRACT FROM AUTHOR]

Misfolded proteins have enhanced formation of toxic oligomers and nonfunctional protein copies lead to recruiting wild‐type protein types. Heat shock protein 90 (HSP90) is a molecular chaperone generated by cells that are involved in many cellular functions through regulation of folding and/or localization of large multi‐protein complexes as well as client proteins. HSP90 can regulate a number of different cellular processes including cell proliferation, motility, angiogenesis, signal transduction, and adaptation to stress. HSP90 makes the mutated oncoproteins able to avoid misfolding and degradation and permits the malignant transformation. As a result, HSP90 is an important factor in several signaling pathways associated with tumorigenicity, therapy resistance, and inhibiting apoptosis. Clinically, the upregulation of HSP90 expression in hepatocellular carcinoma (HCC) is linked with advanced stages and inappropriate survival in cases suffering from this kind of cancer. The present review comprehensively assesses HSP90 functions and its possible usefulness as a potential diagnostic biomarker and therapeutic option for HCC. [ABSTRACT FROM AUTHOR]

Adiponectin, an insulin-sensitizing factor secreted from adipose tissue, is decreased in individuals with type 2 diabetes (T2D) and increased in response to thiazolidinedione (TZD) therapy. Changes in its secretion and assembly into higher-order forms affect insulin sensitivity. To determine the relative potency of TZDs on intra-adipocyte multimerization and secretion of adiponectin, we assessed the impact of in vivo low- or high-dose rosiglitazone treatment alone or combined with metformin in subjects with T2D. T2D subjects received high-dose rosiglitazone (8 mg/day), high-dose metformin (2,000 mg/day), or low-dose combination rosiglitazone-metformin therapy (4 mg + 1,000 mg/day) for 4 mo. All subjects were then switched to high-dose rosiglitazone-metformin combination therapy (8 mg + 2,000 mg/day) for another 4 mo. Low-dose rosiglitazone increased serum adiponectin, whereas the high dose increased both adipocyte content and serum adiponectin levels. TZDs selectively increased the percentage of circulating adiponectin in the potent, high-molecular-weight (HMW) form. No TZD effects were evident on multimer distribution in the cell. Expression of the chaperone protein ERp44, which retains adiponectin within the cell, was decreased by TZD treatment. No changes occurred in Ero1-Lalpha expression. Metformin had no effect on any of these measures. Increases in adiponectin correlated with improvements in insulin sensitivity. In vivo, TZDs have apparent dose-dependent effects on cellular and secreted adiponectin. TZD-mediated improvements in whole body insulin sensitivity are associated with increases in circulating but not cellular levels of the HMW adiponectin multimer. Finally, TZDs promote the selective secretion of HMW adiponectin, potentially, in part, through decreasing the expression of the adiponectin-retaining protein ERp44.

The Hsp70 chaperone protein system is an essential component of the protein folding and homeostasis machinery in E.Coli. Hsp70 is a three domain, 70 kDa protein which functions as an allosteric system cycling between an ADP-bound state where the three domains are loosely coupled via a flexible interdomain linker and an ATP-bound state where they are tightly coupled into a single entity. The structure-function model of this protein proposes an allosteric connection between the 45 kDa Nucleotide Binding Domain (NBD) and the 25 kDa Substrate Binding Domain (SBD) and Lid Domain which operates through the inter NBD-SBD linker. X-Ray crystallography and NMR spectroscopy have provided structures of the end states of the functional cycle of this protein, bound to ADP and ATP. We have used MD simulations to study the transitions between these end states and allosteric communication in this system. Our results largely validate the experimentally derived allosteric model of function, but shed additional light on the flow of allosteric information in the SBD + Lid. Specifically, we find that the Lid domain has a double-hinged structure with the potential for greater conformational flexibility than was hitherto expected.Communicated by Ramaswamy H. Sarma.

Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon (RTN) and cytoskeleton-linking membrane protein 63 (CLIMP-63), respectively. Here, stimulated emission depletion (STED) super-resolution microscopy shows that reticulon4a (RTN4a) and CLIMP-63 also regulate the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP), membrane Sec61βGFP, knock-in calreticulin-GFP, and antibody-labeled ER-resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. RTN4a segregates away from and restricts lumenal blob length, while CLIMP-63 associates with and increases lumenal blob length. RTN4a and CLIMP-63 also regulate the nanodomain distribution of ER-resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that RTN4a and CLIMP-63 regulate dynamic nanoscale lumenal compartmentalization along peripheral ER tubules. RTN4a enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER-shaping proteins RTN and CLIMP-63 therefore regulate lumenal ER nanodomain heterogeneity, interaction with ER-resident proteins, and dynamics in peripheral ER tubules. [ABSTRACT FROM AUTHOR]

Autoantigens are the molecular targets in autoimmune diseases. They are a cohort of seemingly unrelated self-molecules present in different parts of the body, yet they can trigger a similar chain of autoimmune responses such as autoantibody production. We previously reported that dermatan sulfate (DS) can bind self-molecules of dying cells to stimulate autoreactive CD5+ B cells to produce autoantibodies. The formation of autoantigen-DS complexes converts the normally non-antigenic self-molecules to none-self antigens, and thus DS-affinity represents a common underlying biochemical property for autoantigens. This study sought to apply this property to identify potential autoantigens in the kidney. Total proteins were extracted from mouse kidney tissues and loaded onto DS-Sepharose resins. Proteins without affinity were washed off the resins, whereas those with increasing DS-affinity were eluted with step gradients of increasing salt strength. Fractions with strong and moderate DS-affinity were sequenced by mass spectrometry and yielded 25 and 99 proteins, respectively. An extensive literature search was conducted to validate whether these had been previously reported as autoantigens. Of the 124 proteins, 79 were reported autoantigens, and 19 out of 25 of the strong-DS-binding ones were well-known autoantigens. Moreover, these proteins largely fell into the two most common autoantibody categories in autoimmune kidney diseases, including 40 ANA (anti-nuclear autoantibodies) and 25 GBM (glomerular basement membrane) autoantigens. In summary, this study compiles a large repertoire of potential autoantigens for autoimmune kidney diseases. This autoantigen-ome sheds light on the molecular etiology of autoimmunity and further supports our hypothesis DS-autoantigen complexes as a unifying principle of autoantigenicity. [ABSTRACT FROM AUTHOR]

The symbiosis between rhizobia and legumes is characterized by a complex molecular dialogue in which the bacterial NodD protein plays a major role due to its capacity to activate the expression of the nodulation genes in the presence of appropiate flavonoids. These genes are involved in the synthesis of molecules, the nodulation factors (NF), responsible for launching the nodulation process. Rhizobium tropici CIAT 899, a rhizobial strain that nodulates Phaseolus vulgaris, is characterized by its tolerance to multiple environmental stresses such as high temperatures, acidity or elevated osmolarity. This strain produces nodulation factors under saline stress and the same set of CIAT 899 nodulation genes activated by inducing flavonoids are also up-regulated in a process controlled by the NodD2 protein. In this paper, we have studied the effect of osmotic stress (high mannitol concentrations) on the R. tropici CIAT 899 transcriptomic response. In the same manner as with saline stress, the osmotic stress mediated NF production and export was controlled directly by NodD2. In contrast to previous reports, the nodA2FE operon and the nodA3 and nodD1 genes were up-regulated with mannitol, which correlated with an increase in the production of biologically active NF. Interestingly, in these conditions, this regulatory protein controlled not only the expression of nodulation genes but also the expression of other genes involved in protein folding and synthesis, motility, synthesis of polysaccharides and, surprinsingly, nitrogen fixation. Moreover, the non-metabolizable sugar dulcitol was also able to induce the NF production and the activation of nod genes in CIAT 899. [ABSTRACT FROM AUTHOR]

