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The regulation of L-selectin activity by proteolysis

Newman, Andrew 2017. The regulation of L-selectin activity by proteolysis. PhD Thesis, Cardiff University.
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Abstract

L-selectin (CD62L) is a type I transmembrane protein expressed by lymphocytes which directs their migration from the bloodstream into lymph nodes and infected tissues. Stimulation of the T cell receptor (TCR) activates the enzyme A Disintegrin and Metalloproteinase 17 (ADAM 17), which cleaves L-selectin at the ectodomain generating a metalloproteinase product (MP product) comprising of a transmembrane region and a 17-amino acid intracellular domain (ICD). ϒ-secretase is a multi-subunit protease that cleaves up to 90 identified type I transmembrane proteins in the intramembrane region following ectodomain proteolysis by metalloproteinases. Presenilin (PS), the catalytic component of γ-secretase is activated during an intramolecular cleavage called endoproteolysis separating the carboxy (C) and amino (N) termini. The catalytically active C-terminal fragment of PS then induces intramembrane proteolysis of substrates. The aim of my thesis was to firstly determine whether the MP product of L-selectin was a substrate for PS. Subsequently, I analysed whether stimulation of the TCR activates PS, inducing intramembrane proteolysis of the MP product releasing the ICD into the intracellular region. My data showed for the first time that in a resting T-cell, L-selectin forms a multi-component complex with both ADAM 17 and PS. TCR-activation induces ADAM 17 dependent proteolysis of L-selectin generating an MP product. Stimulation of the TCR also causes endoproteolysis of PS, where activated PS then cleaves the bound MP product. After PS cleavage, the released ICD was unstable and therefore difficult to detect, however I was able to block its formation using either PS inhibitor treatment or generating I351W mutated L-selectin, which was resistant to intramembrane proteolysis.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Date of First Compliant Deposit: 22 August 2017
Last Modified: 12 Jun 2019 02:01
URI: http://orca-mwe.cf.ac.uk/id/eprint/103855

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