Disaggregases, molecular chaperones that resolubilize protein aggregates

The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of protein aggregates, an insoluble association of polypeptides that harm cell physiology, either by themselves or in the process of formation. Several biological processes have evolved to prevent and eliminate the existence of non-functional and amyloidogenic aggregates, as they are associated with several human pathologies. Molecular chaperones and heat shock proteins are specialized in controlling the quality of the proteins in the cell, specifically by aiding proper folding, and dissolution and clearance of already formed protein aggregates. The latter is a function of disaggregases, mainly represented by the ClpB/Hsp104 subfamily of molecular chaperones, that are ubiquitous in all organisms but, surprisingly, have no orthologs in the cytosol of metazoan cells. This review aims to describe the characteristics of disaggregases and to discuss the function of yeast Hsp104, a disaggregase that is also involved in prion propagation and inheritance.The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of pro872012731292FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORSEM INFORMAÇÃOSEM INFORMAÇÃOSEM INFORMAÇÃOO processo de enovelamento é um evento importante para o tempo de vida de uma proteína, uma vez que é essencial para a função adequada da proteína e, por conseguinte, para a fisiologia celular. Enovelamento inadequado, ou mau enovelamento, pode não só conWe thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support and fellowship

Identification of Regions Involved in Substrate Binding and Dimer Stabilization within the Central Domains of Yeast Hsp40 Sis1

Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I) deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1) Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2) The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3) The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4) Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Ministerio da Ciencia e Tecnologia/Conselho Nacional de Pesquisa e Desenvolvimento (MCT/CNPq

Vitamin D levels and susceptibility to asthma, elevated immunoglobulin E levels, and atopic dermatitis: A Mendelian randomization study.

BACKGROUND: Low circulating vitamin D levels have been associated with risk of asthma, atopic dermatitis, and elevated total immunoglobulin E (IgE). These epidemiological associations, if true, would have public health importance, since vitamin D insufficiency is common and correctable. METHODS AND FINDINGS: We aimed to test whether genetically lowered vitamin D levels were associated with risk of asthma, atopic dermatitis, or elevated serum IgE levels, using Mendelian randomization (MR) methodology to control bias owing to confounding and reverse causation. The study employed data from the UK Biobank resource and from the SUNLIGHT, GABRIEL and EAGLE eczema consortia. Using four single-nucleotide polymorphisms (SNPs) strongly associated with 25-hydroxyvitamin D (25OHD) levels in 33,996 individuals, we conducted MR studies to estimate the effect of lowered 25OHD on the risk of asthma (n = 146,761), childhood onset asthma (n = 15,008), atopic dermatitis (n = 40,835), and elevated IgE level (n = 12,853) and tested MR assumptions in sensitivity analyses. None of the four 25OHD-lowering alleles were associated with asthma, atopic dermatitis, or elevated IgE levels (p ≥ 0.2). The MR odds ratio per standard deviation decrease in log-transformed 25OHD was 1.03 (95% confidence interval [CI] 0.90-1.19, p = 0.63) for asthma, 0.95 (95% CI 0.69-1.31, p = 0.76) for childhood-onset asthma, and 1.12 (95% CI 0.92-1.37, p = 0.27) for atopic dermatitis, and the effect size on log-transformed IgE levels was -0.40 (95% CI -1.65 to 0.85, p = 0.54). These results persisted in sensitivity analyses assessing population stratification and pleiotropy and vitamin D synthesis and metabolism pathways. The main limitations of this study are that the findings do not exclude an association between the studied outcomes and 1,25-dihydoxyvitamin D, the active form of vitamin D, the study was underpowered to detect effects smaller than an OR of 1.33 for childhood asthma, and the analyses were restricted to white populations of European ancestry. This research has been conducted using the UK Biobank Resource and data from the SUNLIGHT, GABRIEL and EAGLE Eczema consortia. CONCLUSIONS: In this study, we found no evidence that genetically determined reduction in 25OHD levels conferred an increased risk of asthma, atopic dermatitis, or elevated total serum IgE, suggesting that efforts to increase vitamin D are unlikely to reduce risks of atopic disease

