Draft Genome Sequence of the Yeast Rhodotorula sp. Strain CCFEE 5036, Isolated from McMurdo Dry Valleys, Antarctica.

A draft genome sequence was assembled and annotated of the basidiomycetous yeast Rhodotorula sp. strain CCFEE 5036, isolated from Antarctic soil communities. The genome assembly is 19.07 megabases and encodes 6,434 protein-coding genes. The sequence will contribute to understanding the diversity of fungi inhabiting polar regions

MMpI: A widerange of available compounds of matrix metalloproteinase inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the regulation of the extracellular signaling and structural matrix environment of cells and tissues. MMPs are considered as promising targets for the treatment of many diseases. Therefore, creation of database on the inhibitors of MMP would definitely accelerate the research activities in this area due to its implication in above-mentioned diseases and associated limitations in the first and second generation inhibitors. In this communication, we report the development of a new MMpI database which provides resourceful information for all researchers working in this field. It is a web-accessible, unique resource that contains detailed information on the inhibitors of MMP including small molecules, peptides and MMP Drug Leads. The database contains entries of ~3000 inhibitors including ~72 MMP Drug Leads and ~73 peptide based inhibitors. This database provides the detailed molecular and structural details which are necessary for the drug discovery and development. The MMpI database contains physical properties, 2D and 3D structures (mol2 and pdb format files) of inhibitors of MMP. Other data fields are hyperlinked to PubChem, ChEMBL, BindingDB, DrugBank, PDB, MEROPS and PubMed. The database has extensive searching facility with MMpI ID, IUPAC name, chemical structure and with the title of research article. The MMP inhibitors provided in MMpI database are optimized using Python-based Hierarchical Environment for Integrated Xtallography (Phenix) software. MMpI Database is unique and it is the only public database that contains and provides the complete information on the inhibitors of MMP

Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits

Abstract: Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 μmol l−1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 μmol l−1. Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. Significance and Impact of the Study: This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would represent a new alternative to avoid the problems of environmental pollution and antimicrobial resistance.Fil: Díaz, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Luján; ArgentinaFil: Rocha, Gabriela Fernanda. Universidad Nacional de Luján; ArgentinaFil: Kise, Francisco. Universidad Nacional de Luján; ArgentinaFil: Rosso, A. M.. Universidad Nacional de Luján; ArgentinaFil: Guevara, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Parisi, M.G.. Universidad Nacional de Luján; Argentin

AUTOMATED ASSAY OF Caenorhabditis elegans WILD-TYPE AND CYSTATIN MUTANTS THRASHING BEHAVIOUR IN THE PRESENCE OR ABSENCE OF PLANT DERIVED CYSTEINE PROTEINASES (CPs)

The intensive use and over dependence on synthetic anthelmintics for the treatment of nematode infection on only a few drugs with similar mode of action has put pressure on such drug candidates with resultant loss of potency due to development of resistance by target nematodes. Plant materials with promising quality and efficacy to substitute for current anthelmintics include the plant derived cysteine proteinases (CPs). Motility is an important indication of the effectivenes of a drug and is a characteristic of  phenotype useful for high thoroughput screening of chemical and theraputic agents. This study determined the effect of cysteine proteinases on motility of C. elegans strains (wild type and cystatin null mutants) using the worm watcher device. Results show that motility of C. elegans was affected differently in PLS or papain. The effect of CP on motility of C. elegans strains was dependent on CP type, time of incubation and concentration of CP. Generally there was no significant difference (P>0.05) between mean motility of WT, cpi-1 and cpi-2 null mutant C. elegans in PLS when compared with PLS+E64 (control). There was a statistically significant (P ˂0.05) effect of papain dose on all the strains. Enzyme specificity on cuticle structural proteins might be responsible for difference in pattern of attack observed between papain and PLS. CP has potency for use as effective anthelminthic

Biochemical properties of crude extracellular proteases from Chromohalobacter salexigens BKL5 and Micrococcus luteus 11A