Ulcers due to infections with Mycobacterium ulcerans are characterized by complete lack of wound healing processes, painless, an underlying bed of host dead cells and undermined edges due to necrosis. Mycolactone, a macrolide produced by the mycobacterium, is believed to be the toxin responsible. Of interest and relevance is the knowledge that Buruli ulcer (BU) patients remember experiencing trauma previously at the site of the ulcers, suggesting an impairment of wound healing processes, the plausible effect due to the toxin. Wound healing processes involve activation of the blood platelets to release the contents of the dense granules mainly serotonin, calcium ions, and ADP/ATP by exocytosis into the bloodstream. The serotonin release results in attracting more platelets and mast cells to the wound site, with the mast cells also undergoing degranulation, releasing compounds into the bloodstream by exocytosis. Recent work has identified interference in the co-translational translocation of many secreted proteins via the endoplasmic reticulum and cell death involving Wiskott-Aldrich syndrome protein (WASP), Sec61, and angiotensin II receptors (AT2R). We hypothesized that mycolactone by being lipophilic, passively crosses cell membranes and binds to key proteins that are involved in exocytosis by platelets and mast cells, thus inhibiting the initiation of wound healing processes. Based on this, molecular docking studies were performed with mycolactone against key soluble n-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and regulators, namely Vesicle-associated membrane protein (VAMP8), Synaptosomal-associated protein (SNAP23, syntaxin 11, Munc13-4 (its isoform Munc13-1 was used), and Munc18b; and also against known mycolactone targets (Sec61, AT2R, and WASP). Munc18b was shown to be a plausible mycolactone target after the molecular docking studies with binding affinity of -8.5 kcal/mol. Structural studies and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding energy calculations of the mycolactone and Munc18b complex was done with 100 ns molecular dynamics simulations using GROMACS. Mycolactone binds strongly to Munc18b with an average binding energy of -247.571 ± 37.471 kJ/mol, and its presence elicits changes in the structural conformation of the protein. Analysis of the binding interactions also shows that mycolactone interacts with Arg405, which is an important residue of Munc18b, whose mutation could result in impaired granule exocytosis. These findings consolidate the possibility that Munc18b could be a target of mycolactone. The implication of the interaction can be experimentally evaluated to further understand its role in granule exocytosis impairment in Buruli ulcer. [ABSTRACT FROM AUTHOR]

MDA5 belongs to the RIG-I-like receptor family and plays a non-redundant role in recognizing cytoplasmic viral RNA to induce the production of type I IFNs. Upon RNA ligand stimulation, we observed the redistribution of MDA5 from the cytosol to mitochondrial membrane fractions. However, the molecular mechanisms of MDA5 activation remain less understood. Here we show that 14-3-3η is an essential accessory protein for MDA5-dependent type I IFN induction. We found that several 14-3-3 isoforms may interact with MDA5 through the CARDs (N-MDA5), but 14-3-3η was the only isoform that could enhance MDA5-dependent IFNβ promoter activities in a dose-dependent manner. Knock-down of 14-3-3η in Huh7 cells impaired and delayed the kinetics of MDA5 oligomerization, which is a critical step for MDA5 activation. Consequently, the MDA5-dependent IFNβ promoter activities as well as IFNβ mRNA expression level were also decreased in the 14-3-3η knocked-down cells. We also demonstrated that 14-3-3η is essential in boosting the activation of MDA5-dependent antiviral innate immunity during viral infections. In conclusion, our results uncover a novel function of 14-3-3η to promote the MDA5-dependent IFNβ induction pathway by reducing the immunostimulatory potential of viral dsRNA within MDA5 activation signaling pathway. [ABSTRACT FROM AUTHOR]

