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1.
Nat Struct Mol Biol ; 2024 Jul 08.
Article in English | MEDLINE | ID: mdl-38977901

ABSTRACT

Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48-K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48-K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48-K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48-K63-linked Ub, these results suggest a function for K48-K63-branched chains in VCP/p97-related processes.

2.
Sci Adv ; 10(13): eadh0123, 2024 03 29.
Article in English | MEDLINE | ID: mdl-38536929

ABSTRACT

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.


Subject(s)
Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligases , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Ubiquitin/metabolism , Protein Processing, Post-Translational
3.
Cell Rep Methods ; 4(2): 100712, 2024 Feb 26.
Article in English | MEDLINE | ID: mdl-38382522

ABSTRACT

Parkinson's disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Loss-of-function mutations in the gene encoding the ubiquitin E3 ligase Parkin are causal for familial and juvenile PD. Among several therapeutic approaches being explored to treat or improve the prognosis of patients with PD, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high-throughput platforms for the robust and accurate quantification of Parkin activity in vitro. Here, we present two different and complementary Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS)-based approaches for the quantification of Parkin E3 ligase activity in vitro. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.


Subject(s)
Parkinson Disease , Ubiquitin-Protein Ligases , Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Ubiquitin/genetics , Mutation , Parkinson Disease/diagnosis
4.
Front Mol Biosci ; 10: 1184934, 2023.
Article in English | MEDLINE | ID: mdl-37234921

ABSTRACT

The attachment of ubiquitin to a substrate (ubiquitination or ubiquitylation) impacts its lifetime and regulates its function within the cell. Several classes of enzymes oversee the attachment of ubiquitin to the substrate: an E1 activating enzyme that makes ubiquitin chemically susceptible prior to the following stages of conjugation and ligation, respectively mediated by E2 conjugating enzymes (E2s) and E3 ligases (E3s). Around 40 E2s and more than 600 E3s are encoded in the human genome, and their combinatorial and cooperative behaviour dictate the tight specificity necessary for the regulation of thousands of substrates. The removal of ubiquitin is orchestrated by a network of about 100 deubiquitylating enzymes (DUBs). Many cellular processes are tightly controlled by ubiquitylation, which is essential in maintaining cellular homeostasis. Because of the fundamental role(s) of ubiquitylation, there is an interest in better understanding the function and specificity of the ubiquitin machinery. Since 2014, an expanding array of Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Mass Spectrometry (MS) assays have been developed to systematically characterise the activity of a variety of ubiquitin enzymes in vitro. Here we recapitulate how MALDI-TOF MS aided the in vitro characterization of ubiquitin enzymes and the discovery of new and unexpected of E2s and DUBs functions. Given the versatility of the MALDI-TOF MS approach, we foreseen the use of this technology to further expand our understanding of ubiquitin and ubiquitin-like enzymes.

5.
Methods Mol Biol ; 2591: 123-134, 2023.
Article in English | MEDLINE | ID: mdl-36350546

ABSTRACT

Ubiquitylation (or ubiquitination) is the reversible conjugation of a 76-amino-acid polypeptide (ubiquitin) to a target protein to modulate various biological processes. Deubiquitylating enzymes (DUBs) are a class of enzymes that specifically remove ubiquitin from a substrate. In recent years DUBs have garnered significant attention as a new class of targets in multiple therapeutic areas. The recent development of high-throughput Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) has provided new tools to perform drug discovery screening. Here we present a facile and high-throughput step-by-step protocol of the MALDI-TOF MS-based DUB assay for screening the activity of DUBs in vitro. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains. The presented protocol takes advantage of nanoliter dispensing robotics and automated MALDI-TOF MS analysis to screen large and diverse compound libraries.


Subject(s)
Drug Discovery , High-Throughput Screening Assays , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , High-Throughput Screening Assays/methods , Drug Discovery/methods , Ubiquitin/metabolism , Enzyme Assays
6.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Article in English | MEDLINE | ID: mdl-33479176

ABSTRACT

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado-Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.


Subject(s)
Deubiquitinating Enzymes/genetics , Esterases/genetics , Machado-Joseph Disease/genetics , Ubiquitin/genetics , Amino Acids/genetics , Deubiquitinating Enzymes/isolation & purification , Humans , Lysine/genetics , Machado-Joseph Disease/enzymology , Machado-Joseph Disease/pathology , Mass Spectrometry , Protein Processing, Post-Translational/genetics , Ubiquitination/genetics
7.
Nat Protoc ; 15(12): 4034-4057, 2020 12.
Article in English | MEDLINE | ID: mdl-33139956

ABSTRACT

Deubiquitylating enzymes (DUBs) play a vital role in the ubiquitin pathway by editing or removing ubiquitin from their substrate. As breakthroughs within the ubiquitin field continue to highlight the potential of deubiquitylating enzymes as drug targets, there is increasing demand for versatile high-throughput (HT) tools for the identification of potent and selective DUB modulators. Here we present the HT adaptation of the previously published MALDI-TOF-based DUB assay method. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains by DUBs. The method has been specifically developed for use with nanoliter-dispensing robotics to meet drug discovery requirements for the screening of large and diverse compound libraries. Contrary to the most common DUB screening technologies currently available, the MALDI-TOF DUB assay combines the use of physiological substrates with the sensitivity and reliability of the mass spectrometry-based readout.


