Abstract
Uncontrolled matrix degradation by the zinc-dependent matrix metalloproteinases (MMPs) is implicated in tumour metastasis and a wide range of inflammatory diseases. Fibrillar collagens are the most abundant proteins in the extracellular matrix. They consist of tightly-wound triple-helices, arranged into fibrils, which highly resistant to proteases.MMP-1 is one of a select number of proteases able to hydrolyse collagen, in a process termed collagenolysis. MMP-1 consists of a catalytic (CAT) and a hemopexin (HPX) domain, connected by a flexible linker, which allows transient dissociation in solution.
The studies described herein aimed to understand the function of inter domain dynamics in controlling MMP-1’s proteolytic activity. MMP-1 was found to cleave the extracellular region of protease activated receptor-2 (PAR2) in vivo, resulting in down-regulation of MMP expression via the MAPK signalling pathway. This is a novel regulatory mechanism of the collagenolytic MMPs. To understand the role of inter domain flexibility in MMP-1, domainstapled MMP-1(DS) and domain-dislocated MMP-1(DD) variants were engineered. Binding and hydrolysis assays with type I collagen show that, while MMP-1 is able to bind its substrate by multiple routes, the ability to access a compact conformation is required for
unwinding and subsequent hydrolysis of collagen. In carrying out these studies, a new simplified method for measuring enzyme kinetics was developed. Termed EnRootS, the modulation of enzyme concentration as a function of the square root of the substrate allows rapid and accurate determination of kcat and Km of hydrolases, with only 10% of the data required when using constant enzyme concentration. Finally, binding experiments between MMP-1(DS) and the endogenous tissue inhibitor of MMPs (TIMP-1) showed that domain rearrangements within TIMP-1 allow formation of its complex with MMP-1, which is possibly stabilised by interactions between the C-terminal domains of both proteins.
Insights from these investigations improve our understanding MMP-1 regulation, ultimately informing the development of therapeutics specifically targeting its collagenolytic activity in diseases such as arthritis.
| Date of Award | 25 Mar 2020 |
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| Original language | English |
| Supervisor | Andrew Pickford (Supervisor) |