Loss of sacsin, a large 520 kDa multidomain protein, causes autosomal recessive spastic ataxia of the Charlevoix-Saguenay, one of the most common childhood-onset recessive ataxias. A prominent feature is abnormal bundling of neurofilaments in many neuronal populations. This study shows the direct involvement of sacsin domains in regulating intermediate filament assembly and dynamics and identifies important domains for alleviating neurofilament bundles in neurons lacking sacsin. Peptides encoding sacsin internal repeat (SIRPT) 1, J-domains, and ubiquitin-like domain modified neurofilament assembly in vivo. The domains with chaperone homology, the SIRPT and the J-domain, had opposite effects, promoting and preventing filament assembly, respectively. In cultured Sacs-/- motor neurons, both the SIRPT1 and J-domain resolved preexisting neurofilament bundles. Increasing expression of heat shock proteins also resolved neurofilament bundles, indicating that this endogenous chaperone system can compensate to some extent for sacsin deficiency. [ABSTRACT FROM AUTHOR]

Triacylglycerol synthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase-2 (DGAT2). DGAT2 is an integral membrane protein that is localized to the endoplasmic reticulum and interacts with lipid droplets. Using BioId, a method to detect proximal and interacting proteins, we identified calnexin as a DGAT2-interacting protein. Co-immunoprecipitation and proximity ligation assays confirmed this finding. We found that calnexin-deficient mouse embryonic fibroblasts had reduced intracellular triacylglycerol levels and fewer large lipid droplets (>1.0 μm2 area). Despite the alterations in triacylglycerol metabolism, in vitro DGAT2 activity, localization and protein stability were not affected by the absence of calnexin. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To promote a comprehensive insight into the molecular basis of EHEC O157:H7 physiology and pathogenesis, the combined proteome of EHEC O157:H7 strains, Clade 8 and Clade 6 isolated from cattle in Argentina, and the standard EDL933 (clade 3) strain has been analyzed. From shotgun proteomic analysis a total of 2,644 non-redundant proteins of EHEC O157:H7 were identified, which correspond approximately 47% of the predicted proteome of this pathogen. Normalized spectrum abundance factor analysis was performed to estimate the protein abundance. According this analysis, 50 proteins were detected as the most abundant of EHEC O157:H7 proteome. COG analysis showed that the majority of the most abundant proteins are associated with translation processes. A KEGG enrichment analysis revealed that Glycolysis / Gluconeogenesis was the most significant pathway. On the other hand, the less abundant detected proteins are those related to DNA processes, cell respiration and prophage. Among the proteins that composed the Type III Secretion System, the most abundant protein was EspA. Altogether, the results show a subset of important proteins that contribute to physiology and pathogenicity of EHEC O157:H7. [ABSTRACT FROM AUTHOR]

The marine bacterium Photobacterium damselae subsp. damselae (Pdd) is a generalist and facultative pathogen that causes disease in a wide range of marine animals including fish species of importance in aquaculture. Disease outbreaks in fish farms have been correlated with an increased water temperature during summer months. In this study, we have used RNA sequencing to analyze the transcriptome of Pdd RM-71 cultured at two different temperatures, which simulated temperature conditions experienced during free swimming lifestyle at mid latitudes in winter months (15°C) and during outbreaks in aquaculture in warm summer months (25°C). The enhanced bacterial growth of Pdd observed at 25°C in comparison to 15°C suggests that an elevated seawater temperature contributes to the build-up of a sufficient bacterial population to cause disease. In comparison to growth at 15°C, growth at 25°C resulted in the upregulation of genes involved in DNA synthesis, nutrient uptake, chemotaxis, flagellar motility, secretion systems and antimicrobial resistance. Plasmid-encoded virulence factors, which include a putative adhesin/invasin OmpU, a transferrin receptor and a serum resistance protein, were also upregulated. Transcription factor RpoS, genes involved in cold shock response, modulation of cell envelope and amino acid metabolism, as well as genes of yet unknown function were downregulated at 25°C. Notably, the gene encoding damselysin cytotoxin (Dly) was among the most highly transcribed genes at the two assayed temperatures, at levels comparable to the most highly expressed housekeeping genes. This study contributes to our understanding of the regulatory networks and biology of a generalist marine bacterial pathogen, and provides evidence that temperature regulates multiple physiological and virulence-related functions in Pdd. [ABSTRACT FROM AUTHOR]