Subject(s)
Drug Discovery/methods , Enzyme Assays/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Ubiquitination , Humans
8.
Sci Rep ; 8(1): 13146, 2018 09 03.
Article in English | MEDLINE | ID: mdl-30177856

ABSTRACT

Prostate cancer (PCa), a leading cause of cancer-related death in men, becomes resistant to androgen deprivation therapy by inducing androgen receptor (AR) activity, which is known as castration-resistant PCa (CRPC). Enzalutamide is an approved drug that inhibits AR activity and increases overall survival. However, resistance to enzalutamide develops rapidly often by increasing AR activity, suggesting that new therapies are required for CRPC. We investigated whether betulinic acid (BA), a small molecule from plants that inhibits multiple deubiquitinases (DUBs), reduces AR, and selectively kills PCa cells, can provide an adjuvant strategy for CRPC. Our data indicated that BA reduced AR protein stability and mRNA expression, making it an attractive agent for CRPC. BA decreased AR mRNA possibly by inhibiting a histone 2A DUB thereby increasing ubiquitinated histone 2A, a transcriptional repressor. We identified multiple and specific DUBs inhibited by BA either in PCa cells or using recombinant DUBs. Similar results were obtained using another multi-DUB inhibitor WP1130, suggesting that these DUB inhibitors can decrease AR expression and increase PCa-specific death. Our results also suggest that combining multi-DUB inhibitors BA or WP1130 with enzalutamide may provide a novel strategy for CRPC by further decreasing AR expression and increasing apoptotic cell death.


Subject(s)
Androgen Receptor Antagonists/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Deubiquitinating Enzymes/genetics , Gene Expression Regulation, Neoplastic , Prostate/drug effects , Receptors, Androgen/genetics , Triterpenes/pharmacology , Animals , Benzamides , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Cyanoacrylates/pharmacology , Deubiquitinating Enzymes/antagonists & inhibitors , Deubiquitinating Enzymes/metabolism , Drug Combinations , Drug Synergism , Histones/genetics , Histones/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mice , Mice, Transgenic , Nitriles , Pentacyclic Triterpenes , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Stability/drug effects , Pyridines/pharmacology , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Androgen/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Betulinic Acid
9.
Cell Chem Biol ; 25(9): 1117-1127.e4, 2018 09 20.
Article in English | MEDLINE | ID: mdl-30017913

ABSTRACT

Due to their role in many diseases, enzymes of the ubiquitin system have recently become interesting drug targets. Despite efforts, primary screenings of compound libraries targeting E2 enzymes and E3 ligases have been strongly limited by the lack of robust and fast high-throughput assays. Here we report a label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry. The MALDI-TOF E2/E3 assay allows testing E2 enzymes and E3 ligases for their ubiquitin transfer activity, identifying E2/E3 active pairs, inhibitor potency and specificity and screening compound libraries in vitro without chemical or fluorescent probes. We demonstrate that the MALDI-TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared with standard biochemical assays.


Subject(s)
Drug Discovery/methods , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , High-Throughput Screening Assays/methods , Humans , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitination/drug effects
10.
Mol Cell ; 70(1): 150-164.e6, 2018 04 05.
Article in English | MEDLINE | ID: mdl-29576527

ABSTRACT

Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.


Subject(s)
Cell Nucleus/enzymology , DNA Damage , Deubiquitinating Enzymes/metabolism , Genomic Instability , Polyubiquitin/metabolism , Binding Sites , Cell Survival , Deubiquitinating Enzymes/chemistry , Deubiquitinating Enzymes/genetics , HEK293 Cells , HeLa Cells , Humans , Jurkat Cells , Lysine , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Structure-Activity Relationship , Substrate Specificity , Ubiquitination
11.
Nat Chem Biol ; 13(12): 1207-1215, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28967922

ABSTRACT

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.


Subject(s)
Antineoplastic Agents/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Thiophenes/pharmacology , Ubiquitin Thiolesterase/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/metabolism , Animals , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Molecular Structure , Mutation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Protein Kinase Inhibitors/chemistry , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Thiophenes/chemistry , Tumor Cells, Cultured , Ubiquitin/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , fms-Like Tyrosine Kinase 3/genetics
12.
Cell Chem Biol ; 24(4): 458-470.e18, 2017 Apr 20.
Article in English | MEDLINE | ID: mdl-28343940

ABSTRACT

USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3ß-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types.


