In the model yeast Saccharomyces cerevisiae, Trk1 is the main K+ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K+ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K+ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb+ . Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K+ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li+ ions.
- MeSH
- biologický transport MeSH
- draslík metabolismus MeSH
- fylogeneze MeSH
- proteiny přenášející kationty * genetika MeSH
- Saccharomyces cerevisiae - proteiny * metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- Yarrowia * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.
- MeSH
- DNA-helikasy * genetika metabolismus MeSH
- genetická transkripce MeSH
- lidé MeSH
- multifunkční enzymy genetika metabolismus MeSH
- neurodegenerativní nemoci MeSH
- R-smyčka MeSH
- regulace genové exprese MeSH
- RNA-helikasy * metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- terminace genetické transkripce * MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.
- MeSH
- DNA vazebné proteiny * genetika metabolismus MeSH
- DNA-topoisomerasy I genetika metabolismus MeSH
- endonukleasy * genetika metabolismus MeSH
- fosfodiesterasy * genetika metabolismus MeSH
- oprava DNA MeSH
- poškození DNA MeSH
- Saccharomyces cerevisiae - proteiny * genetika metabolismus MeSH
- Saccharomyces cerevisiae MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In baker's yeast (Saccharomyces cerevisiae), Trk1, a member of the superfamily of K-transporters (SKT), is the main K+ uptake system under conditions when its concentration in the environment is low. Structurally, Trk1 is made up of four domains, each similar and homologous to a K-channel α subunit. Because most K-channels are proteins containing four channel-building α subunits, Trk1 could be functional as a monomer. However, related SKT proteins TrkH and KtrB were crystallised as dimers, and for Trk1, a tetrameric arrangement has been proposed based on molecular modelling. Here, based on Bimolecular Fluorescence Complementation experiments and single-molecule fluorescence microscopy combined with molecular modelling; we provide evidence that Trk1 can exist in the yeast plasma membrane as a monomer as well as a dimer. The association of monomers to dimers is regulated by the K+ concentration.
- MeSH
- biologický transport MeSH
- buněčná membrána metabolismus MeSH
- draslík metabolismus MeSH
- fungální proteiny metabolismus MeSH
- proteiny přenášející kationty * genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny * genetika metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- translokace genetická MeSH
- transportní proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Chitin exists in yeast cells both as free and bound in a complex with β-1,3/β-1,6-glucan. The formation of covalent links between chitin and β-glucans is catalyzed by the enzymes Crh1 and Crh2, acting as transglycosylases. We found that N-acetyl-chito-oligosaccharides, as well as laminarioligosaccharides, the respective products of partial hydrolysis of chitin, and β-1,3-glucan, interfered with reactions catalyzed by Crh1p and Crh2p in vitro. However, the N-acetyl-chito-oligosaccharides did not influence the growth rate of the yeast, neither did they affect the yeast phenotype, but they prolonged the lag phase. Inhibition of Crh1 and Crh2 in vivo with oligosaccharides derived from chitin leads to an increase of alkali-soluble chitin and a decrease in the amount of chitin linked to β-glucans. In addition, yeast cells growing in the presence of N-acetyl-D-chito-oligosaccharides accumulated more chitin than control cells.
Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.
- MeSH
- DNA-helikasy metabolismus MeSH
- lidé MeSH
- proteosyntéza MeSH
- ribozomy metabolismus MeSH
- Saccharomyces cerevisiae - proteiny * genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika metabolismus MeSH
- transkripční faktory metabolismus MeSH
- transportní proteiny * metabolismus MeSH
- ubikvitinace MeSH
- ubikvitinligasy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Previously, the Nine amino acid TransActivation Domain (9aaTAD) was identified in the Gal4 region 862-870 (DDVYNYLFD). Here, we identified 9aaTADs in the distal Gal4 orthologs by our prediction algorithm and found their conservation in the family. The 9aaTAD function as strong activators was demonstrated. We identified adjacent Gal4 region 871-811 (DEDTPPNPKKE) as a natural 9aaTAD inhibitory domain located at the extreme Gal4 terminus. Moreover, we identified conserved Gal4 region 172-185 (FDWSEEDDMSDGLP), which was capable to reverse the 9aaTAD inhibition. In conclusion, our results uncover the existence of the cryptic inhibitory domains, which need to be carefully implemented in all functional studies with transcription factors to avoid incorrect conclusions.
