Mitochondrial HSP70 Chaperone System—The Influence of Post-Translational Modifications and Involvement in Human Diseases

Since their discovery, heat shock proteins (HSPs) have been identified in all domains of life, which demonstrates their importance and conserved functional role in maintaining protein homeostasis. Mitochondria possess several members of the major HSP sub-families that perform essential tasks for keeping the organelle in a fully functional and healthy state. In humans, the mitochondrial HSP70 chaperone system comprises a central molecular chaperone, mtHSP70 or mortalin (HSPA9), which is actively involved in stabilizing and importing nuclear gene products and in refolding mitochondrial precursor proteins, and three co-chaperones (HSP70-escort protein 1—HEP1, tumorous imaginal disc protein 1—TID-1, and Gro-P like protein E—GRPE), which regulate and accelerate its protein folding functions. In this review, we summarize the roles of mitochondrial molecular chaperones with particular focus on the human mtHsp70 and its co-chaperones, whose deregulated expression, mutations, and post-translational modifications are often considered to be the main cause of neurological disorders, genetic diseases, and malignant growth.     

赤子爱胜蚓蛋白磷酸酶2A类似激活剂样蛋白cDNA和氨基酸序列分析

目的分析赤子爱胜蚓蛋白磷酸酶2A(protein phosphatase 2A,PP2A)类似激活剂样蛋白的cDNA和氨基酸序列。方法从赤子爱胜蚓cDNA文库中随机测序得到目标cDNA序列,应用DNAMAN、NCBI ORF finder、BLAST、Conserved Domains、GOR、SWISS-MODEL、PDB等基因和蛋白质分析软件进行该目标基因测序及氨基酸序列分析。结果赤子爱胜蚓PP2A类似激活剂样蛋白cDNA序列长547 bp,编码87个氨基酸残基;与该蛋白基因序列相似度较高的多是不同物种中PP2A类似激活剂蛋白基因序列,其中与Crassostrea virginica serine/threonine-protein phosphatase 2A activator-like mRNA核苷酸相似度为67%;与该蛋白氨基酸序列相似度较高的多是不同物种中的PP2A类似激活剂蛋白氨基酸序列,其中与serine/threonine-protein phosphatase 2A activator(Crassostrea gigas)氨基酸相似度为70%;目标序列中存在一段PTPA超家族保守结构域,蛋白质二级结构以α螺旋以及无规则卷曲为主,三级预测结果与二级结构一致。结论本实验分析的赤子爱胜蚓PP2A类似激活剂样蛋白与牡蛎PP2A类似激活剂蛋白及存在于所有真核生物中且高度保守的PTPA同源度较高,可能具有特异性激活肿瘤抑制因子PP2A的间接抑制肿瘤作用。     

The Multifaceted Role of HSF1 in Pathophysiology: Focus on Its Interplay with TG2

The cellular environment needs to be strongly regulated and the maintenance of protein homeostasis is crucial for cell function and survival. HSF1 is the main regulator of the heat shock response (HSR), the master pathway required to maintain proteostasis, as involved in the expression of the heat shock proteins (HSPs). HSF1 plays numerous physiological functions; however, the main role concerns the modulation of HSPs synthesis in response to stress. Alterations in HSF1 function impact protein homeostasis and are strongly linked to diseases, such as neurodegenerative disorders, metabolic diseases, and different types of cancers. In this context, type 2 Transglutaminase (TG2), a ubiquitous enzyme activated during stress condition has been shown to promote HSF1 activation. HSF1-TG2 axis regulates the HSR and its function is evolutionary conserved and implicated in pathological conditions. In this review, we discuss the role of HSF1 in the maintenance of proteostasis with regard to the HSF1-TG2 axis and we dissect the stress response pathways implicated in physiological and pathological conditions.     

Differential Expression of Copper-Zinc Superoxide Dismutase Gene of Polygonum sibiricum Leaves, Stems and Underground Stems, Subjected to High-Salt Stress

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5' untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3' untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1-16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO(3). The different mRNA levels' expression of PS-CuZnSOD show the gene's different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.     

