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Nascent proteomes in peripheral blood mononuclear cells as a novel source for biomarker discovery in human stroke.

ABSTRACT: The proteome of newly synthesized proteins (nascent proteome) in peripheral blood mononuclear cells (PBMCs) can be a novel source of stroke biomarkers. Changes in the PBMC nascent proteome after stroke reflect the dynamic response-in-action not detectable in the total proteome (all existing proteins) in blood. Here, we test the application of nascent proteomics as a novel approach for stroke biomarker discovery.The PBMC nascent proteome in human blood was determined by metabolic labeling of fresh PBMC cultures with azidohomoalanine (an azide-containing methionine surrogate), followed by mass spectrometry detection and quantification of azidohomoalanine-labeled proteins. The PBMC nascent and total proteomes were compared between patients with stroke and matched controls.Both PBMC nascent and total proteomes showed differences between stroke patients and controls. Results of hierarchical clustering analysis of proteomic data revealed greater changes in the nascent than in the total PBMC proteomes, supporting the usefulness of the PBMC nascent proteome as a novel source of stroke biomarkers.Nascent proteomes in PBMC can be a novel source for biomarker discovery in human stroke.

SUBMITTER: Bian F

PROVIDER: S-EPMC3992918 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Nascent proteomes in peripheral blood mononuclear cells as a novel source for biomarker discovery in human stroke.

Bian Fang F Simon Roger P RP Li Yun Y David Larry L Wainwright Jolita J Hall Casey L CL Frankel Michael M Zhou An A

Stroke 20140220 4

<h4>Background and purpose</h4>The proteome of newly synthesized proteins (nascent proteome) in peripheral blood mononuclear cells (PBMCs) can be a novel source of stroke biomarkers. Changes in the PBMC nascent proteome after stroke reflect the dynamic response-in-action not detectable in the total proteome (all existing proteins) in blood. Here, we test the application of nascent proteomics as a novel approach for stroke biomarker discovery.<h4>Methods</h4>The PBMC nascent proteome in human blo ...[more]

PMID: 24558093

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Project description:BackgroundMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious and complex physical illness that affects all body systems with a multiplicity of symptoms, but key hallmarks of the disease are pervasive fatigue and 'post-exertional malaise', exacerbation after physical and/or mental activity of the intrinsic fatigue and other symptoms that can be highly debilitating and last from days to months. Although the disease can vary widely between individuals, common symptoms also include pain, cognitive deficits, sleep dysfunction, as well as immune, neurological and autonomic symptoms. Typically, it is a very isolating illness socially, carrying a stigma because of the lack of understanding of the cause and pathophysiology.MethodsTo gain insight into the pathophysiology of ME/CFS, we examined the proteomes of peripheral blood mononuclear cells (PBMCs) by SWATH-MS analysis in a small well-characterised group of patients and matched controls. A principal component analysis (PCA) was used to stratify groups based on protein abundance patterns, which clearly segregated the majority of the ME/CFS patients (9/11) from the controls. This majority subgroup of ME/CFS patients was then further compared to the control group.ResultsA total of 60 proteins in the ME/CFS patients were differentially expressed (P < 0.01, Log10 (Fold Change) > 0.2 and < -0.2). Comparison of the PCA selected subgroup of ME/CFS patients (9/11) with controls increased the number of proteins differentially expressed to 99. Of particular relevance to the core symptoms of fatigue and post-exertional malaise experienced in ME/CFS, a proportion of the identified proteins in the ME/CFS groups were involved in mitochondrial function, oxidative phosphorylation, electron transport chain complexes, and redox regulation. A significant number were also involved in previously implicated disturbances in ME/CFS, such as the immune inflammatory response, DNA methylation, apoptosis and proteasome activation.ConclusionsThe results from this study support a model of deficient ATP production in ME/CFS, compensated for by upregulation of immediate pathways upstream of Complex V that would suggest an elevation of oxidative stress. This study and others have found evidence of a distinct pathology in ME/CFS that holds promise for developing diagnostic biomarkers.

Project description:BACKGROUND:Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease with various clinical manifestations. MicroRNAs (miRNAs) and immunometabolism are recognized as key elements in SLE pathogenesis; however, the relationship between miRNAs in peripheral blood mononuclear cells (PBMCs) and metabolism in SLE remains unclear. METHODS:We detected PBMC miRNA and mRNA profiles from 3 pooled SLE patients and 3 healthy controls (HCs) using next-generation sequencing, predicted miRNA targets in dysregulated mRNAs, predicted functions and interactions of differentially expressed genes using bioinformatics analysis, validated candidate miRNAs using qRT-PCR, and investigated the association between the expression of candidate miRNAs and SLE clinical characteristics. Moreover, we validated the direct and transcriptional regulatory effect of NovelmiRNA-25 on adenosine monophosphate deaminase 2 (AMPD2) using a dual-luciferase reporter assay and western blot and confirmed AMPD2 mRNA and protein expression in PBMCs using qRT-PCR and western blot, respectively. RESULTS:Multilayer integrative analysis of microRNA and mRNA regulation showed that 10 miRNAs were down-regulated and 19 miRNAs were up-regulated in SLE patient PBMCs compared with HCs. Bioinformatics analysis of regulatory networks between miRNAs and mRNAs showed that 19 miRNAs were related to metabolic processes. Two candidate miRNAs, NovelmiRNA-25 and miR-1273h-5p, which were significantly increased in the PBMCs of SLE patients (P?<?0.05), represented diagnostic biomarkers with sensitivities of 94.74% and 89.47%, respectively (area under the curve?=?0.574 and 0.788, respectively). NovelmiRNA-25 expression in PBMCs was associated with disease activity in SLE patients, in both active and stable groups (P?<?0.05). NovelmiRNA-25 overexpression downregulated AMPD2 expression in HEK293T cells through direct targeting of the AMPD2 3'UTR (P?<?0.01), while inhibition of NovelmiRNA-25 activity led to increased AMPD2 expression (P?<?0.01). NovelmiRNA-25 overexpression also downregulated AMPD2 protein expression in HEK293T cells; AMPD2 protein expression in SLE patient PBMCs was decreased. Our results show that differentially expressed miRNAs play an important role in SLE. CONCLUSIONS:Our data demonstrate a novel mechanism in SLE development that involves the targeting of AMPD2 expression by NovelmiRNA-25. miRNAs may serve as novel biomarkers for the diagnosis and evaluation of disease activity of SLE and represent potential therapeutic targets for this disease.

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Nascent proteomes in peripheral blood mononuclear cells as a novel source for biomarker discovery in human stroke.

Publications

Nascent proteomes in peripheral blood mononuclear cells as a novel source for biomarker discovery in human stroke.

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