The fungal cell wall, comprised primarily of protein and polymeric carbohydrate, maintains cell structure, provides protection from the environment, and is an important antifungal drug target. Pir proteins (proteins with internal repeats) are linked to cell wall β-1,3-glucan and are best studied in Saccharomyces cerevisiae. Sequential deletion of S. cerevisiae PIR genes produces strains with increasingly notable cell wall damage. However, a true null mutant lacking all five S. cerevisiae PIR genes was never constructed. Because only two PIR genes (PIR1, PIR32) were annotated in the Candida albicans genome, the initial goal of this work was to construct a true Δpir/Δpir null strain in this species. Unexpectedly, the phenotype of the null strain was almost indistinguishable from its parent, leading to the search for other proteins with Pir function. Bioinformatic approaches revealed nine additional C. albicans proteins that share a conserved Pir functional motif (minimally DGQ). Examination of the protein sequences revealed another conserved motif (QFQFD) toward the C-terminal end of each protein. Sequence similarities and presence of the conserved motif(s) were used to identify a set of 75 proteins across 16 fungal species that are proposed here as Pir proteins. The Pir family is greatly expanded in C. albicans and C. dubliniensis compared to other species and the orthologs are known to have specialized function during chlamydospore formation. Predicted Pir structures showed a conserved core of antiparallel beta-sheets and sometimes-extensive loops that contain amino acids with the potential to form linkages to cell wall components. Pir phylogeny demonstrated emergence of specific ortholog groups among the fungal species. Variation in gene expression patterns was noted among the ortholog groups during growth in rich medium. PIR allelic variation was quite limited despite the presence of a repeated sequence in many loci. Results presented here demonstrate that the Pir family is larger than previously recognized and lead to new hypotheses to test to better understand Pir proteins and their role in the fungal cell wall.
Yeast glucan particles (GPs) are promising agents for the delivery of biologically active compounds as drugs. GPs possess their own biological activities and can act synergistically with their cargo. This study aimed to determine how incorporating artemisinin, ellagic acid, (-)-epigallocatechin gallate, morusin, or trans-resveratrol into GPs affects their anti-inflammatory and antioxidant potential in vitro. Two different methods - slurry evaporation and spray drying - were used to prepare composites (GPs + bioactive compound) and the anti-inflammatory and antioxidative properties of the resultant products were compared. Several of the natural compounds showed the beneficial effects of being combined with GPs. The materials prepared by spray drying showed greater activity than those made using a rotary evaporator. Natural compounds incorporated into yeast GPs showed greater anti-inflammatory potential in vitro than simple suspensions of these compounds as demonstrated by their inhibition of the activity of transcription factors NF-κB/AP-1 and the secretion of the pro-inflammatory cytokine TNF-α.
- MeSH
- antiflogistika farmakologie MeSH
- antioxidancia farmakologie MeSH
- artemisininy farmakologie MeSH
- cytokiny MeSH
- flavonoidy chemie farmakologie MeSH
- glukany chemie farmakologie MeSH
- katechin analogy a deriváty chemie farmakologie MeSH
- kyselina ellagová chemie farmakologie MeSH
- monocyty účinky léků MeSH
- NF-kappa B MeSH
- resveratrol chemie farmakologie MeSH
- Saccharomyces cerevisiae - proteiny MeSH
- Saccharomyces cerevisiae MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions via changes in nuclear gene expression. The most comprehensive picture of components and regulation of retrograde signaling has been obtained in Saccharomyces cerevisiae, where retrograde-target gene expression is regulated by RTG genes. In this chapter, we describe methods to measure mitochondrial retrograde pathway activation at the level of mRNA and protein products in yeast model systems, including cell suspensions and microcolonies. In particular, we will focus on three major procedures: mRNA levels of RTG-target genes, such as those encoding for peroxisomal citrate synthase (CIT2), aconitase, and NAD+-specific isocitrate dehydrogenase subunit 1 by real-time PCR; expression analysis of CIT2-gene protein product (Cit2p-GFP) by Western blot and fluorescence microscopy; the phosphorylation status of transcriptional factor Rtg1/3p which controls RTG-target gene transcription.
- MeSH
- akonitáthydratasa genetika metabolismus MeSH
- buněčné jádro genetika metabolismus MeSH
- citrátsynthasa genetika metabolismus MeSH
- fosforylace MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- isocitrátdehydrogenasa genetika metabolismus MeSH
- mitochondrie metabolismus patologie MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- signální transdukce MeSH
- transkripční faktory BHLH-Zip metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
