Investigating the production and secretion of PEPITEM, a regulator of leukocyte trafficking

Nathan, Poppy Ysabel ORCID: 0000-0002-1987-3165 (2024). Investigating the production and secretion of PEPITEM, a regulator of leukocyte trafficking. University of Birmingham. Ph.D.

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Abstract

Leukocyte trafficking is an essential part of the inflammatory response. Immune cells need to migrate to areas of inflammation to initiate pathogen clearance and mediate tissue repair. It is vital that this process is tightly regulated, as aberrant lymphocyte recruitment contributes to immune-mediated inflammatory diseases (IMIDs). PEPITEM (PEPtide Inhibitor of TransEndothelial Migration) is an immuno-regulatory peptide secreted by adiponectin-stimulated B-cells and acts to suppress T-cell transendothelial migration. It is known that PEPITEM is derived from the parent protein 14-3-3ζ, however the mechanisms behind PEPITEM production and secretion are currently unknown. In this thesis, we explored proteases involved in PEPITEM production. Using genetic silencing and blockade with inhibitors, we identified the matrix metalloproteinases MMP-2 and MMP-9 are both required for cleavage of 14-3-3ζ into functional peptide. We also found inflamed endothelial cells upregulate MMP-9 expression and MMP-2 activity in parallel, with a concomitant downregulation of expression of their endogenous inhibitors, TIMP1 and TIMP2. This represents a novel mechanism of regulation of the PEPITEM pathway in inflammatory conditions.
We explored mechanisms of PEPITEM secretion and identified B-cells secrete whole 14-3-3ζ protein in response to adiponectin, which is then proteolytically cleaved once in the extracellular environment. We investigated extracellular vesicle release from B-cells and observed constitutive release of vesicles that was not regulated by adiponectin. Importantly, these vesicles did not contain 14-3-3ζ and therefore do not represent the mechanism of 14-3-3ζ release from B-cells. Despite 14-3-3ζ lacking the signal recognition particle required for trafficking to the endoplasmic reticulum, we identified inhibition of the classical secretory pathway suppressed 14-3-3ζ secretion. Therefore, it is likely 14-3-3ζ is transported out of the cell coupled with a classically secreted protein.
Thus, we report that 14-3-3ζ is secreted via the classical secretory pathway and subsequently cleaved by MMP-2 and MMP-9 to yield PEPITEM once in the extracellular milieu. Data from this thesis offer a novel understanding of the mechanisms and regulation of PEPITEM production, which will support the development of PEPITEM as a therapeutic agent.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):