Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

Abstract

PurposeTo elucidate functions of wild-type myocilin, a secreted glycoprotein associated with glaucoma.MethodsLysates of mouse eyes were used for immunoprecipitation with affinity-purified antibodies against mouse myocilin. Shotgun proteomic analysis was used for the identification of proteins interacting with myocilin. Colocalization of myocilin and tissue inhibitor of metalloproteinases 3 (TIMP3) in different eye structures was investigated by a multiplex fluorescent in situ hybridization and immunofluorescent labeling with subsequent confocal microscopy. Matrix metalloproteinase 2 (MMP2) activity assay was used to test effects of myocilin on TIMP3 inhibitory action.ResultsTIMP3 was identified by a shotgun proteomic analysis as a protein that was coimmunoprecipitated with myocilin from eye lysates of wild-type and transgenic mice expressing elevated levels of mouse myocilin but not from lysates of transgenic mice expressing mutated mouse myocilin. Interaction of myocilin and TIMP3 was confirmed by coimmunoprecipitation of myocilin and TIMP3 from HEK293 cells transiently transfected with cDNAs encoding these proteins. The olfactomedin domain of myocilin is essential for interaction with TIMP3. In the eye, the main sites of myocilin and TIMP3 colocalization are the trabecular meshwork, sclera, and choroid. Using purified proteins, it has been shown that myocilin markedly enhanced the inhibitory activity of TIMP3 toward MMP2.ConclusionsMyocilin may serve as a modulator of TIMP3 activity via interactions with the myocilin olfactomedin domain. Our data imply that in the case of MYOCILIN null or some glaucoma-causing mutations, inhibitory activity of TIMP3 toward MMP2 might be reduced, mimicking deleterious mutations in the TIMP3 gene.

Active synthesis of type I collagen homotrimer in Dupuytren’s fibrosis is unaffected by anti-TNF-α treatment

“…Insufficient myocilin in Dupuytren's tissue may therefore promote pro-fibrotic Wnt signalling. TIMP3 has been shown to interact with myocilin to regulate MMP2 activity (Joe et al, 2017) and a dearth of myocilin in Dupuytren's tissue may result in a corresponding increase in MMP2 activity.…”

Physiological function of myocilin and its role in the pathogenesis of glaucoma in the trabecular meshwork (Review)

“…Although the physiological function of the intracellular proteolytic processing of myocilin remains unknown, the amount of proteolytic myocilin may be associated with the regulation of the normal TM structure through extracellular proteins, including fibrillin-1 (45), secreted protein acidic and rich in cysteine (SPARC) (34), hevin (34,46), collagen (45), optimedin (47), decorin (45), fibronectin (48) and laminin (45,49), which contribute to the regulation of aqueous humor outflow that can affect IOP (23). The identification of proteins interacting with myocilin is a possible approach to elucidating its functions, since interacting proteins are typically involved in the same physiological and pathological processes (50).…”

“…Recently, the crystal structure of myocilin-OLF was elucidated, and it indicated that myocilin-OLF belongs to the five-bladed β-propeller family (108), which is a known hub for protein-protein interactions. To date, a number of extracellular proteins interacting with myocilin have been identified, including tissue inhibitor of matrix metalloproteinase (TIMP3) (8,50), fibronectin (107), flotillin-1 (109) and hevin (46).…”

“…Changes in MMP activity are involved in the pathogenesis of glaucoma (112). Notably, MMP2 activity may be associated with the regulation of IOP, which may be predominantly associated with the TM, where myocilin and TIMP3 coexist (50). MMP2, which is abundant in the TM (8), is involved in the breakdown of extracellular matrix (52), which facilitates aqueous humor outflow (8).…”

Another Piece of the Puzzle: MYOC and Myocilin Glaucoma

“…In this issue of Investigative Ophthalmology & Visual Science , Joe et al . 3 report use of a shotgun proteomic approach for discovery of new MYOC interacting partners. They identify TIMP3 as the third MYOC OLF binding protein.…”