Identification and Characterization of Human Lon Protease-Associated Proteins, Hsp60 and mtHsp70, by Shotgun Proteomics Approach
Date Issued
2012
Date
2012
Author(s)
Fang, Wei-Cheng
Abstract
Human Lon protease (hLon), an ATP-stimulated mitochondrial matrix protein, is a multifunctional protein that catalyzes the degradation of oxidized mitochondrial proteins, chaperons the assembly of matrix proteins and regulates gene expression of mitochondrial DNA (mtDNA) due to its ability to bind mtDNA and interact with mtDNA binding proteins. Recent studies indicated that dys-regulation of hLon relates to tumorigenesis, aging, diabetes, and neurodegeneration. Although hLon appears to play an essential role in regulating physiological functions of mitochondria, its physiological binding-proteins and molecular mechanism are not fully characterized. As hLon is overexpressed in various types of cancer, in this report, we used a shotgun proteomic approach to investigate the interactome of the protein using the cells overexpressing hLon.
The shotgun proteomics strategy, based on digesting proteins into peptides and sequencing them using tandem mass spectrometry (MS/MS), has widely used to investigate protein-protein interactions. In the present study, we designed a strategy connecting co-immunoprecipitation (Co-IP) with in-solution digestion and liquid chromatography tandem mass spectrum (LTQ-Orbitrap) to study hLon-associated proteins by using AD293 cells which stably overexpressed hLon with myc tag (called 293M cells). Here we overcame the restriction that Co-IP samples must be separated by SDS-PAGE to remove detergents and to avoid antibodies disturbance, both dramatically interfere with the result of mass spectrometry, before applying to shotgun proteomics analysis.
Using this method, we totally identified 245 hLon-associated proteins including proteins participated in mitochondrial chaperone system, cellular metabolism and even cytoskeleton proteins which recently have been indicated that may associate with the stability of mtDNA. To verify the credibility of the result, we further confirmed candidate proteins by Co-IP/western blotting and in vivo immunofluorescence assay. We thus confirmed that Hsp60 and mtHsp70 are hLon-associated proteins. Moreover, we found that hLon may utilize its chaperone activity to influence the stability of Hsp60 and mtHsp70 and suggested that, under oxidative stress, overexpressed hLon inhibits apoptosis due to its ability to stabilize Hsp60 and mtHsp70.
Subjects
human Lon protease
Hsp60
mtHsp70
Shotgun proteomics
Co-immunoprecipitation
SDGs
Type
thesis
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