Abstract Chaperone proteins are required to maintain the overall fold and function of proteins in the cell. As part of the Hsp70 family, Ssa1 acts to maintain cellular proteostasis through a variety of diverse pathways aimed to preserve the native conformation of target proteins, thereby preventing aggregation and future states of cellular toxicity. Studying the structural dynamics of Ssa1 in vitro is essential to determining their precise mechanisms and requires the development of purification methods that result in highly pure chaperones. Current methods of expressing and purifying Ssa1 utilize affinity tagged constructs expressed in Escherichia coli , however, expression in an exogenous source produces proteins that lack post-translational modifications leading to undesired structural and functional effects. Current protocols to purify Ssa1 from Saccharomyces cerevisiae require large amounts of starting material, multiple steps of chromatography, and result in low yield. Our objective was to establish a small-scale purification of Ssa1 expressed from its endogenous source, Saccharomyces cerevisiae , with significant yield and purity. We utilized a protein A affinity tag that was previously used to purify large protein complexes from yeast, combined with magnetic Dynabeads that are conjugated with rabbit immunoglobulin G (IgG). Our results show that we can produce native, highly pure, active Ssa1 via this one-step purification with minimal amounts of starting material, and this Ssa1-protein A fusion does not alter cellular phenotypes. This methodology is a significant improvement in Ssa1 purification and will facilitate future experiments that will elucidate the biochemical and biophysical properties of Hsp70 chaperones. Highlights • One-step purification increases simplicity and efficiency of purification. • Highly pure, native protein containing post-translational modifications. • Native Ssa1 chaperone protein from its endogenous source that can be used for downstream assays. [ABSTRACT FROM AUTHOR]

Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness.

A variety of mammals employ torpor as an energy-saving strategy in environments of marginal or severe stress either on a daily basis during their inactive period or on a seasonal basis during prolonged multi-day hibernation. Recently, a few Madagascar lemur species have been identified as the only primates that exhibit torpor; one of these is the gray mouse lemur (Microcebus murinus). To explore the regulatory mechanisms that underlie daily torpor in a primate, we analyzed the expression of 28 selected genes that represent crucial survival pathways known to be involved in squirrel and bat hibernation. Array-based real-time PCR was used to compare gene expression in control (aroused) versus torpid lemurs in five tissues including the liver, kidney, skeletal muscle, heart, and brown adipose tissue. Significant differences in gene expression during torpor were revealed among genes involved in glycolysis, fatty acid metabolism, antioxidant defense, apoptosis, hypoxia signaling, and protein protection. The results showed upregulation of select genes primarily in liver and brown adipose tissue. For instance, both tissues showed elevated gene expression of peroxisome proliferator activated receptor gamma (ppargc), ferritin (fth1), and protein chaperones during torpor. Overall, the data show that the expression of only a few genes changed during lemur daily torpor, as compared with the broader expression changes reported for hibernation in ground squirrels. These results provide an indication that the alterations in gene expression required for torpor in lemurs are not as extensive as those needed for winter hibernation in squirrel models. However, identification of crucial genes with altered expression that support lemur torpor provides key targets to be explored and manipulated toward a goal of translational applications of inducible torpor as a treatment option in human biomedicine.

Prolonged heat stress is one of the harsh conditions Lactobacillus casei strains encounter as non-starter lactic acid bacteria in dairy product manufacture. To understand the physiological and molecular mechanisms through which Lb. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (log2-fold change of 7) at 45°C. We identified a novel SecB homolog as a protein export chaperone putatively involved in posttranslational translocation systems, which was down-regulated as growth temperature increased and where the modelled 3D-structure shared architectural similarities with the Escherichia coli SecB protein. Membrane lipid analyses revealed temporal changes in fatty acid composition, cyclization of oleic acid to cyclopropane and novel cyclopentenyl moieties, and reduced synthesis of vaccenic acid, at higher temperatures. An 18kDa α-crystallin domain, Hsp20 family heat shock protein was more highly up-regulated in response to heat stress compared to other molecular chaperones, suggesting this protein could be a useful biomarker of prolonged heat stress in Lb. casei GCRL163. [ABSTRACT FROM AUTHOR]

