Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.
- MeSH
- "flap" endonukleasy genetika metabolismus terapeutické užití MeSH
- enzymy opravy DNA genetika MeSH
- exodeoxyribonukleasy genetika MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- lidé MeSH
- nádorový supresorový protein p53 * genetika metabolismus MeSH
- nádory * farmakoterapie genetika MeSH
- oprava DNA MeSH
- PARP inhibitory farmakologie MeSH
- poškození DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Until recently, glycosidases, naturally hydrolyzing carbohydrate-active enzymes, have found few synthetic applications in industry, being primarily used for cleaving unwanted carbohydrates. With the establishment of glycosynthase and transglycosidase technology by genetic engineering, the view of glycosidases as industrial biotechnology tools has started to change. Their easy production, affordability, robustness, and substrate versatility, added to the possibility of controlling undesired side hydrolysis by enzyme engineering, have made glycosidases competitive synthetic tools. Current promising applications of engineered glycosidases include the production of well-defined chitooligomers, precious galactooligosaccharides or specialty chemicals such as glycosylated flavonoids. Other synthetic pathways leading to human milk oligosaccharides or remodeled antibodies are on the horizon. This work provides an overview of the synthetic achievements to date for glycosidases, emphasizing the latest trends and outlining possible developments in the field.
Until recently, glycosidases, naturally hydrolyzing carbohydrate-active enzymes, have found few synthetic applications in industry, being primarily used for cleaving unwanted carbohydrates. With the establishment of glycosynthase and transglycosidase technology by genetic engineering, the view of glycosidases as industrial biotechnology tools has started to change. Their easy production, affordability, robustness, and substrate versatility, added to the possibility of controlling undesired side hydrolysis by enzyme engineering, have made glycosidases competitive synthetic tools. Current promising applications of engineered glycosidases include the production of well-defined chitooligomers, precious galactooligosaccharides or specialty chemicals such as glycosylated flavonoids. Other synthetic pathways leading to human milk oligosaccharides or remodeled antibodies are on the horizon. This work provides an overview of the synthetic achievements to date for glycosidases, emphasizing the latest trends and outlining possible developments in the field.
- MeSH
- glykosidhydrolasy * genetika metabolismus MeSH
- glykosylace MeSH
- hydrolýza MeSH
- lidé MeSH
- oligosacharidy * MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.
- MeSH
- buněčné jádro metabolismus MeSH
- Caenorhabditis elegans genetika metabolismus MeSH
- DNA metabolismus MeSH
- dvouřetězcové zlomy DNA * MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- oprava DNA fyziologie MeSH
- poly-ADP-ribosylace MeSH
- polyadenosindifosfátribosa metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- zárodečné buňky MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair.
- MeSH
- adenosindifosfát ribosa chemie MeSH
- chromatin chemie MeSH
- fibroblasty MeSH
- genový knockout MeSH
- glykosidhydrolasy genetika MeSH
- HEK293 buňky MeSH
- histony chemie MeSH
- jednořetězcové zlomy DNA * MeSH
- lidé MeSH
- mutace * MeSH
- nádorové buněčné linie MeSH
- neurodegenerativní nemoci genetika MeSH
- oprava DNA * MeSH
- protein XRCC1 genetika MeSH
- regulace genové exprese MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rutinosidases (α-l-rhamnosyl-β-d-glucosidases) catalyze the cleavage of the glycosidic bond between the aglycone and the disaccharide rutinose (α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranose) of specific flavonoid glycosides such as rutin (quercetin 3-O-rutinoside). Microbial rutinosidases are part of the rutin catabolic pathway, enabling the microorganism to utilize rutin and related plant phenolic glycosides. Here, we report the first three-dimensional structure of a rutinosidase determined at 1.27-Å resolution. The rutinosidase from Aspergillus niger K2 (AnRut), a member of glycoside hydrolase family GH-5, subfamily 23, was heterologously produced in Pichia pastoris. The X-ray structure of AnRut is represented by a distorted (β/α)8 barrel fold with its closest structural homologue being an exo-β-(1,3)-glucanase from Candida albicans (CaExg). The catalytic site is located in a deep pocket with a striking structural similarity to CaExg. However, the entrance to the active site of AnRut has been found to be different from that of CaExg - a mostly unstructured section of ~ 40 residues present in CaExg is missing in AnRut, whereas an additional loop of 13 amino acids partially covers the active site of AnRut. NMR analysis of reaction products provided clear evidence for a retaining reaction mechanism of AnRut. Unexpectedly, quercetin 3-O-glucoside was found to be a better substrate than rutin, and thus, AnRut cannot be considered a typical diglycosidase. Mutational analysis of conserved active site residues in combination with in silico modeling allowed identification of essential interactions for enzyme activity and helped to reveal further details of substrate binding. The protein sequence of AnRut has been revised. DATABASES: The nucleotide sequence of the rutinosidase-encoding gene is available in the GenBank database under the accession number MN393234. Structural data are available in the PDB database under the accession number 6I1A. ENZYME: α-l-Rhamnosyl-β-d-glucosidase (EC 3.2.1.168).
