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Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

ABSTRACT: Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase C? (PKC?). DMF blocked the association of PKC? with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKC?-CD28 interactions and T cell activation, designating the C2 domain of PKC? as a key functional, electrophile-sensing module important for T cell biology.

SUBMITTER: Blewett MM

PROVIDER: S-EPMC5068918 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

Blewett Megan M MM Xie Jiji J Zaro Balyn W BW Backus Keriann M KM Altman Amnon A Teijaro John R JR Cravatt Benjamin F BF

Science signaling 20160913 445

Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity ...[more]

PMID: 27625306

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Project description:Molecular profiling of human primary cell types is essential for holistic understanding of human biology. Here we present a map of 28 human primary cell types with comprehensive measurements of expressed transcripts and proteins. Three major clusters of epithelial, endothelial, and mesenchymal cell types were observed both at transcriptome and proteome level along with discovery of several cell type specific proteins. Among them, we characterized the novel epithelial specific protein C1orf116 which was further validated with immunohistochemistry across various human tissues. Comprehensive proteome profiling across cell types allowed characterization of ten orphan proteins including TMEM252 and TM4SF18 which lacked protein level evidence. Exhaustive protein database search considering 27 biologically relevant post-translational modifications and 12 chemical modifications was performed, thanks to the increased performance of cloud-based search engine. This, for the first time, provided the catalog of post-translational modifications across cell types, which serve as a resource of PTMs for future studies. We characterized proteins such as protein EEF1A1with various types of PTMs and understudied PTMs including histidine methylation, and O-acetylation of serine. Interestingly, the unexpected higher frequency of dioxidation on tryptophan than that of methionine led us to characterize oxidative mitochondria complex subunit proteins modified through oxidative phosphorylation in mitochondria, which was mostly considered as chemical artifact so far. Overall, these indicated that considering various PTMs during protein database search from non-enriched samples could be routinely used in proteomics experiments. Further, protein database search strategy considering alternative translational start sites, splice junctions, and translational readthrough provided a unique opportunity to refine genome annotation with their proteomic evidence. We believe that our data with the large catalog of transcriptome and proteome of normal human primary cells provide a cornerstone for holistic understanding of cell biology.

Project description:To study whether dimethyl fumarate is superior to placebo in decreasing CSF concentrations of neurofilament light chain (NFL) in patients with primary progressive MS (PPMS). In the double-blind, placebo-controlled phase 2 study dimethyl FUMArate treatment in Progressive Multiple Sclerosis (FUMAPMS), patients with PPMS were randomly assigned to treatment with 240 mg dimethyl fumarate or placebo in a 1:1 ratio for 48 weeks. The primary endpoint was change in concentration of NFL in the CSF. Secondary endpoints included other CSF biomarkers and clinical and MRI measures. Efficacy was evaluated for the full data set by multiple imputations to account for missing data. Safety was assessed for the full data set. Fifty-four patients (mean age 54.9 years [SD 6.1], median Expanded Disability Status Scale 4.0 [nterquartile range 4.0-6.0], disease duration 14.1 [SD 9.4], and 21 [39%] female) were randomized to either placebo (n = 27) or dimethyl fumarate (n = 27) therapy. At screening CSF concentrations, adjusted for age and sex, of NFL, myelin basic protein (MBP), soluble CD27, chitinase 3-like 1, and B-cell maturation antigen were higher than in a group of symptomatic controls. Twenty-six patients (96%) in the dimethyl fumarate group and 24 patients (89%) in the placebo group completed the randomized phase. Mean change in CSF concentrations of NFL did not differ between groups (mean difference 99 ng/L; 95% CI -292 to 491 ng/L). MBP in CSF decreased in the treatment group (-182 ng/L, 95% CI -323 to -41 ng/L compared with placebo). The difference observed in the multiple imputation data set was not significant in a per protocol analysis. This was nominally significant in the multiple imputation data set but not in the per protocol analysis This was not found in the per protocol analysis Other secondary and tertiary outcomes were not affected. Various infections, lymphopenia, flushing, and gastrointestinal side effects were more frequent in the dimethyl fumarate group. Serious adverse events were similar between groups. Dimethyl fumarate treatment for 48 weeks had no effect on any of the investigated efficacy measures in patients with PPMS. We did not observe adverse events not anticipated for dimethyl fumarate treatment. Clinicaltrials.gov identifier NCT02959658. This study provides Class I evidence that for patients with PPMS, dimethyl fumarate treatment has no effect on CSF NFL levels compared with placebo treatment.

Dataset Information

Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

Publications

Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

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