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Coupling proteomics and metabolomics for the unsupervised identification of protein-metabolite interactions in Chaetomium thermophilum.

ABSTRACT: Protein-metabolite interactions play an important role in the cell's metabolism and many methods have been developed to screen them in vitro. However, few methods can be applied at a large scale and not alter biological state. Here we describe a proteometabolomic approach, using chromatography to generate cell fractions which are then analyzed with mass spectrometry for both protein and metabolite identification. Integrating the proteomic and metabolomic analyses makes it possible to identify protein-bound metabolites. Applying the concept to the thermophilic fungus Chaetomium thermophilum, we predict 461 likely protein-metabolite interactions, most of them novel. As a proof of principle, we experimentally validate a predicted interaction between the ribosome and isopentenyl adenine.

SUBMITTER: Li Y

PROVIDER: S-EPMC8270407 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Coupling proteomics and metabolomics for the unsupervised identification of protein-metabolite interactions in Chaetomium thermophilum.

Li Yuanyue Y Kuhn Michael M Zukowska-Kasprzyk Joanna J Hennrich Marco L ML Kastritis Panagiotis L PL O'Reilly Francis J FJ Phapale Prasad P Beck Martin M Gavin Anne-Claude AC Bork Peer P

PloS one 20210709 7

Protein-metabolite interactions play an important role in the cell's metabolism and many methods have been developed to screen them in vitro. However, few methods can be applied at a large scale and not alter biological state. Here we describe a proteometabolomic approach, using chromatography to generate cell fractions which are then analyzed with mass spectrometry for both protein and metabolite identification. Integrating the proteomic and metabolomic analyses makes it possible to identify pr ...[more]

PMID: 34242379

Similar Datasets

Proteometabolomics for unsupervised identification of protein–metabolite interactions

Project description:Protein–metabolite interactions play an important role in the cell’s metabolism and many methods have been developed to screen them in vitro. However, few methods can be applied at a large scale and not alter biological state. Here we describe a proteometabolomic approach, using chromatography to generate cell fractions which are then analyzed with mass spectrometry for both protein and metabolite identification. Integrating the proteomic and metabolomic analyses makes it possible to identify protein-bound metabolites. Applying the concept to the thermophilic fungus Chaetomium thermophilum, we predict many likely protein-metabolite interactions, most of them novel. As a proof of principle, we experimentally validate a predicted interaction between the ribosome and isopentenyl adenine.

2021-04-27 | PXD025571 | Pride

Identification and characterization of sugar-regulated promoters in Chaetomium thermophilum.

Project description:The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.

| S-EPMC10329369 | biostudies-literature

Identification and characterization of sugar regulated promoters in Chaetomium thermophilum

Project description:The thermophilic fungus Chaetomium thermophilum has been successfully used in the past for biochemical and high resolution structural studies of protein complexes, but subsequent functional analysis of these assemblies were hindered due to the lack of genetic tools in this thermophile, which are typically amenable in several other mesophilic eukaryotic model organisms, in particular the yeast Saccharomycers cerevisiae. Hence, we aimed to develop a regulatable gene-expression system in C. thermophilum, which might facilitate such in vivo studies, based on what we know about the galactose-inducible GAL promoter in yeast. To identify sugar-regulatable promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes induced by xylose but repressed by glucose. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of the thermostable YFP (yellow fluorescent protein) reporter. In this way, we could demonstrate xylose-dependent YFP expression by either western blotting or life cell imaging fluorescence microscopy. Prompted by these results, we finally expressed a well-characterized dominant-negative ribosome assembly factor mutant, rsa4 E117>D, under the control of the XDH promoter, which allowed us to induce a nuclear export defect of the pre-60S subunit when C. thermophilum cells were grown in xylose but not glucose containing medium. Altogether, our study recognized xylose-regulatable promoters in Chaetomium thermophilum, which may foster functional studies of genes of interest in this thermophilic eukaryotic model organism.

2022-09-26 | GSE214043 | GEO

Structural characterisation of the Chaetomium thermophilum Chl1 helicase.

Project description:Chl1 is a member of the XPD family of 5'-3' DNA helicases, which perform a variety of roles in genome maintenance and transmission. They possess a variety of unique structural features, including the presence of a highly variable, partially-ordered insertion in the helicase domain 1. Chl1 has been shown to be required for chromosome segregation in yeast due to its role in the formation of persistent chromosome cohesion during S-phase. Here we present structural and biochemical data to show that Chl1 has the same overall domain organisation as other members of the XPD family, but with some conformational alterations. We also present data suggesting the insert domain in Chl1 regulates its DNA binding.

| S-EPMC8109800 | biostudies-literature

Biochemical characterization of a GDP-mannose transporter from Chaetomium thermophilum.

