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Kazushige Katsura, Yuri Tomabechi, Takayoshi Matsuda, Mayumi Yonemochi, Junko Mikuni, Noboru Ohsawa, Takaho Terada, Shigeyuki Yokoyama, Mutsuko Kukimoto-Niino, Chie Takemoto, Mikako Shirouzu
Since phosphorylation is involved in various physiological events, kinases and interacting factors can be potential targets for drug discovery. For the development and improvement of inhibitors from the point of view of mechanistic enzymology, a cell-free protein synthesis system would be advantageous, since it could prepare mutant proteins easily. However, especially in the case of protein kinase, product solubility remains one of the major challenges. To overcome this problem, we prepared a chaperone-supplemented extract from Escherichia coli BL21 cells harboring a plasmid encoding a set of chaperone genes, dnaK, dnaJ, and grpE...
May 21, 2018: Protein Expression and Purification
Samantha Reddy, Oliver T Zishiri
Virulence-associated genes have been recognised and detected in Campylobacter species. The majority of them have been proven to be associated with pathogenicity. This study aimed to detect the presence of virulence genes associated with pathogenicity and responsible for invasion, expression of adherence, colonisation and production of the cytolethal distending toxin (cdt) in Campylobacter jejuni and Campylobacter coli. Commercial chicken faecal samples were randomly sampled from chicken farms within the Durban metropolitan area in South Africa...
February 15, 2018: Onderstepoort Journal of Veterinary Research
Sirikul Kulanuwat, Watip Tangjittipokin, Prapaporn Jungtrakoon, Chutima Chanprasert, Jatuporn Sujjitjoon, Ninareeman Binnima, Pa-Thai Yenchitsomanus, Nattachet Plengvidhya
Type 2 diabetes mellitus (T2D) is a heterogeneous disease, with certain cases presenting an autosomal dominant type. The rare coding variants of disease‑causing genes in T2D remain mostly unclear. The present study aimed to identify the disease‑causing gene conducting whole exome sequencing in a Thai T2D family with an autosomal dominant transmission of T2D with no evidence of mutations in known maturity‑onset diabetes of the young (MODY) genes. Candidate variants were selected according to certain criteria of mutation prediction programs, followed by segregation analysis with diabetes in the family...
May 14, 2018: International Journal of Molecular Medicine
Samantha J Sojourner, Willie M Graham, Aurellia M Whitmore, Jana S Miles, Devon Freeny, Hernan Flores-Rozas
Doxorubicin, a highly effective therapeutic agent against several types of cancer, is associated with serious side-effects, particularly cardiotoxicity. In addition, drug resistance leads to unsuccessful outcomes in many patients. There are no current biomarkers to indicate doxorubicin treatment response in patients. To understand the mechanisms of toxicity of doxorubicin, a whole-genome sensitivity screen was performed in the yeast S. cerevisiae . A deletion mutant of the yeast DNAJ (YDJ1), a J-domain heat-shock protein 40 (HSP40) was among the most sensitive strains...
2018: Journal of Nature and Science
Mengfei Song, Qingzhen Wei, Jing Wang, Wenyuan Fu, Xiaodong Qin, Xiumei Lu, Feng Cheng, Kang Yang, Lu Zhang, Xiaqing Yu, Ji Li, Jinfeng Chen, Qunfeng Lou
Leaf color mutants in higher plants are ideal materials for investigating the structure and function of photosynthetic system. In this study, we identified a cucumber vyl (virescent-yellow leaf) mutant in the mutant library, which exhibited reduced pigment contents and delayed chloroplast development process. F2 and BC1 populations were constructed from the cross between vyl mutant and cucumber inbred line 'Hazerd' to identify that the vyl trait is controlled by a simply recessive gene designated as CsVYL. The CsVYL gene was mapped to a 3...
2018: Frontiers in Plant Science
Bin Zhang, Hanlin Qiu, Donghai Qu, Ying Ruan, Dong-Hong Chen
HSP40s or DnaJ/J-proteins are evolutionarily conserved in all the organisms as cochaperones of molecular chaperone HSP70s that mainly participate in maintaining cellular protein homeostasis, such as protein folding, assembly, stabilization, and translocation under normal conditions as well as refolding and degradation under environmental stresses. It has been reported that Arabidopsis J-proteins are classified into four classes (type A~D) according to domain organization, but their phylogenetic relationships are unknown...
April 5, 2018: Genome Génome / Conseil National de Recherches Canada
Meng-Dan Yang, Ying Zhao, Peng-da Ma
The study is aimed to construct high quality Salvia miltiorrhiza cDNA library and obtain the SmJAZ8 gene of S. miltiorrhiza by yeast two-hybrid system. In this study, full-length cDNA was synthesized from roots, stems, leaves, flowers and hairy roots of S. miltiorrhiza. The full-length cDNA library was synthesized by SMART method and constructed with DSN homogenization technique. The results showed that the library capacity was 1.45×10⁶, the recombination rate was 100%, and the average size of the insert was 500-2 000 bp...
