title: Protein networks involved in vesicle fusion, transport, and storage revealed by array-based proteomics.
abstract: Secretagogin is a calcium-binding protein whose expression is characterised in neuroendocrine, pancreatic, and retinal cells. We have used an array-based proteomic approach with the prokaryotically expressed human protein array (hEx1) and the eukaryotically expressed human protein array (Protoarray) to identify novel calcium-regulated interaction networks of secretagogin. Screening of these arrays with fluorophore-labelled secretagogin in the presence of Ca(2+) ions led to the identification of 12 (hEx1) and 6 (Protoarray) putative targets. A number of targets were identified in both array screens. The putative targets from the hEx1 array were expressed, purified, and subjected to binding analysis using surface plasmon resonance. This identified binding affinities for nine novel secretagogin targets with equilibrium dissociation constants in the 100 pM to 10 nM range. Six of the novel target proteins have important roles in vesicle trafficking; SNAP-23, ARFGAP2, and DOC2alpha are involved in regulating fusion of vesicles to membranes, kinesin 5B and tubulin are essential for transport of vesicles in the cell, and rootletin builds up the rootlet, which is believed to function as scaffold for vesicles. Among the targets are two enzymes, DDAH-2 and ATP-synthase, and one oncoprotein, myeloid leukaemia factor 2. This screening method identifies a role for secretagogin in secretion and vesicle trafficking interacting with several proteins integral to these processes.
authors.affiliation: Biophysical Chemistry and Biochemistry, Lund University, Lund, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Bauer
authors.forename: Magdalena
authors.initials: M
authors.lastname: Maj
authors.forename: Ludwig
authors.initials: L
authors.lastname: Wagner
authors.forename: Dolores J
authors.initials: DJ
authors.lastname: Cahill
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
authors.forename: David J
authors.initials: DJ
authors.lastname: O'Connell
mesh_terms: D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D006801:Humans; D040081:Protein Array Analysis / Q000379:methods*; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D022142:Secretory Vesicles / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D002118:Calcium