An overview of tissue engineering approaches for management of spinal cord injuries

Severe spinal cord injury (SCI) leads to devastating neurological deficits and disabilities, which necessitates spending a great deal of health budget for psychological and healthcare problems of these patients and their relatives. This justifies the cost of research into the new modalities for treatment of spinal cord injuries, even in developing countries. Apart from surgical management and nerve grafting, several other approaches have been adopted for management of this condition including pharmacologic and gene therapy, cell therapy, and use of different cell-free or cell-seeded bioscaffolds. In current paper, the recent developments for therapeutic delivery of stem and non-stem cells to the site of injury, and application of cell-free and cell-seeded natural and synthetic scaffolds have been reviewed

CpG binding protein (CFP1) occupies open chromatin regions of active genes, including enhancers and non-CpG islands

Additional file 1. Fig. S1: Analysis of CFP1 binding at individual loci and CpG islands (CGIs). (A-B) Analysis of CFP1 binding at the human α-globin locus in expressing and non-expressing cells. (A) Real-Time PCR analysis of immunoprecipitated chromatin using CFP1 antibody in human erythroblasts (red) and B-lymphocytes (blue). The y-axis represents enrichment over the input DNA, normalised to a control sequence in the human 18S gene. The x-axis represents the positions of Taqman probes used. The coding sequence is represented by the three exons (Promoter/Ex1, Ex2, Ex3) of the α-globin genes. 218 and hBact denote control sequences adjacent to the CpG islands of the human LUC7L (218) and ACTB promoters. Error bars correspond to ± 1 SD from at least two independent ChIPs. (B) Real-Time PCR analysis of immunoprecipitated chromatin using the CFP1 antibody indicated in humanised erythroblasts (normal, +MCS-R2 (left) and mutant, MCS-R2 (right). The y-axis represents enrichment over the input DNA, normalised to a control sequence in the mouse GAPDH gene. CpG Act denotes additional control sequence at the CGI of the mouse ACTB gene. The amplicons highlighted in red represent deleted regions in the humanised mice, for which no PCR signal is observed. Error bars correspond to ± 1 SD from at least two independent ChIPs. (C) CFP1 ChIP signal intensity in the top 200 peaks, by antibody and by cell type. Abcam, ab56035 antibody. Roeder, main antibody used in this study. (D) Analysis of CGI (green) and non-CGI (blue) transcription start sites (1-kb window, centred on TSS). Gene symbols shown with CpG content of individual loci in parentheses. Greek letters represent individual globin genes. Fig. S2: Peak overlaps of CFP1 and marks of active and repressed chromatin in transcription start sites (TSSs). Peaks were detected by MACS2. Venn diagrams show that CFP1 peaks within 1-kb of TSSs are strongly associated with H3K4me3 histone mark and poorly associated with H3K27me3 repressive histone mark. Cell types are (A) ERY and (B) EBV. Public data sets: * NCBI GEO GSE36985, ** NCBI GEO GSE50893. Fig. S3: UCSC tracks showing CFP1 and other ChIP signals in gene loci in erythroblasts (ERY) and EBV-transformed B-lymphoblasts (EBV). Hg38 coordinates for multiple genes, CpG islands (CGI, green boxes), and putative regulatory regions (blue boxes) are shown. CFP1 signals are shown in dark reds, inputs in grey, histone H3 signals in blues and open chromatin marks in greens. All ChIP pileups are scaled to 1x coverage genome-wide and shown in a range 0–50, except CFP1 (Roeder) is shown with extended range and H3K27me3 graphs scaled by 2x. (A) Tissue-specific binding