Aggrecan imparts resilience to articular cartilage, enabling cartilage to withstand compression, thereby protecting the bony surfaces of joints. Aggrecan's protective properties emanate from its molecular structure which is assembled via a complicated cellular pathway. A critical element early in that pathway is quality control, imposed by molecular chaperones which assist aggrecan core protein to properly fold before it can continue on its route. Until now, the molecular chaperones responsible for assisting aggrecan core protein to fold have not been identified; recent studies in this lab have implicated chaperone Hsp25 as a principal participant in that process. It is postulated that Hsp25 binds to the nascent core protein's G3 domain in the cytosol and accompanies the core protein into the lumen of the endoplasmic reticulum where it assists G3 folding; once folded, G3, as an intramolecular chaperone, binds to the core protein's nascent G1 domain, releasing Hsp25 which recycles to the cytosol; G3, while adhering to G1, enters the Golgi zones where glycosaminoglycan chains are added, followed by secretion of completed aggrecan into the matrix. The long term objective of this proposal is to comprehend the molecular details of this proposed aggrecan-chaperone pathway; critical features of Hsp25-G3 association and dissociation will be investigated, including diversion of Hsp25/G3 complexes into the nucleus and culture medium under certain circumstances.
Three specific aims will focus on: 1) which amino acids in nascent G3 interact with Hsp25 and vice-versa; 2) the nature of the interactions of G1, G3 and Hsp25; 3) which isoform(s) of Hsp25 interact with G3. Cellular transfection with informative constructs will be used to explore each specific aim; normal and mutant Hsp25 and G3 proteins will be expressed, as will hybrid proteins formed between G1, G3, Hsp25 and unique colored isoforms of green fluorescent protein (GFP). GFP labeling will enable tracking of expressed proteins through intracellular compartments; simultaneous tracking of two different proteins will be accomplished as they will be emitting separate colors. Other studies will utilize expressed G1, G3 and Hsp25 proteins containing a polyhistidine tag; proteins will be labeled using radioactive precursors, then harvested and purified by immunocapture and/or by nickel chelation. The composite results will further define a unique system for aggrecan core protein folding and routing. Cartilage repair, including aggrecan production, is impaired in arthritic disease. Understanding the chaperone-based pathway of aggrecan formation and its regulation will potentially provide new insights into the way in which arthritic diseases alter cartilage physiology.
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