Molecular Cloning and Expression Analysis of P-Selectin from Zebrafish (Danio rerio)

The glycoprotein P-selectin belongs to the selectin family of cell adhesion molecules. In this study, we cloned the full-length cDNA of P-selectin from zebrafish (Danio rerio) by the method of rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Zebrafish P-selectin cDNA is 2,800 bp and encodes a putative 868 amino acid protein with a theoretical molecular weight of 122.36 kDa and isoelectric point of 6.27. A signal peptide of 25 amino acids is predicted at the N-terminus of the putative protein. All structural domains involved in P-selectin function are conserved in the putative protein. The amino acid sequence of zebrafish P-selectin is 37% to 39% identical to that of mammalian P-selectins. Real-time quantitative PCR and whole-mount in situ hybridization analysis revealed that P-selectin was expressed in early embryonic development, the expression increased from 0.2 hpf (1-cell stage) to 72 hpf, and the expression significantly upregulated within 30 minutes of ADP induction. The results indicate that the structure of P-selectin protein is highly conserved among species and zebrafish P-selectin plays an important role in early embryonic development and probably has similar biological function to mammalian P-selectins.     

幽门螺杆菌热休克蛋白A基因的克隆和表达

目的　扩增幽门杆菌热休克蛋白A 35 1bp的DNA片段 ,并将其克隆到pET - 2 8a(+ )质粒中高效表达。方法　用PCR方法扩增的片段经测序后 ,用GoldKey分析软件进行序列分析 ,应用pET - 2 8a(+ )系统在受体菌BL2 1(DE3)pLysS中表达热休克蛋白。结果　该序列表达的蛋白与已报道的热休克蛋白A序列相似。重组菌株用IPTG诱导后 ,经SDS PAGE和薄层扫描分析 ,表达的外源蛋白的相对分子质量为 180 0 0 ,以包涵体的形式存在 ,表达产物占菌体总蛋白的 6 4%。结论　HspA是最有可能做为H .p疫苗中的候选抗原成分     

Phenotypic Identification of the Redox Dye Methylene Blue as an Antagonist of Heat Shock Response Gene Expression in Metastatic Melanoma Cells

Repurposing approved and abandoned non-oncological drugs is an alternative developmental strategy for the identification of anticancer therapeutics that has recently attracted considerable attention. Due to the essential role of the cellular heat shock response in cytoprotection through the maintenance of proteostasis and suppression of apoptosis, small molecule heat shock response antagonists can be harnessed for targeted induction of cytotoxic effects in cancer cells. Guided by gene expression array analysis and a phenotypic screen interrogating a collection of 3,7-diamino-phenothiazinium derivatives, we have identified the redox-drug methylene blue (MB), used clinically for the infusional treatment of methemoglobinemia, as a negative modulator of heat shock response gene expression in human metastatic melanoma cells. MB-treatment blocked thermal (43 °C) and pharmacological (celastrol, geldanamycin) induction of heat shock response gene expression, suppressing Hsp70 (HSPA1A) and Hsp27 (HSPB1) upregulation at the mRNA and protein level. MB sensitized melanoma cells to the apoptogenic activity of geldanamycin, an Hsp90 antagonist known to induce the counter-regulatory upregulation of Hsp70 expression underlying cancer cell resistance to geldanamycin chemotherapy. Similarly, MB-cotreatment sensitized melanoma cells to other chemotherapeutics (etoposide, doxorubicin). Taken together, these data suggest feasibility of repurposing the non-oncological redox drug MB as a therapeutic heat shock response antagonist for cancer cell chemosensitization.     

Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7-8.5, with an optimum pH of 7.5, and at temperatures in the range 30-45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.     

