Identification of paracrine neuroprotective candidate proteins by a functional assay-driven proteomics approach

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Fältnamn	Indikatorer	Metadata
000		03781naa a2200349 4500
001		oai:lup.lub.lu.se:a035ab04-a2b9-4fa1-8903-ce5e6adf3d0d
003		SwePub
008		160401s2008 \| \|\|\|\|\|\|\|\|\|\|\|000 \|\|eng\|
024	7	a https://lup.lub.lu.se/record/12569692 URI
024	7	a https://doi.org/10.1074/mcp.M700456-MCP2002 DOI
040		a (SwePub)lu
041		a engb eng
042		9 SwePub
072	7	a art2 swepub-publicationtype
072	7	a ref2 swepub-contenttype
100	1	a Hauck, Stefanie M.4 aut
245	1 0	a Identification of paracrine neuroprotective candidate proteins by a functional assay-driven proteomics approach
264	1	c 2008
520		a Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Toward this goal we first isolated conditioned medium (CM) from primary porcine RMGs cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins toward their identification consists of several fractionation steps combined with tests for biological function. Here we combined 1) size fractionation, 2) ion exchange chromatography, 3) reverse phase liquid chromatography, and 4) mass spectrometry (Q-TOF MS/MS or MALDI MS/MS) for protein identification. As a result of this integrated work flow, the insulin-like growth factor-binding proteins IGFBP5 and IGFBP7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7, significantly increased photoreceptor survival when secreted from HEK293 cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multifactorial with IGFBP5 and CTGF as an integral part of this activity.
650	7	a MEDICIN OCH HÄLSOVETENSKAPx Klinisk medicinx Oftalmologi0 (SwePub)302172 hsv//swe
650	7	a MEDICAL AND HEALTH SCIENCESx Clinical Medicinex Ophthalmology0 (SwePub)302172 hsv//eng
700	1	a Gloeckner, Christian J.4 aut
700	1	a Harley, Margaret E.4 aut
700	1	a Schoeffmann, Stephanie4 aut
700	1	a Boldt, Karsten4 aut
700	1	a Ekström, Peru Lund University,Lunds universitet,Oftalmologi, Lund,Sektion IV,Institutionen för kliniska vetenskaper, Lund,Medicinska fakulteten,Ophthalmology, Lund,Section IV,Department of Clinical Sciences, Lund,Faculty of Medicine4 aut0 (Swepub:lu)zoof-pek
700	1	a Ueffing, Marius4 aut
710	2	a Oftalmologi, Lundb Sektion IV4 org
773	0	t Molecular & Cellular Proteomicsg 7:7, s. 1349-1361q 7:7<1349-1361x 1535-9484
856	4	u http://dx.doi.org/10.1074/mcp.M700456-MCP200y FULLTEXT
856	4 8	u https://lup.lub.lu.se/record/1256969
856	4 8	u https://doi.org/10.1074/mcp.M700456-MCP200

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Av författaren/redakt...: Hauck, Stefanie ...; Gloeckner, Chris ...; Harley, Margaret ...; Schoeffmann, Ste ...; Boldt, Karsten; Ekström, Per; visa fler...; Ueffing, Marius; visa färre...

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MEDICIN OCH HÄLSOVETENSKAP: MEDICIN OCH HÄLS ...; och Klinisk medicin; och Oftalmologi

Artiklar i publikationen: Molecular & Cell ...

Av lärosätet: Lunds universitet

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