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Tools for functional dissection of site-specific O-GlcNAcylation.

ABSTRACT: Protein O-GlcNAcylation is an abundant post-translational modification of intracellular proteins with the monosaccharide N-acetylglucosamine covalently tethered to serines and threonines. Modification of proteins with O-GlcNAc is required for metazoan embryo development and maintains cellular homeostasis through effects on transcription, signalling and stress response. While disruption of O-GlcNAc homeostasis can have detrimental impact on cell physiology and cause various diseases, little is known about the functions of individual O-GlcNAc sites. Most of the sites are modified sub-stoichiometrically which is a major challenge to the dissection of O-GlcNAc function. Here, we discuss the application, advantages and limitations of the currently available tools and technologies utilised to dissect the function of O-GlcNAc on individual proteins and sites in vitro and in vivo. Additionally, we provide a perspective on future developments required to decipher the protein- and site-specific roles of this essential sugar modification.

SUBMITTER: Gorelik A

PROVIDER: S-EPMC8386111 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Tools for functional dissection of site-specific O-GlcNAcylation.

Gorelik Andrii A van Aalten Daan M F DMF

RSC chemical biology 20200612 3

Protein O-GlcNAcylation is an abundant post-translational modification of intracellular proteins with the monosaccharide <i>N</i>-acetylglucosamine covalently tethered to serines and threonines. Modification of proteins with O-GlcNAc is required for metazoan embryo development and maintains cellular homeostasis through effects on transcription, signalling and stress response. While disruption of O-GlcNAc homeostasis can have detrimental impact on cell physiology and cause various diseases, littl ...[more]

PMID: 34458751

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Project description:UnlabelledHerpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes.ImportanceHerpesviruses include up to 170 genes in their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when viral gene transcripts are alternatively spliced, leading to the expression of several proteins from a single gene. In this study, we showed, as a proof of principle, that each protein product of an alternatively spliced gene can be eliminated individually by splice site mutagenesis. Mutant viruses lacking individual protein products displayed different phenotypes, demonstrating that the products of alternatively spliced genes have nonredundant functions.

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Tools for functional dissection of site-specific O-GlcNAcylation.

Publications

Tools for functional dissection of site-specific O-GlcNAcylation.

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