The mealybug, Phenacoccus manihoti, is a leading pest of cassava (Manihot esculenta Crantz), damaging this crop globally. Although the biological control of this mealybug using natural predators has been established, resistance breeding remains an important means of control. Understanding plant responses to insect herbivory, by determining and identifying differentially expressed genes (DEGs), is a vital step towards the understanding of molecular mechanisms of defence responses in plants and the development of resistant cultivars by gene editing. Morphological and molecular analysis confirmed the mealybug identity as Phenacoccus manihoti (Matile-Ferrero). The transcriptome response of the green mite resistant cassava genotype AR23.1 was compared to P40/1 with no known resistance at 24 and 72 hours of mealybug infestation compared to non-infested mock. A total of 301 and 206 genes were differentially expressed at 24 and 72 of mealybug infestation for AR23.1 and P40/1 genotypes respectively, using a log2 fold change and P-value ≤ 0.05. Gene ontology functional classification revealed an enrichment of genes in the secondary metabolic process category in AR23.1 in comparison with P40/1, while genes in the regulation of molecular function, cellular component biogenesis and electron carrier categories were more significantly enriched in P40/1 than in AR23.1. Biological pathway analysis, based on KEGG, revealed a significant enrichment of plant-pathogen interaction and plant hormonal signal transduction pathways for a cohort of up-regulated and down-regulated DEGs in both genotypes. Defence-related genes such as 2-oxogluterate, gibberellin oxidase and terpene synthase proteins were only induced in genotype AR23.1 and not in P40/1, and subsequently validated by RT-qPCR. The study revealed a difference in response to mealybug infestation in the two genotypes studied, with AR23.1 showing a higher number of differentially expressed transcripts post mealybug infestation at 24 and 72 hours. Candidate defence-related genes that were overexpressed in the AR23.1 genotype post mealybug infestation will be useful in future functional studies towards the control of mealybugs. [ABSTRACT FROM AUTHOR]

Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios. [ABSTRACT FROM AUTHOR]

The “Bioénergétique et Ingénierie des Protéines (BIP)” laboratory, CNRS (France), organized its first French workshop on molecular chaperone proteins and protein folding in November 2017. The goal of this workshop was to gather scientists working in France on chaperone proteins and protein folding. This initiative was a great success with excellent talks and fruitful discussions. The highlights were on the description of unexpected functions and post-translational regulation of known molecular chaperones (such as Hsp90, Hsp33, SecB, GroEL) and on state-of-the-art methods to tackle questions related to this theme, including Cryo-electron microscopy, Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR), simulation and modeling. We expect to organize a second workshop in two years that will include more scientists working in France in the chaperone field. [ABSTRACT FROM AUTHOR]

Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the Chlamydiaceae, in the absence of which most bacteria failed to establish a successful infection. CT622 is abundant in the infectious form of the bacteria, in which it associates with CT635, a putative novel chaperone protein. We show that CT622 is translocated into the host cytoplasm via type three secretion throughout the developmental cycle of the bacteria. Two separate domains of roughly equal size have been identified within CT622 and a 1.9 Å crystal structure of the C-terminal domain has been determined. Genetic disruption of ct622 expression resulted in a strong bacterial growth defect, which was due to deficiencies in proliferation and in the generation of infectious bacteria. Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. They reveal that genetic disruption of a single effector can deeply affect bacterial fitness. [ABSTRACT FROM AUTHOR]