of CFP1 to CGI promoters of tissue-specifically expressed genes. Left (chr16), CGI promoters of active genes in alpha globin locus are CFP1-bound in ERY, and unbound in EBV. Flanking regions are included, with known tissue-specific enhancers. Right (chr6), first seven exons of IRF4 locus, active in EBV and inactive in ERY, with CFP1 binding to CGI promoter in EBV only. (B) CGI promoters of housekeeping genes are CFP1 bound and unmarked by H3K27me3. Left (chr7), ACTB locus. Right (chr16), LUC7L locus. (C) CGI promoter of RHBDF1 locus (chr16) has H3K27me3 mark and the absence of CFP1 binding in both ERY and EBV. Fig. S4: Western blot analysis of CGBP (CFP1) expression in mouse and human erythroid and human lymphoid cell types. Whole cell extracts (20 µg) were loaded in each lane (1) mouse ES, (2) U-MEL, (3) I-MEL, (4) mouse primary erythroblasts and (5) human primary T lymphocytes and (6) human primary erythroblasts and separated on a 10% SDS-polyacrylamide gel. CFP1 antibody was used at a 1:1000 dilution. Fig. S5: Similar cell type-specific CFP1 read depth at CGI TSS of HBA1 gene and non-CGI TSS of HBB gene. Upper two tracks use the main antibody, and second two tracks use the commercial antibody. Coordinates are from the hg38 human genome build. Read depths are averaged in 50 bp bins and normalised to 1x genome-wide coverage. Blue boxes, known regulatory regions; green box, CGI. Fig. S6: Distribution of TrxG components in erythroid cells. Green indicates CGI and blue indicates other putative regulatory regions. All loci transcribed right to left. Pileups are shown scaled to 1x genome coverage, with full scale 0–50x depth. (A) Housekeeping genes ACTB, left (chr7), and LUC7L, right (chr16). (B) β-globin locus (chr11), (C) Non-expressed RHBDF1 locus (chr16). Fig. S7: Overlap of TrxG subunit ChIP peaks in a high-confidence subset of regions. SET1A complexes are represented by CFP1-SET1A colocalisation. MLL1/2 complexes are represented by Menin, and MLL3/4 complexes are represented by UTX, respectively. HCF1 is found in SET1A/B and MLL1/2 complexes, and RBBP5 is a member of SET1A/B and MLL1/2/3/4 complexes. Red outline (4220 peaks) shows strong colocalisation of Menin and CFP1-SET1A, accounting for the vast majority (99.5%) of 4242 CFP1-SET1A and half (50.0%) of 8432 Menin peak regions. Majority (87.0%, 2089/2400 peaks) of HCF1 (blue region) is accounted for by approximately half (49.5%, 2089/4220) of regions of Menin-SET1A-CFP1 colocalisation. Regions where either SET1A-CFP1 or Menin or both are colocalised with HCF1 (blue dashed line) accounts for nearly all (99.6%, 2390/2400) HCF1 regions, suggesting that HCF1 bound to DNA is primarily present as part of SET1A/B or MLL1/2 complexes. Fig. S8: Chromatin accessibility in TSSs and enhancers in erythroid cells as measured by ATAC-seq and DNase-seq. 1x-normalised, input-subtracted signals from ATAC-seq and DNase were averaged in a 2-kb window about TSSs and putative enhancers. Z-score transformed values for ATAC-seq and DNase-seq at a given locus were averaged. Fig. S9: Relationship of CFP1 signal to three predictive factors in top-decile open chromatin regions. A linear combination of CpG density and SET1A and H3K4me3 ChIP signals explains a substantial fraction of variation in CFP1 ChIP signal. Table S1: Bias of CFP1 for CGI TSSs in cell types and gene classes. Table S2: Bias of CFP1 for housekeeping gene TSSs. Table S3: Motifs associated with CFP1 peaks. Table S4: Dependence of CFP1 ChIP signal in erythroid cells on covariates putatively associated with its binding. Table S5: Analysis of variance of CFP1 signal in top-decile open chromatin regions surrounding TSSs and putative enhancers