In this work, we have reported an enzymatic activity and biochemical properties of extracellular proteases from Chromohalobacter salexigens BKL5 and Micrococcus luteus 11A. C. salexigens BKL5 and M. luteus 11A were previously isolated from Bledug Kuwu mud volcano and dietary industry wastewater treatment, respectively. Both bacterial strains were able to produce extracellular proteases, when grown on minimal agar medium supplemented with 1% of skim milk. Proteolytic indexes of C. salexigens BKL5 and M. luteus 11A were 2.5±0.14 and 2.9±0.42, respectively. Both extracellular proteases exhibited optimum enzymatic activity at pH 7, with specific activity of C. salexigens BKL5 was 13.3% higher than that of M. luteus 11A. Optimum temperature for enzymatic activity of both proteases was 45°C. Metal cofactor preferences assay showed that extracellular protease from C. salexigens BKL5 preferred Zn2+, meanwhile extracellular protease from M. luteus 11A mainly preferred Ca2+ ion. Metal cofactor preferences assay also suggested that crude extracellular protease from C. salexigens BKL5 was categorized as metalloprotease, meanwhile crude extracellular protease of M. luteus 11A was common neutral protease. The enzymatic stability assay against various salt concentrations showed that crude extracellular protease from C. salexigens BKL5 was more stable than that of M. luteus 11A

Novel pseudo-aspartic peptidase from the midgut of the tick Rhipicephalus microplus

The characterization of Rhipicephalus microplus tick physiology can support efforts to develop and improve the efficiency of control methods. A sequence containing a domain with similarity to one derived from the aspartic peptidase family was isolated from the midgut of engorged female R. microplus. The lack of the second catalytic aspartic acid residue suggest that it may be a pseudoaspartic peptidase, and it was named RmPAP. In this work we confirm the lack of proteolytic activity of RmPAP and investigate it’s non-proteolytic interaction with bovine hemoglobin by Surface Plasmon Resonance and phage display. Moreover we carried out RNAi interference and artificial feeding of ticks with anti-RmPAP antibodies to assess it’s possible biological role, although no changes were observed in the biological parameters evaluated. Overall, we hypothesize that RmPAP may act as a carrier of hemoglobin/heme between the tick midgut and the ovaries

Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley

Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.España, MINECO AGL2014-52028-R and AGL2017-82447-

Bioinformatics analysis and molecular cloning of an extracellular serine protease from acinetobacter baumannii

Drug resistant Acinetobacter baumannii topped the list for antibiotic resistant ‘critical’ pathogens that was released by the World Health Organisation (WHO) in February 2017. The list was intended to guide and promote research and development (R&D) of antibiotics. One of the factors that may contribute to A. baumannii virulence are the secretory proteases that this bacteria produces. In order to design effective antibiotocs and treatment targeting secretory proteases from A. baumannii, the gene coding for the proteases needed to be cloned to produce recombinant form of the proteins that can be easily expressed and purified in the quantities and purity suitable for functional and structural studies. A secreted serine protease was identified from A. baumannii, termed as “SPSFQ”. Bioinformatics analysis using BLAST and multiple sequence alignment indicated that the enzyme belonged to serine endopeptidases (E.C. 3.4.21.-) family with a predicted catalytic triad motif of D130/H163/S315. Structure of SPSFQ modelled using the homology modeling software, I-TASSER revealed that the enzyme folding was highly conserved to keratinase 5WSL with seven stranded parallel β sheets flanking by six α helices and four β sheets made of two anti-parallel strands. SPSFQ with 1104 bp coding for 368 amino acids was subcloned into pET-22b(+) between BamH1 and Sal1 and expressed in periplasmic fraction of E. coli BL21 (DE3). Total cell protein with induction condition at 16 ºC and 25 ºC with 1mM IPTG showed two distinct bands around 40 kDa (proenzyme form) and 30 kDa (active form) in western blot. Cell lysate did not show any activity during enzymatic assay probably because of SPSFQ was expressed in inclusion form. As a conclusion, SPSFQ was successfully sub-cloned and expressed in E. coli BL21 (DE3). Further study will focus on purification and characterization of SPSFQ in order to identify the cellular importance of SPSFQ towards A. baumannii

マアジ骨格筋における致死誘導性細胞生物学的変動に関する研究

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 潮 秀樹, 東京大学教授 金子 豊二, 東京大学教授 浅川 修一, 東京大学教授 松永 茂樹, 東京大学准教授 木下 滋晴University of Tokyo(東京大学