The Tasmanian devil (Sarcophilus harrisii), the largest extant carnivorous marsupial and endemic to Tasmania, is at the verge of extinction due to the emergence of a transmissible cancer known as devil facial tumour disease (DFTD). DFTD has spread over the distribution range of the species and has been responsible for a severe decline in the global devil population. To protect the Tasmanian devil from extinction in the wild, our group has focused on the development of a prophylactic vaccine. Although this work has shown that vaccine preparations using whole DFTD tumour cells supplemented with adjuvants can induce anti-DFTD immune responses, alternative strategies that induce stronger and more specific immune responses are required. In humans, heat shock proteins (HSPs) derived from tumour cells have been used instead of whole-tumour cell preparations as a source of antigens for cancer immunotherapy. As HSPs have not been studied in the Tasmanian devil, this study presents the first characterisation of HSPs in this marsupial and evaluates the suitability of these proteins as antigenic components for the enhancement of a DFTD vaccine. We show that tissues and cancer cells from the Tasmanian devil express constitutive and inducible HSP. Additionally, this study suggests that HSP derived from DFTD cancer cells are immunogenic supporting the future development of a HSP-based vaccine against DFTD. [ABSTRACT FROM AUTHOR]

Background: Maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) causes an economically important disease in maize and other gramineous crops in Iran. MIMV negative-sense RNA genome sequence of 12,426 nucleotides has recently been completed. Maize Genetics and Genomics database shows that 39,498 coding genes and 4,976 non-coding genes of maize have been determined, but still some transcripts could not be annotated. The molecular host cell responses of maize to MIMV infection including differential gene expression have so far not been elucidated. Methodology/Principal findings: Complementary DNA libraries were prepared from total RNA of MIMV-infected and mock-inoculated maize leaves and sequenced using Illumina HiSeq 2500. Cleaned raw transcript reads from MIMV-infected maize were mapped to reads from uninfected maize and to a maize reference genome. Differentially expressed transcripts were characterized by gene ontology and biochemical pathway analyses. Transcriptome data for selected genes were validated by real-time quantitative PCR. Conclusion/Significance: Approximately 42 million clean reads for each treatment were obtained. In MIMV-infected maize compared to uninfected plants, 1689 transcripts were up-regulated and 213 transcripts were down-regulated. In response to MIMV infection, several pathways were activated in maize including immune receptor signaling, metabolic pathways, RNA silencing, hormone-mediated pathways, protein degradation, protein kinase and ATP binding activity, and fatty acid metabolism. Also, several transcripts including those encoding hydrophobic protein RCI2B, adenosylmethionine decarboxylase NAC transcription factor and nucleic acid binding, leucine-rich repeat, heat shock protein, 26S proteasome, oxidoreductases and endonuclease activity protein were up-regulated. These data will contribute to the identification of genes and pathways involved in plant-virus interactions that may serve as future targets for improved disease control. [ABSTRACT FROM AUTHOR]

Objective. Although generally classified within the group of inflammatory myopathies, JDM displays many pathological features of vasculitis. Previous work has shown that AECA are abundant in other forms of vasculitis. We therefore investigated whether such antibodies might also be detected in JDM. Methods. We screened plasma from children with JDM for the presence of AECA by western blotting and 2D gel electrophoresis (2DE) using proteins extracted from human aortic endothelial cells as the substrate. We performed mass spectrometry to identify candidate antigens from 2DE gels and used ELISA to confirm the presence of specific antibodies. Results. We identified 22 candidate target autoantigens for AECA probed with JDM plasma. Interestingly, 17 of these 22 target antigens were proteins associated with antigen processing and protein trafficking. ELISA confirmed the presence of antibodies to heat shock cognate 71 kDa protein in JDM plasma, par- ticularly in children with active, untreated disease. Conclusion. Children with JDM express antibodies to autoantigens in endothelial cells. The clinical and pathological significance of such autoantibodies require further investigation. [ABSTRACT FROM AUTHOR]