Project description:Nucleotide Sugar Transporters (NSTs) belong to the SLC35 family (human solute carrier) of membrane transport proteins and are crucial components of the glycosylation machinery. NSTs are localized in the ER and Golgi apparatus membranes, where they accumulate nucleotide sugars from the cytosol for subsequent polysaccharide biosynthesis. Loss of NST function impacts the glycosylation of cell surface molecules. Mutations in NSTs cause several developmental disorders, immune disorders, and increased susceptibility to infection. Atomic resolution structures of three NSTs have provided a blueprint for a detailed molecular interpretation of their biochemical properties. In this work, we have identified, cloned, and expressed 18 members of the SLC35 family from various eukaryotic organisms in Saccharomyces cerevisiae. Out of 18 clones, we determined Vrg4 from Chaetomium thermophilum (CtVrg4) is a GDP-mannose transporter with an enhanced melting point temperature (Tm) of 56.9°C, which increases with the addition of substrates, GMP and GDP-mannose. In addition, we report-for the first time-that the CtVrg4 shows an affinity to bind to phosphatidylinositol lipids.

| S-EPMC10118193 | biostudies-literature

Structure of the catalytic domain of Mre11 from Chaetomium thermophilum.

Project description:Together with the Rad50 ATPase, the Mre11 nuclease forms an evolutionarily conserved protein complex that plays a central role in the repair of DNA double-strand breaks (DSBs). Mre11-Rad50 detects and processes DNA ends, and has functions in the tethering as well as the signalling of DSBs. The Mre11 dimer can bind one or two DNA ends or hairpins, and processes DNA endonucleolytically as well as exonucleolytically in the 3'-to-5' direction. Here, the crystal structure of the Mre11 catalytic domain dimer from Chaetomium thermophilum (CtMre11(CD)) is reported. CtMre11(CD) crystals diffracted to 2.8 Å resolution and revealed previously undefined features within the dimer interface, in particular fully ordered eukaryote-specific insertion loops that considerably expand the dimer interface. Furthermore, comparison with other eukaryotic Mre11 structures reveals differences in the conformations of the dimer and the capping domain. In summary, the results reported here provide new insights into the architecture of the eukaryotic Mre11 dimer.

| S-EPMC4461342 | biostudies-literature

Global Transcriptome Characterization and Assembly of thermophilic ascomycete Chaetomium thermophilum

Project description:A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome of Chaetomium thermophilum has been reported previously, but it is limited to open reading frames (ORFs) of genes and contains only a few noncoding transcripts. In this study, we identified and annotated by deep RNA sequencing full-length transcripts of C.thermophilum. We identified 7044 coding genes and a large number of noncoding genes (n=4567). Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help understanding the gene regulatory landscape in C.thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species. The new genome annotation of the GTF file can be found here.

2019-12-31 | GSE116834 | GEO

Expression, purification and crystallization of Chaetomium thermophilum Cu,Zn superoxide dismutase.

Project description:Cu,Zn superoxide dismutase (Cu,ZnSOD) from the thermophilic fungus Chaetomium thermophilum was expressed in Pichia pastoris and purified. Crystals were grown in over 120 conditions but only those produced with 1.4 M sodium potassium phosphate pH 8.2 as precipitant were suitable for structural studies. Data were collected to 1.9 A resolution at 100 K from a single crystal using a synchrotron-radiation source. The crystals belonged to space group P6(1)/P6(5), with unit-cell parameters a=90.2, c=314.5 A and eight molecules in the asymmetric unit. Elucidation of the crystal structure will provide insights into the active site of the enzyme and a better understanding of the structure-activity relationship, assembly and thermal stability of Cu,ZnSODs.

| S-EPMC2935235 | biostudies-literature

Global Transcriptome Characterization and Assembly of the Thermophilic Ascomycete Chaetomium thermophilum.

Project description:A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome of Chaetomium thermophilum has been reported previously, but it is essentially limited to open reading frames (ORFs) of protein coding genes and contains only a few noncoding transcripts. In this study, we identified and annotated full-length transcripts of C. thermophilum by deep RNA sequencing. We annotated 7044 coding genes and 4567 noncoding genes. Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes as well as 2878 novel noncoding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help to understand the gene regulatory landscape in C. thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species.

| S-EPMC8535861 | biostudies-literature

Structural Characterization of Glycerol Kinase from the Thermophilic Fungus Chaetomium thermophilum.

Project description:Glycerol is an organic compound that can be utilized as an alternative source of carbon by various organisms. One of the ways to assimilate glycerol by the cell is the phosphorylative catabolic pathway in which its activation is catalyzed by glycerol kinase (GK) and glycerol-3-phosphate (G3P) is formed. To date, several GK crystal structures from bacteria, archaea, and unicellular eukaryotic parasites have been solved. Herein, we present a series of crystal structures of GK from Chaetomium thermophilum (CtGK) in apo and glycerol-bound forms. In addition, we show the feasibility of an ADP-dependent glucokinase (ADPGK)-coupled enzymatic assay to measure the CtGK activity. New structures described in our work provide structural insights into the GK catalyzed reaction in the filamentous fungus and set the foundation for understanding the glycerol metabolism in eukaryotes.

| S-EPMC7765489 | biostudies-literature

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