Biosynthetic Short Neuropeptides: A Rational Theory Based on Experimental Results for the Missing Pain-Relief Opioid Endomorphin Precursor Gene

Endomorphins are neuropeptides that bind strongly to μ-opioid receptors and are considered to play important roles in pain modulation and other biological functions. Two endomorphins have been identified, to date, endomorphine-1 and -2; both are tetrapeptides and differ by only a single amino acid in the third position. Both peptides were isolated from bovine brains; however, their precursor genes have not been identified. In this study, a nucleotide sequence corresponding to the endomorphin-1 peptide in an expressed sequence tag database has been found and a preproendomorphin-like precursor peptide from human brain complementary DNA (cDNA) has been cloned. The cDNA consists of nucleotide sequences of two already annotated predicted genes, and the putative peptide differs by one amino acid from the isolated endomorphin peptides. It is proposed herein that there is the possibility of unknown short proteins or peptide precursors being missed by automated gene prediction programs based on similarities of known protein sequences. A novel concept of how to produce endomorphins from a similar peptide is described. The oxidatively modified base might provide a clue for understanding discrepancies between nucleotide sequences on the genome and those on cDNAs.     

Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional regulation of fatty acyl Δ6 desaturase of Atlantic cod (Gadus morhua L.)

This study reports the cloning, functional characterization, tissue expression, and nutritional regulation of a Δ6 fatty acyl desaturase of Atlantic cod (Gadus morhua). PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from cod liver, with full-length cDNA obtained by rapid amplification of cDNA ends. The cDNA for the putative desaturase was shown to comprise 1980 bp, including a 261-bp 5′-UTR, a 375-bp 3′-UTR, and an ORF of 1344 bp that specified a protein of 447 amino acids. The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b₅ domain containing the heme-binding motif HPGG, all characteristic of microsomal fatty acyl desaturases. The cDNA displayed Δ6 desaturase activity in a yeast expression system. Quantitative real-time PCR assay of gene expression in cod showed that the Δ6 desaturase gene was expressed highly in brain, to a slightly lesser extent in liver, kidney, intestine, red muscle, and gill, and at much lower levels in white muscle, spleen, and heart. The expression of the Δ6 desaturase gene did not appear to be under significant nutritional regulation, with levels in liver and intestine being barely altered in fish fed a vegetable oil blend, in comparison with levels in fish fed fish oil. This was reflected in enzyme activity, as hepatocytes or enterocytes showed very little highly unsaturated FA biosynthesis activity irrespective of diet. Further studies are required to determine why the Δ6 desaturase appears to be barely functional in cod under the conditions tested.     

小鼠激活素受体相互作用蛋白5的基因克隆

目的克隆新的小鼠激活素受体相互作用蛋白5(ActRIP5)基因。方法应用酵母双杂交技术,筛选出与激活素ⅡA型受体(ActRⅡA)相互作用蛋白的基因片段,再以此基因片段作为探针,从小鼠脑cDNA文库中钓取ActRIP5cDNA。采用哺乳动物细胞双杂交系统,进一步确定ActRIP5与ActRⅡA的相互作用。采用RT-PCR检测ActRIP5mRNA在组织中的转录。结果克隆的ActRIP5全编码基因长1839bp,编码145个氨基酸残基,与ActRⅡA具有特异结合作用。ActRIP5mR-NA在小鼠多种组织中均可检出。结论ActRIP5属于ActRIP家族新成员,可以与ActRⅡA相互作用。     

Silent mutations in secondary Shine-Dalgarno sequences in the cDNA of human serum amyloid A4 promotes expression of recombinant protein in Escherichia coli

The serum amyloid A (SAA) superfamily comprises a number ofdifferentially expressed genes with a high degree of homologyin mammalian species. SAA4, an apolipoprotein constitutivelyexpressed only in humans and mice, is associated almost entirelywith lipoproteins of the high-density range. The presence ofSAA4 mRNA and protein in macrophage-derived foam cells of coronaryand carotid arteries suggested a specific role of human SAA4during inflammation including atherosclerosis. Here we underlinethe importance of ribosome binding site (rbs)-like sequences(also known as Shine–Dalgarno sequences) in the SAA4 cDNAfor expression of recombinant SAA4 protein in Escherichia coli.In contrast to rbs sequences coded by the expression vectors,rbs-like sequences in the cDNA of target protein(s) are knownto interfere with protein translation via binding to the small16S ribosome subunit, yielding low or even no expression. Herewe show that PCR mutations of two rbs-like sequences in thehuman SAA4 cDNA promote expression of considerable amounts ofrecombinant SAA4 in E.coli.