February 2018: Zhongguo Zhong Yao za Zhi, Zhongguo Zhongyao Zazhi, China Journal of Chinese Materia Medica
Christopher A G Söderberg, Cecilia Månsson, Katja Bernfur, Gudrun Rutsdottir, Johan Härmark, Sreekanth Rajan, Salam Al-Karadaghi, Morten Rasmussen, Peter Höjrup, Hans Hebert, Cecilia Emanuelsson
The remarkably efficient suppression of amyloid fibril formation by the DNAJB6 chaperone is dependent on a set of conserved S/T-residues and an oligomeric structure, features unusual among DNAJ chaperones. We explored the structure of DNAJB6 using a combination of structural methods. Lysine-specific crosslinking mass spectrometry provided distance constraints to select a homology model of the DNAJB6 monomer, which was subsequently used in crosslink-assisted docking to generate a dimer model. A peptide-binding cleft lined with S/T-residues is formed at the monomer-monomer interface...
March 26, 2018: Scientific Reports
Lior Doron, Pierre Goloubinoff, Michal Shapira
Photosynthesis is performed by large complexes, composed of subunits encoded by the nuclear and chloroplast genomes. Assembly is assisted by general and target-specific chaperones, but their mode of action is yet unclear. We formerly showed that ZnJ2 is an algal chaperone resembling BSD2 from land plants. In algae, it co-migrates with the rbcL transcript on chloroplast polysomes, suggesting it contributes to the de-novo synthesis of RbcL (Doron et al., 2014). ZnJ2 contains four CXXCXGXG motifs, comprising a canonical domain typical also of DnaJ-type I (DNAJA)...
2018: Frontiers in Molecular Biosciences
Judit Perales-Calvo, David Giganti, Guillaume Stirnemann, Sergi Garcia-Manyes
It is well established that chaperones modulate the protein folding free-energy landscape. However, the molecular determinants underlying chaperone-mediated mechanical folding remain largely elusive, primarily because the force-extended unfolded conformation fundamentally differs from that characterized in biochemistry experiments. We use single-molecule force-clamp spectroscopy, combined with molecular dynamics simulations, to study the effect that the Hsp70 system has on the mechanical folding of three mechanically stiff model proteins...
February 2018: Science Advances
Elena R Toenshoff, Peter D Fields, Yann X Bourgeois, Dieter Ebert
The planktonic freshwater crustacean of the genus Daphnia are a model system for biomedical research and, in particular, invertebrate-parasite interactions. Up until now, no virus has been characterized for this system. Here we report the discovery of an iridovirus as the causative agent of White Fat Cell Disease (WFCD) in Daphnia WFCD is a highly virulent disease of Daphnia that can easily be cultured under laboratory conditions. Although it has been studied from sites across Eurasia for more than 60 years, its causative agent had not been described, nor had an iridovirus been connected to WFCD before now...
March 28, 2018: G3: Genes—Genomes—Genetics
Duncan C Krause, Songye Chen, Jian Shi, Ashley J Jensen, Edward S Sheppard, Grant J Jensen
The Mycoplasma pneumoniae terminal organelle functions in adherence and gliding motility and is comprised of at least eleven substructures. We used electron cryotomography to correlate impaired gliding and adherence function with changes in architecture in diverse terminal organelle mutants. All eleven substructures were accounted for in the prkC, prpC and P200 mutants, and variably so for the HMW3 mutant. Conversely, no terminal organelle substructures were evident in HMW1 and HMW2 mutants. The P41 mutant exhibits a terminal organelle detachment phenotype and lacked the bowl element normally present at the terminal organelle base...
May 2018: Molecular Microbiology
M Seo, S A Islam, S-S Moon
The hypothalamus is the regulatory centre of both appetite and energy balance and endoplasmic reticulum (ER) stress in the hypothalamus is involved in the pathogenesis of obesity. Recently, inhibition of 11 β hydroxysteroid dehydrogenase type1 (11β-HSD1) was reported to have an anti-obesity effect by reducing fat mass. However, the link between the role of 11β-HSD1 in the hypothalamus and obesity has yet to be determined. In the present study, embryonal primary hypothalamic neurones and high-fat diet (HFD) fed mice were used to investigate the anorexigenic effects of 11β-HSD1 inhibitors both in vitro and in vivo...