- MeSH
- Aspergillus niger enzymologie MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- glykosidhydrolasy chemie genetika metabolismus MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- mutace MeSH
- oxidace-redukce MeSH
- rutin chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AbstractDiglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-β-glucosidase I and II (αRβG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-β-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRβG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRβG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRβG I. On the other hand, αRβG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRβG I showed exclusive specificity toward 7-O-β-rutinosyl flavonoids, whereas αRβG II hydrolyzed both 7-O-β-rutinosyl- and 3-O-β-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-β-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRβG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.
- MeSH
- Acremonium enzymologie genetika MeSH
- flavonoidy metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- fylogeneze MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- molekulová hmotnost MeSH
- Pichia genetika MeSH
- rekombinantní proteiny metabolismus MeSH
- sekvenční analýza DNA MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-β-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-l-rhamnopyranosyl-β-d-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4-hydroxyphenyl-β-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-β-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 °C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.
Soil microorganisms are important mediators of carbon cycling in nature. Although cellulose- and hemicellulose-degrading bacteria have been isolated from Algerian ecosystems, the information on the composition of soil bacterial communities and thus the potential of their members to decompose plant residues is still limited. The objective of the present study was to describe and compare the bacterial community composition in Algerian soils (crop, forest, garden, and desert) and the activity of cellulose- and hemicellulose-degrading enzymes. Bacterial communities were characterized by high-throughput 16S amplicon sequencing followed by the in silico prediction of their functional potential. The highest lignocellulolytic activity was recorded in forest and garden soils whereas activities in the agricultural and desert soils were typically low. The bacterial phyla Proteobacteria (in particular classes α-proteobacteria, δ-proteobacteria, and γ-proteobacteria), Firmicutes, and Actinobacteria dominated in all soils. Forest and garden soils exhibited higher diversity than agricultural and desert soils. Endocellulase activity was elevated in forest and garden soils. In silico analysis predicted higher share of genes assigned to general metabolism in forest and garden soils compared with agricultural and arid soils, particularly in carbohydrate metabolism. The highest potential of lignocellulose decomposition was predicted for forest soils, which is in agreement with the highest activity of corresponding enzymes.
- MeSH
- Bacteria klasifikace enzymologie genetika izolace a purifikace MeSH
- bakteriální proteiny genetika metabolismus MeSH
- celulasa genetika metabolismus MeSH
- ekosystém MeSH
- fylogeneze MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- lesy MeSH
- půda chemie MeSH
- půdní mikrobiologie * MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Alžírsko MeSH
The concept of operational taxonomic units (OTUs), which constructs "mathematically" defined taxa, is widely accepted and applied to describe bacterial communities using amplicon sequencing of 16S rRNA gene. OTUs are often used to infer functional traits since they are considered to fairly represent of community members. However, the link between molecular taxa, real taxa, and OTUs seems to be much more complicated. Strains of the same bacterial species (ideally belonging to the same OTU) typically only share some genes (the core genome), while other genes are strain-specific and unique. It is thus unclear to what extent are important functional traits homogeneous within an OTU and how correctly can functional traits be inferred for individual OTU members. Here, we have tested in silico the similarity of all genes and, more specifically, the set of genes encoding for glycoside hydrolases (GH) in bacterial genomes that belong to the same OTU. Genome similarity varied among OTUs, but as many as 5-78% of genes were not shared between the two bacterial genomes in the pair. The complement of GH families (the presence of gene families and the number of genes per family) differed in 95% of OTUs. In average, 43% of GH families either differed in gene counts or were present in one genome and absent in the other. These results show a serious limitation of the OTU-based approaches when used to infer the functional traits of bacterial communities and open the questions how to link environmental sequencing data and microbial functions.
- MeSH
- Bacteria klasifikace genetika MeSH
- bakteriální geny genetika MeSH
- databáze nukleových kyselin MeSH
- DNA bakterií genetika MeSH
- fylogeneze MeSH
- genetická variace MeSH
- genom bakteriální genetika MeSH
- glykosidhydrolasy genetika MeSH
- metagenomika * MeSH
- mikrobiota MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
