Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry.

Hauck, SM
·
Dietter, J
·
Kramer, RL
·
Hofmaier, F
·
Zipplies, JK
·
Amann, B
·
Feuchtinger, A
·
Deeg, CA
·
Ueffing, M

Abstract: Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Müller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis.

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Publication Date: 2010-07-04 PubMed ID: 20601722PubMed Central: PMC2953921DOI: 10.1074/mcp.M110.001073Google Scholar: Lookup

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MeSH
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Journal Article
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Non-U.S. Gov't
Validation Study

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The paper discusses a study on the molecular processes in the tissue targeted by autoimmune uveitis, a blinding disease, using a label-free quantitative mass spectrometry method. The research focussed on understanding the changes in membrane protein expression patterns during the disease to provide insight into disease initiation processes.

Objective of the Research

The main objective of the research was to explore the molecular events that enable T cells to invade tissues and instigate autoimmune uveitis. The study particularly analysed the changes in membrane protein expression patterns between the stages of disease and health, citing that the initial events of the disease most likely occur at the membranes.

Research Methodology

The research overcame the limitation of serum-derived proteins masking potential tissue-related changes through the use of membrane-enriched fractions. These were derived from retinas of the only known spontaneous animal model for the disease, equine recurrent uveitis.
The expression levels in the derived fractions were then compared to healthy control samples using a label-free LC-MSMS-based strategy.

Research Outcomes

A total of 893 equine proteins were identified in the process. Of these, 57% of the proteins were connected to the Gene Ontology project term “membrane,” implying their broad involvement in cellular processes occurring at or in proximity to cell membranes.
Approximately 179 proteins were found to exhibit differential expressions in equine recurrent uveitis tissues.
Pathway enrichment analyses revealed an increase in proteins associated with antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions, and focal adhesions.
A decrease of vision-indicating proteins reflective of the loss of retina-specific proteins, and an increase in Müller glial cell-specific proteins indicating heightened glial reactivity were also observed.

Validation of Findings

Selected protein candidates (caveolin 1, integrin alpha 1, and focal adhesion kinase) uptake and their expression were validated through immunohistochemistry and tissue staining.
An increase in these proteins was noted on the outer limiting membrane, part of the outer blood-retinal barrier.
The validation suggested that the adopted methodology of membrane enrichment combined with LC-MSMS-based label-free quantification, significantly increased the sensitivity of the comparative tissue profiling.

In conclusion, the study effectively used comparative tissue profiling and was successful in detecting new molecular pathways associated with equine recurrent uveitis.

Cite This Article

APA

Hauck SM, Dietter J, Kramer RL, Hofmaier F, Zipplies JK, Amann B, Feuchtinger A, Deeg CA, Ueffing M. (2010). Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry. Mol Cell Proteomics, 9(10), 2292-2305. https://doi.org/10.1074/mcp.M110.001073

Publication

Molecular & cellular proteomics : MCP

ISSN: 1535-9484

NlmUniqueID: 101125647

Country: United States

Language: English

Volume: 9

Issue: 10

Pages: 2292-2305

Researcher Affiliations

Hauck, Stefanie M

Department of Protein Science, Helmholtz Zentrum Mu00f9nchen - German Research Center for Environmental Health, Neuherberg, Germany. hauck@helmholtz-muenchen.de

Dietter, Johannes

Kramer, Roxane L

Hofmaier, Florian

Zipplies, Johanna K

Amann, Barbara

Feuchtinger, Annette

Deeg, Cornelia A

Ueffing, Marius

MeSH Terms

Animals
Autoimmune Diseases / metabolism
Chromatography, Liquid
Disease Models, Animal
Horses
Immunohistochemistry
Mass Spectrometry / methods
Membrane Proteins / metabolism
Protein Binding
Tandem Mass Spectrometry
Uveitis / metabolism

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Huber MC, Falkenberg N, Hauck SM, Priller M, Braselmann H, Feuchtinger A, Walch A, Schmitt M, Aubele M. Cyr61 and YB-1 are novel interacting partners of uPAR and elevate the malignancy of triple-negative breast cancer.. Oncotarget 2016 Jul 12;7(28):44062-44075.
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Bakshi MV, Azimzadeh O, Merl-Pham J, Verreet T, Hauck SM, Benotmane MA, Atkinson MJ, Tapio S. In-Utero Low-Dose Irradiation Leads to Persistent Alterations in the Mouse Heart Proteome.. PLoS One 2016;11(6):e0156952.
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Carella P, Merl-Pham J, Wilson DC, Dey S, Hauck SM, Vlot AC, Cameron RK. Comparative Proteomics Analysis of Phloem Exudates Collected during the Induction of Systemic Acquired Resistance.. Plant Physiol 2016 Jun;171(2):1495-510.
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Deeg CA, Eberhardt C, Hofmaier F, Amann B, Hauck SM. Osteopontin and fibronectin levels are decreased in vitreous of autoimmune uveitis and retinal expression of both proteins indicates ECM re-modeling.. PLoS One 2011;6(12):e27674.
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