March 2018: Journal of Neuroendocrinology
Anna Wanecka, Jarosław Król, Jan Twardoń, Jacek Mrowiec, Jacek Bania, Agnieszka Korzeniowska-Kowal, Anna Tobiasz
The aim of this paper is to describe a novel subpopulation of Staphylococcus haemolyticus isolated from intramammary gland infections (IMI) in cattle. In total, eight isolates originating from milk samples from two unrelated dairy farms were examined phenotypically (using the ID 32 STAPH system) and genotypically. These isolates had almost identical sequences of each of the housekeeping genes examined (dnaJ, rpoB and sodA) but these sequences displayed similarity of only ∼92.5%, 95.0% and 96.8%, respectively, with known S...
February 2018: Veterinary Microbiology
Katharina B Beer, Jennifer Rivas-Castillo, Kenneth Kuhn, Gholamreza Fazeli, Birgit Karmann, Jeremy F Nance, Christian Stigloher, Ann M Wehman
Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans However, no proteins that inhibit ectocytosis upstream of TAT-5 were known...
January 24, 2018: Proceedings of the National Academy of Sciences of the United States of America
Monica Modesto, Samanta Michelini, Kaihei Oki, Bruno Biavati, Koichi Watanabe, Paola Mattarelli
In our previous study based on hsp60 PCR-restriction fragment length polymorphism and 16S rRNA gene sequencing, we stated that the bifidobacterial strains isolated from the individual faecal samples of five baby common marmosets constituted different phylogenetically isolated groups of the genus Bifidobacterium. In that study, we also proposed that these isolated groups potentially represented novel species of the genus Bifidobacterium. Out of them, Bifidobacterium aesculapii, Bifidobacterium myosotis, Bifidobacterium tissieri and Bifidobacterium hapali, have been described recently...
February 2018: International Journal of Systematic and Evolutionary Microbiology
Yongliang Shang, Jie Yan, Wenhao Tang, Chao Liu, Sai Xiao, Yueshuai Guo, Li Yuan, Liang Chen, Hui Jiang, Xuejiang Guo, Jie Qiao, Wei Li
Acephalic spermatozoa syndrome has been reported for many decades; it is characterized by very few intact spermatozoa and tailless sperm heads in the semen and causes severe male infertility. The only gene in which mutations have been found to be associated with this syndrome encodes Sad1 and UNC84 domain-containing 5 ( SUN5 ), a testis-specific nuclear envelope protein. The functional role of SUN5 has been well-studied in mouse models, but the molecular basis for the pathogenic effects of mutations in the human SUN5 gene remains elusive...
February 16, 2018: Journal of Biological Chemistry
Roman Kityk, Jürgen Kopp, Matthias P Mayer
Efficient targeting of Hsp70 chaperones to substrate proteins depends on J-domain cochaperones, which in synergism with substrates trigger ATP hydrolysis in Hsp70s and concomitant substrate trapping. We present the crystal structure of the J-domain of Escherichia coli DnaJ in complex with the E. coli Hsp70 DnaK. The J-domain interacts not only with DnaK's nucleotide-binding domain (NBD) but also with its substrate-binding domain (SBD) and packs against the highly conserved interdomain linker. Mutational replacement of contacts between J-domain and SBD strongly reduces the ability of substrates to stimulate ATP hydrolysis in the presence of DnaJ and compromises viability at heat shock temperatures...
January 18, 2018: Molecular Cell
Marcia Y Maier, Lisanne Luks, Oliver R Baudendistel, Valentin Wittmann, Daniel R Dietrich
Propiverine, a frequently-prescribed pharmaceutical for the treatment of symptoms associated with overactive bladder syndrome, provoked massive intranuclear and cytosolic protein inclusions in rat proximal tubule epithelium, primarily consisting of the peroxisomal targeting signal 1 (PTS1) containing protein d-amino acid oxidase (DAAO). As this type of nephropathy was also observed for other drugs, the aim was to determine whether propiverine interferes with trafficking and/or import of peroxisomal proteins...
February 1, 2018: Chemico-biological Interactions
Xibing Xu, Ke Liang, Yulong Niu, Yan Shen, Xuedong Wan, Haiyan Li, Yi Yang
In Escherichia coli, the DnaK/DnaJ chaperone can control the stability and activity of σ32 , which is the key factor in heat shock response. Heterologous expression of eukaryotic molecular chaperones protects E. coli from heat stress. Here, we show that BAH1, an E3 ligase from plant that has a similar zinc finger domain to DnaJ, can perform block the effect of DnaK on σ32 in Escherichia coli. By constructing a chimeric DnaJ protein, with the J-domain of DnaJ fused to BAH1, we found BAH1 could partially compensate for the DnaJ' zinc finger domain in vivo, and that it was dependent on the zinc finger domain of BAH1...
April 2018: Current Microbiology
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