Angiotensin-Converting Enzyme 2 ACE2) Is A Novel Substrate For γSecretase-Dependent Intramembrane Proteolysis
AUTHORS: Harte, J.V.1,2, McCarthy J.V.3
1 Department of Biological Sciences, Cork Institute of Technology, Rossa Avenue, Bishopstown, Cork, Ireland. 2 School of Biochemistry and Cell Biology, University College Cork, College Road, Cork, Ireland. 3 Signal Transduction Laboratory, School of Biochemistry and Cell Biology, ABCRF, 3.41 Western Gateway Building, Western Road, University College Cork, Cork, Ireland.
Introduction: The renin-angiotensin system (RAS) is a critical component of the response to hypovolaemia and hypotension; however, RAS dysfunction is an appreciable mechanism of disease1. The recently discovered angiotensin-converting enzyme 2 (ACE2) is known to counterregulate the potentially destructive effects of uncontrolled angiotensinergic activity2. Subversion of ACE2 by pathophysiological conditions, including infection by zoonotic coronaviruses, can contribute to inappropriate activation of the RAS and accentuate inflammatory and fibrotic disease. Although ACE2 is known to undergo ectodomain shedding, a feature shared by many substrates of γ-secretase-dependent regulated intramembrane proteolysis3, no studies have investigated the subsequent cleavage of the ACE2 carboxyl-terminal fragment (CTF).
Materials and Methods: The molecular mechanism of ACE2 cleavage following ectodomain shedding was investigated by Western blot analysis in cultured cells expressing exogenous or endogenous ACE2 using pharmacological inhibitors and genetic deficiency of presenilin. The subcellular localization of γ-secretase-dependent proteolysis was also investigated using inhibitors of clathrin-dependent endocytosis.
Results: In this study, we show that following ectodomain shedding, the membrane-bound ACE2 CTF is subsequently cleaved by γ-secretase, and that specific biochemical and genetic loss of γsecretase activity independently compromise sequential cleavage of ACE2. Pharmacological inhibition of proteasomal degradation also revealed a putative γ-secretase generated ACE2 intracellular domain (ICD). Moreover, we show that clathrin-mediated internalization of ACE2 is necessary for γ-secretase-dependent proteolysis.
Conclusions: These observations demonstrate that ACE2 is a novel γ-secretase substrate and the formation of a potentially biologically active intracellular domain may be partly responsible for the counter-regulation of the “classical” RAS. Internalization-dependent cleavage may also partly explain the uptake of zoonotic coronaviruses. Thus, the identification of ACE2 as a γ-secretase substrate may facilitate translational approaches for manipulating RAS activity in vivo, with potential biomedical significance in terms of health and disease.
1. Forrester S, Booz G, Sigmund C, Coffman T, Kawai T, Rizzo V, et al. Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology. Physiological Reviews. 2018;98(3):1627-1738.
2. Paz Ocaranza, M, Riquelme, J, García, L, et al. Counter-regulatory renin–angiotensin system in cardiovascular disease. Nature Reviews Cardiology. 2020;17(2):116-129.
3. McCarthy, A, Coleman-Vaughan, C, and McCarthy, J. Regulated intramembrane proteolysis: emergent role in cell signalling pathways. Biochemical Society Transactions. 2017; 45(6); 1185– 1202.
James Harte is a graduate of the BSc (Honours) in Biomedical
Science from Cork Institute of Technology and University
College Cork, and is currently completing his Diploma in
Clinical Laboratory Practice in Cork University Hospital.
EVALUATION OF A FAECAL CALPROTECTIN ASSAY FOR INTRODUCTION INTO ROUTINE TESTING IN BIOCHEMISTRY LABORATORY, UHL
Authors: (1) Lalor C, McGrath M, Conmy B. (1) Riversdale, North Circular Road, Limerick.
Institution: Dept. of Biochemistry, University Hospital Limerick in collaboration with Dept. of Biopharmaceutical and Medical Science, Galway-Mayo Institute of Technology.
Introduction: Faecal calprotectin (FC) is a routinely used, non-invasive biomarker for the diagnosis and monitoring of Inflammatory Bowel Disease (IBD) . The primary aim of this study is to evaluate the Bühlmann fCAL faecal calprotectin turbo assay in order to bring FC testing into routine testing on site in University Hospital Limerick (UHL).
Material and Methods: The performance characteristics of fCAL turbo assay were verified on the Abbott Architect C16000 analyser. Precision was carried out by analysing two levels (low and high) of Internal Quality Control (IQC) in three replicates, daily for five days. Accuracy was measured by analysing nine EQA samples. Additionally, a comparison study was performed which involved the measurement of 20 patient samples on both the Abbott Architect C16000 and Phadia 250 instrument.
Results: The precision study confirmed consistency with the manufacturer’s claims and revealed a total coefficient of variation (CV) of 1.53% and 3.8%, respectively. A high percentage inaccuracy of 21.83% and significant negative bias was observed for accuracy studies. A percentage bias of 88.9% and Passing-Bablok analysis revealed poor agreement between the two methods, further emphasised by a p value of 0.007 (α=0.05). The null hypothesis was thereby rejected.
Conclusion: The fCAL turbo assay performed in accordance with the manufacturer’s specifications of precision. However, accuracy and method comparison results were unsatisfactory for the verification of the assay. Further analysis is required before introduction of the assay into routine testing. Once the analytical performance is evaluated successfully, the assay will be implemented on site, drastically reducing turnaround times, decreasing referral costs and improving service to users.
References:  Seenan JP, Thomson F, Rankin K, Smith K, Gaya DR. Are we exposing patients with a mildly elevated FC to unnecessary investigations? Frontline Gastroenterology. 6th ed. 2015; 156-160.
Clodagh Lalor graduated in 2019 with a BSc. in Medical
Science from GMIT. She completed her clinical
laboratory placement in University Hospital Limerick
(UHL). Following this, She majored in Clinical
Chemistry and Medical Microbiology and is now
working in the Biochemistry Laboratory, UHL.