Functionele regulatie van MMP-9 door posttranslationele modificaties

Proteases are enzymes catalysing the breakdown of proteins into polypeptides or single amino acids, a process called proteolysis. They are key molecules involved in different physiological processes, ranging from digestion of food proteins to development and host protection against pathologies. However, they also contribute to pathological processes including autoimmune diseases and cancer. Matrix metalloproteinases (MMPs) are an important family of extracellular matrix (ECM) modulators. By cleaving ECM proteins, MMPs regulate tissue architecture, create space for cell migration, produce protein fragments with distinct biological functions and modify the activity of signaling molecules. Additionally, MMPs cleave and modulate cytokines, as well as membrane-bound and intracellular proteins. Moreover, MMPs also carry out non-proteolytic functions, e.g. inducing cellular signaling events. From the entire MMP family, MMP-9 (also known as gelatinase-B) is the most complex member. MMP-9 is a modular enzyme that contains catalytic domains and domains mediating interactions with enzyme regulators and cell surface receptors. The protease is secreted as an inactive form containing a propeptide (proMMP-9) that interacts with the catalytic zinc ion blocking MMP-9 activity. Activation of proMMP-9 (actMMP-9) is a consequence of disruption of this interaction, by proteolysis or chemical modifications. Proteins are produced by transcription of DNA into RNA and further translation of RNA codons into amino acids, the basic components of proteins. After protein production, amino acid residues may be altered, a process referred to as posttranslational modification. In this way, a single protein may be converted into many different modified forms. Within my doctoral project, I studied the functional roles of several posttranslational modifications of MMP-9, specifically proteolytic activation and glycosylation. Moreover, we discovered citrullination as a new modification of MMP-9. First, we studied glycosylation of the human MMP-9 propeptide. Protein glycosylation is a well-described posttranslational modification that consists of the addition of sugar side-chains onto specific amino acids. By comparing different glycosylated forms of proMMP-9, we discovered that glycosylation of the propeptide and the consequent galectin-3 binding to proMMP-9, reduce the processing of the propeptide, thereby decreasing proMMP-9 activation. Valorising this theory, we observed a higher activation rate of proMMP-9 in patients with a congenital disease of glycosylation, who have defective protein glycosylation compared to healthy controls. With these results, we suggest a role of MMP-9 glycosylation in fine-tuning its proteolytic activation. Second, we discovered citrullination as a new posttranslational modification of MMPs, with MMP-9 as an excellent example. Citrullination consist of the conversion of arginine into citrulline residues. This modification in several domains of proMMP-9 changes critical functions. Citrullination of MMP-9 increases the affinity to its substrate gelatin and ensures a faster activation rate of the enzyme compared to control proMMP-9. High MMP-9 activity has been linked to airway damage (in cystic fibrosis). Additionally, we showed higher levels of hypercitrullinated MMP-9 internalization in U87 cells, inducing differential gene expression compared to control MMP-9. Interestingly, in a pilot experiment we detected a citrullinated form of MMP-9 in samples from patients with cystic fibrosis. Last, we observed higher levels of MMP-9 in patients with achalasia, an esophageal autoimmune disease, compared to control samples. In serum samples of achalasia patients, higher MMP-9/MMP-2 and actMMP-9/proMMP-9 ratios were measured in comparison with control samples. Additionally, we provided evidence for a role of MMP-9 and the REGA (Remnant Epitopes Generate Autoimmunity) model in the pathogenesis of achalasia. In conclusion, we described functional regulation by posttranslational modifications, specifically the effects by N-glycosylation and citrullination on the activation, proteolytic activity and interactions of MMP-9 with natural regulators. Based on the new findings from my doctoral research, it becomes clear that behind the gross transcriptional regulatory networks, yielding proteomes, we see the glimpse of many levels of biological fine-tuning by posttranslational modifications.status: publishe