Disaggregases, Molecular Chaperones That Resolubilize Protein Aggregates

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The process of folding is a seminal event in the life of a protein, as it is essential for proper protein function and therefore cell physiology. Inappropriate folding, or misfolding, can not only lead to loss of function, but also to the formation of protein aggregates, an insoluble association of polypeptides that harm cell physiology, either by themselves or in the process of formation. Several biological processes have evolved to prevent and eliminate the existence of non-functional and amyloidogenic aggregates, as they are associated with several human pathologies. Molecular chaperones and heat shock proteins are specialized in controlling the quality of the proteins in the cell, specifically by aiding proper folding, and dissolution and clearance of already formed protein aggregates. The latter is a function of disaggregases, mainly represented by the ClpB/Hsp104 subfamily of molecular chaperones, that are ubiquitous in all organisms but, surprisingly, have no orthologs in the cytosol of metazoan cells. This review aims to describe the characteristics of disaggregases and to discuss the function of yeast Hsp104, a disaggregase that is also involved in prion propagation and inheritance.872S12731292Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Hsp78 (78 kDa Heat Shock Protein), a Representative AAA Family Member Found in the Mitochondrial Matrix of Saccharomyces cerevisiae

ATPases associated with diverse cellular activities (AAA+) form a superfamily of proteins involved in a variety of functions and are characterized by the presence of an ATPase module containing two conserved motifs known as Walker A and Walker B. ClpB and Hsp104, chaperones that have disaggregase activities, are members of a subset of this superfamily, known as the AAA family, and are characterized by the presence of a second highly conserved motif, known as the second region of homology (SRH). Hsp104 and its homolog Hsp78 (78 kDa heat shock protein) are representatives of the Clp family in yeast. The structure and function of Hsp78 is reviewed and the possible existence of other homologs in metazoans is discussed

Characterization of the Hsp100 disaggregase from sugarcane (SHsp101) for chaperone like activity in a yeast system

FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe Hsp104/ClpB protein subfamily is unique in its ability to reactivate protein aggregates, which are implicated in several human and animal diseases. However, when compared to the unicellular yeast Hsp104 and bacterial ClpB disaggregases, the multicellu26478487FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2012/50161-8We thank Claudio Masuda (UFRJ) for gifting the BY4741 yeast strain and isogenic Hsp104 knockout, and Doug Cyr (University of North Carolina) for the Rnq-mRFP yeast expression plasmid, and the W303 [RNQ+] yeast strai

Heterologous Expression Studies of Saccharomyces cerevisiae Reveal Two Distinct Trypanosomatid CaaX Protease Activities and Identify Their Potential Targets▿ †

The CaaX tetrapeptide motif typically directs three sequential posttranslational modifications, namely, isoprenylation, proteolysis, and carboxyl methylation. In all eukaryotic systems evaluated to date, two CaaX proteases (Rce1 and Ste24/Afc1) have been identified. Although the Trypanosoma brucei genome also encodes two putative CaaX proteases, the lack of detectable T. brucei Ste24 activity in trypanosome cell extracts has suggested that CaaX proteolytic activity within this organism is solely attributed to T. brucei Rce1 (J. R. Gillespie et al., Mol. Biochem. Parasitol. 153:115-124. 2007). In this study, we demonstrate that both T. brucei Rce1 and T. brucei Ste24 are enzymatically active when heterologously expressed in yeast. Using a-factor and GTPase reporters, we demonstrate that T. brucei Rce1 and T. brucei Ste24 possess partially overlapping specificities much like, but not identical to, their fungal and human counterparts. Of interest, a CaaX motif found on a trypanosomal Hsp40 protein was not cleaved by either T. brucei CaaX protease when examined in the context of the yeast a-factor reporter but was cleaved by both in the context of the Hsp40 protein itself when evaluated using an in vitro radiolabeling assay. We further demonstrate that T. brucei Rce1 is sensitive to small molecules previously identified as inhibitors of the yeast and human CaaX proteases and that a subset of these compounds disrupt T. brucei Rce1-dependent localization of our GTPase reporter in yeast. Together, our results suggest the conserved presence of two CaaX proteases in trypanosomatids, identify an Hsp40 protein as a substrate of both T. brucei CaaX proteases, support the potential use of small molecule CaaX protease inhibitors as tools for cell biological studies on the trafficking of CaaX proteins, and provide evidence that protein context influences T. brucei CaaX protease specificity