scholarly journals Involvement of the Saccharomyces cerevisiae Hydrolase Ldh1p in Lipid Homeostasis

2011 ◽  
Vol 10 (6) ◽  
pp. 776-781 ◽  
Author(s):  
Mykhaylo O. Debelyy ◽  
Sven Thoms ◽  
Melanie Connerth ◽  
Günther Daum ◽  
Ralf Erdmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lipid Droplets ◽  
Functional Characterization ◽  
State Level ◽  
Lipid Homeostasis ◽  
Nonpolar Lipid ◽  
Triacylglycerol Lipase ◽  
Content Type ◽  
Nonpolar Lipids

ABSTRACTHere, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of theSaccharomyces cerevisiaeΔldh1strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels.

scholarly journals The Putative Saccharomyces cerevisiae Hydrolase Ldh1p Is Localized to Lipid Droplets

2011 ◽  
Vol 10 (6) ◽  
pp. 770-775 ◽  
Author(s):  
Sven Thoms ◽  
Mykhaylo O. Debelyy ◽  
Melanie Connerth ◽  
Günther Daum ◽  
Ralf Erdmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Subcellular Localization ◽  
Lipid Droplets ◽  
Peroxisomal Enzyme ◽  
Content Type ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Nonpolar Lipids ◽  
Peroxisomal Targeting Signal ◽  
Hydrolase Family

ABSTRACT Here, we report the identification of a novel hydrolase in Saccharomyces cerevisiae . Ldh1p (systematic name, Ybr204cp) comprises the typical GXSXG-type lipase motif of members of the α/β-hydrolase family and shares some features with the peroxisomal lipase Lpx1p. Both proteins carry a putative peroxisomal targeting signal type1 (PTS1) and can be aligned with two regions of homology. While Lpx1p is known as a peroxisomal enzyme, subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets, the storage compartment of nonpolar lipids. Ldh1p is not required for the function and biogenesis of peroxisomes, and targeting of Ldh1p to lipid droplets occurs independently of the PTS1 receptor Pex5p.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Brooke D. Esquivel ◽  
Jeffrey M. Rybak ◽  
Katherine S. Barker ◽  
Jarrod R. Fortwendel ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Substrate Specificity ◽  
Abc Transporter ◽  
Drug Susceptibility ◽  
Antifungal Drugs ◽  
Efflux Transporters ◽  
Content Type ◽  
Background Strain

ABSTRACT This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable. IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae

Biochimie ◽  
1996 ◽  
Vol 78 (2) ◽  
pp. 144-152 ◽  
Author(s):  
M. Góra ◽  
A. Chaciñska ◽  
J. Rytka ◽  
R. Labbe-Bois
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Characterization

scholarly journals Functional Characterization of AbaQ, a Novel Efflux Pump Mediating Quinolone Resistance inAcinetobacter baumannii

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
María Pérez-Varela ◽  
Jordi Corral ◽  
Jesús Aranda ◽  
Jordi Barbé
Keyword(s):  
Acinetobacter Baumannii ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Functional Characterization ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Nosocomial Pathogen ◽  
Content Type ◽  
Mfs Transporter

ABSTRACTAcinetobacter baumanniihas emerged as an important multidrug-resistant nosocomial pathogen. In previous work, we identified a putative MFS transporter, AU097_RS17040, involved in the pathogenicity ofA. baumannii(M. Pérez-Varela, J. Corral, J. A. Vallejo, S. Rumbo-Feal, G. Bou, J. Aranda, and J. Barbé, Infect Immun 85:e00327-17, 2017,https://doi.org/10.1128/IAI.00327-17). In this study, we analyzed the susceptibility to diverse antimicrobial agents ofA. baumanniicells defective in this transporter, referred to as AbaQ. Our results showed that AbaQ is mainly involved in the extrusion of quinolone-type drugs inA. baumannii.

Use of Monoclonal Antibodies in the Functional Characterization of the Saccharomyces Cerevisiae Sepl Protein

1995 ◽  
Vol 231 (2) ◽  
pp. 329-336 ◽  
Author(s):  
Anita Holler ◽  
Vladimir I. Bashkirov ◽  
Jachen A. Solinger ◽  
Ursula Reinhart ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Monoclonal Antibodies ◽  
Functional Characterization

scholarly journals A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Minyeong Yoo ◽  
Gwenaelle Bestel-Corre ◽  
Christian Croux ◽  
Antoine Riviere ◽  
Isabelle Meynial-Salles ◽  
...  
Keyword(s):  
Steady State ◽  
Metabolic Engineering ◽  
Clostridium Acetobutylicum ◽  
Functional Characterization ◽  
Scale Model ◽  
Primary Metabolism ◽  
Scale Analysis ◽  
Content Type ◽  
Commercial Process

ABSTRACTEngineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacteriumClostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model,iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering ofC. acetobutylicumto develop a commercial process for the production ofn-butanol.IMPORTANCECurrently, there is a resurgence of interest inClostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to producen-butanol for use both as a bulk chemical and as a renewable alternative transportation fuel. To develop a commercial process for the production ofn-butanol via a metabolic engineering approach, it is necessary to better characterize both the primary metabolism ofC. acetobutylicumand its regulation. Here, we apply a quantitative system-scale analysis to acidogenic, solventogenic, and alcohologenic steady-stateC. acetobutylicumcells and report for the first time quantitative transcriptomic, proteomic, and fluxomic data. This approach allows for a better understanding of the regulation of primary metabolism and for the functional characterization of numerous enzymes involved in primary metabolism.

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