Subject(s)
Cyclin D1/metabolism , Endopeptidases/metabolism , G1 Phase Cell Cycle Checkpoints/drug effects , Lithocholic Acid/analogs & derivatives , Catalytic Domain , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin D1/antagonists & inhibitors , Cyclin D1/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cycloheximide/chemistry , Cycloheximide/pharmacology , Down-Regulation/drug effects , Endopeptidases/chemistry , Endopeptidases/genetics , Glycogen Synthase Kinase 3 beta/metabolism , HCT116 Cells , Humans , Lithocholic Acid/pharmacology , MCF-7 Cells , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Signal Transduction/drug effects , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin Thiolesterase
13.
Cell Rep ; 16(4): 1180-1193, 2016 07 26.
Article in English | MEDLINE | ID: mdl-27425610

ABSTRACT

Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.


Subject(s)
Deubiquitinating Enzymes/genetics , Deubiquitinating Enzymes/metabolism , Phosphorylation/genetics , Ubiquitin/genetics , Ubiquitin/metabolism , Binding Sites/genetics , Humans , Serine/genetics , Serine/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
14.
PLoS Pathog ; 12(6): e1005735, 2016 06.
Article in English | MEDLINE | ID: mdl-27352036

ABSTRACT

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently ('specialised') or non-covalently ('cargo' effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a 'core' T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery.


Subject(s)
Bacterial Proteins/metabolism , Serratia marcescens/pathogenicity , Type VI Secretion Systems/metabolism , Amino Acid Sequence , Chromatography, Affinity , Coculture Techniques , Escherichia coli , Immunoblotting , Mass Spectrometry , Proteomics , Serratia marcescens/metabolism
15.
Appl Environ Microbiol ; 80(7): 2176-85, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24487536

ABSTRACT

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30 to 76% of the cases of neonatal meningitis. In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low-, and non-biofilm-forming strains, and to facilitate interpretation of data. This protocol was used to screen the biofilm-forming abilities of 366 GBS clinical isolates from pregnant women and from neonatal infections of different serotypes in relation to medium composition and pH. The results identified a subset of isolates of serotypes III and V that formed strong biofilms under acidic conditions. Importantly, the best biofilm formers belonged to serotype III hypervirulent clone ST-17. Moreover, the abilities of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm initiation and contribute to biofilm structural stability.


Subject(s)
Biofilms/growth & development , Streptococcus agalactiae/drug effects , Streptococcus agalactiae/physiology , Bacterial Proteins/metabolism , Bacteriological Techniques/methods , Endopeptidase K/metabolism , Female , Humans , Hydrogen-Ion Concentration , Infant, Newborn , Mass Screening/methods , Pregnancy , Proteolysis , Streptococcal Infections/microbiology , Streptococcus agalactiae/isolation & purification
16.
FEBS J ; 281(1): 175-90, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24164706

ABSTRACT

The COP9 signalosome (CSN) is a highly conserved eukaryotic protein complex which regulates the cullin RING family of ubiquitin ligases and carries out a deneddylase activity that resides in subunit 5 (CSN5). Whereas CSN activity is essential for the development of higher eukaryotes, several unicellular fungi including the budding yeast Saccharomyces cerevisiae can survive without a functional CSN. Nevertheless, the budding yeast CSN is biochemically active and deletion mutants of each of its subunits exhibit deficiency in cullins deneddylation, although the biological context of this activity is still unknown in this organism. To further characterize CSN function in budding yeast, we present here a transcriptomic and proteomic analysis of a S. cerevisiae strain deleted in the CSN5/RRI1 gene (hereafter referred to as CSN5), coding for the only canonical subunit of the complex. We show that Csn5 is involved in modulation of the genes controlling amino acid and lipid metabolism and especially ergosterol biosynthesis. These alterations in gene expression correlate with the lower ergosterol levels and increased intracellular zinc content which we observed in csn5 null mutant cells. We show that some of these regulatory effects of Csn5, in particular the control of isoprenoid biosynthesis, are conserved through evolution, since similar transcriptomic and/or proteomic effects of csn5 mutation were previously observed in other eukaryotic organisms such as Aspergillus nidulans, Arabidopsis thaliana and Drosophila melanogaster. Our results suggest that the diverged budding yeast CSN is more conserved than was previously thought.


Subject(s)
Cullin Proteins/metabolism , Gene Expression Profiling , Lipid Metabolism , Metalloendopeptidases/metabolism , Multiprotein Complexes/metabolism , Peptide Hydrolases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transition Elements/metabolism , Biomarkers/metabolism , Blotting, Western , COP9 Signalosome Complex , Chromatography, Gas , Chromatography, Liquid , Ergosterol/metabolism , Metalloendopeptidases/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Tandem Mass Spectrometry , Zinc/metabolism
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