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Spontaneous propeptide processing of mini-stromelysin-1 mutants blocked by APMA ((4-Aminophenyl)mercuric acetate)
G Galazka1, L J Windsor, H Birkedal-Hansen
1Research Center in Oral Biology, Oral Cancer Research Center, and Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Abstract:
Human stromelysin-1 (SL-1) is a member of the stromelysin subfamily of matrix metalloproteinases (MMPs). The MMPs play a major role in the degradation of the extracellular matrix (ECM) during normal and pathological conditions. SL-1 like the other MMPs can be activated in vitro by the stepwise removal of the propeptide that contains a single unpaired cysteine which coordinates the active site zinc. Other residues in the propeptide also play a role in maintaining the latency of the enzymes. Deletion mutants and single-site amino acid replacements within the propeptide of a carboxyl-terminally truncated stromelysin-1 (mini-SL-1) were constructed and expressed in Escherichia coli to further examine what amino acids within the propeptide of SL-1 are important for maintaining latency. While the natural enzyme displayed some limited tendency to spontaneously (autolytically) convert to lower Mr in a stepwise manner and finally to the fully processed form, all of the truncation mutants of more than 19 amino acids generated in E. coli showed greatly accelerated self-cleavage indicative of diminished stability and/or resistance to proteolysis of the residual propeptide. Mutant Delta63 as well as other mutants in which most of the propeptide had been deleted no longer responded to exposure to the organomercurial APMA by accelerated autolytic processing. Rather, APMA inhibited the autolytic processing in these mutants, further confirming the complexity of the action of this organomercurial in the activation of pro-MMPs.
Insights
Human stromelysin-1 (SL-1), a matrix metalloproteinase (MMP), requires its propeptide for latency. Truncation mutants showed accelerated self-cleavage, indicating key roles for propeptide residues in maintaining enzyme stability and activation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Human stromelysin-1 (SL-1) is a matrix metalloproteinase (MMP) involved in extracellular matrix degradation.
- MMPs are crucial in physiological and pathological processes.
- Enzyme activation involves propeptide removal, coordinated by a cysteine residue and other propeptide elements.
Purpose of the Study:
- To investigate the role of specific amino acids within the propeptide of stromelysin-1 (SL-1) in maintaining enzyme latency.
- To analyze the impact of deletion mutants and single-site amino acid replacements on SL-1 stability and activation.
Main Methods:
- Construction and expression of deletion mutants and single-site mutants of carboxyl-terminally truncated SL-1 (mini-SL-1) in Escherichia coli.
- Analysis of spontaneous (autolytic) processing and activation of mutants.
- Assessment of the effect of organomercurial 4-aminophenylmercuric acetate (APMA) on mutant processing.
Main Results:
- Truncation mutants with deletions exceeding 19 amino acids exhibited accelerated self-cleavage, suggesting reduced stability.
- Mutants with significant propeptide deletions (e.g., Delta63) showed inhibited autolytic processing upon APMA exposure.
- These findings highlight the complex role of the propeptide in both maintaining latency and responding to activation signals.
Conclusions:
- Specific amino acid residues within the SL-1 propeptide are critical for maintaining enzyme latency and stability.
- The propeptide's structure influences the enzyme's susceptibility to autolytic processing and its response to activation agents like APMA.
- Understanding these mechanisms is vital for comprehending MMP regulation in health and disease.