Edwardsiella ictaluri is a Gram-negative facultative anaerobic rod and the causative agent of enteric septicemia of channel catfish (ESC), which is one of the most prevalent diseases of catfish, causing significant economic losses in the catfish industry. E. ictaluri is resistant to complement system and macrophage killing, which results in rapid systemic septicemia. However, mechanisms of E. ictaluri stress responses under conditions of host environment are not studied well. Therefore, in this work, we report E. ictaluri stress responses during hydrogen peroxide, low pH, and catfish serum stresses as well as during catfish invasion. E. ictaluri stress responses were characterized by identifying expression of 13 universal stress protein (USP) genes (usp01-usp13) and seven USP-interacting protein genes (groEL, groES, dnaK, grpE, and clpB, grpE, relA). Data indicated that three usp genes (usp05, usp07, and usp13) were highly expressed in all stress conditions. Similarly, E. ictaluri heat shock proteins groEL, groES, dnaK, grpE, and clpB were highly expressed in oxidative stress. Also, E. ictaluri grpE and relA were highly expressed in catfish spleen and head kidney. These findings contribute to our understanding of stress response mechanisms in E. ictaluri stress response, and stress-related proteins that are essential for E. ictaluri could be potential targets for live attenuated vaccine development against ESC. [ABSTRACT FROM AUTHOR]

Werner syndrome (WS) is a premature aging disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domain. Mice lacking the helicase domain of the Wrn protein orthologue exhibit transcriptomic and metabolic alterations, some of which are reversed by vitamin C. Recent studies on these animals indicated that the mutant protein is associated with enriched endoplasmic reticulum (ER) fractions of tissues resulting in an ER stress response. In this study, we identified proteins that exhibit actual level differences in the ER enriched fraction between the liver of wild type and Wrn mutant mice using quantitative proteomic profiling with label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Multiple Reaction Monitoring (MRM) and immunoblotting were performed to validate findings in a secondary independent cohort of wild type and Wrn mutant mice. DAVID 6.7 (NIH) was used for functional annotation analysis and indicated that the identified proteins exhibiting level changes between untreated wild type, Wrn mutant, and vitamin C treated Wrn mutant mice (ANOVA P–value < 0.05) were involved in fatty acid and steroid metabolism pathways (Bonferroni P-value = 0.0137). Finally, when we compared the transcriptomic and the proteomic data of our mouse cohorts only ~7% of the altered mRNA profiles encoding for ER gene products were consistent with their corresponding protein profiles measured by the label-free quantification methods. These results suggest that a great number of ER gene products are regulated at the post-transcriptional level in the liver of Wrn mutant mice exhibiting an ER stress response. [ABSTRACT FROM AUTHOR]

The GroE chaperonin system, which comprises GroEL and GroES, assists protein folding in vivo and in vitro. It is conserved in all prokaryotes except in most, but not all, members of the class of mollicutes. In Escherichia coli, about 60 proteins were found to be obligatory clients of the GroE system. Here, we describe the properties of the homologs of these GroE clients in mollicutes and the evolution of chaperonins in this class of bacteria. Comparing the properties of these homologs in mollicutes with and without chaperonins enabled us to search for features correlated with the presence of GroE. Interestingly, no sequence-based features of proteins such as average length, amino acid composition and predicted folding/disorder propensity were found to be affected by the absence of GroE. Other properties such as genome size and number of proteins were also found to not differ between mollicute species with and without GroE. Our data suggest that two clades of mollicutes re-acquired the GroE system, thereby supporting the view that gaining the system occurred polyphyletically and not monophyletically, as previously debated. Our data also suggest that there might have been three isolated cases of lateral gene transfer from specific bacterial sources. Taken together, our data indicate that loss of GroE does not involve crossing a high evolutionary barrier and can be compensated for by a small number of changes within the few dozen client proteins. [ABSTRACT FROM AUTHOR]

Abstract: Approaching protein structural dynamics and protein–protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site‐directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide‐based spin labels for in‐cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido‐proxyl‐based spin label (M‐TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M‐TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells. [ABSTRACT FROM AUTHOR]

The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington’s disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in sse1Δ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance. [ABSTRACT FROM AUTHOR]