Date of Defense
9-4-2025 1:00 PM
Location
Yanah Theatre
Document Type
Dissertation Defense
Degree Name
Doctor of Philosophy in Public Health
College
CMHS
Department
Genetics & Genomics
First Advisor
Bassam R. Ali
Keywords
ACE2, B0AT1, Genetic variants, sub-cellular localization, Endoplasmic reticulum, Molecular modulators, Drug screening, Proteins interactions, Biochemical analysis.
Abstract
Angiotensin-converting enzyme 2 (ACE2) and the amino acid transporter B0AT1 are essential for blood pressure regulation, amino acid absorption, and viral entry. Their interaction is crucial in both normal physiology and disease, including hypertension, Hartnup disease, and SARS-CoV-2 infection. However, the effects of genetic variants on their biogenesis, trafficking, and function remain poorly understood. This PhD thesis investigates how specific ACE2 and B0AT1 variants influence their subcellular localization and interactions. A multidisciplinary approach, incorporating site-directed mutagenesis, confocal microscopy, Western blotting, and in silico modelling, was used to examine 39 ACE2 variants and 18 B0AT1 mutations. Results showed that wild-type ACE2 reaches the plasma membrane within 10 hours. While most genetic variants had no major impact on intracellular trafficking or membrane targeting, disruption of the signal peptide completely blocked trafficking. Drug screening revealed that ACE2 maturation is generally rapid and robust. Of 23 tested compounds, 8 significantly reduced ACE2 maturation levels, with 3 causing an approximate 20% decrease. Screening of trafficking inhibitors demonstrated strong effects from most molecular modulators, mild effects from proposed COVID-19 drugs, and no effects from statins. Given that altering ACE2 levels can be beneficial or detrimental depending on the context, therapeutic modulation requires careful evaluation. For B0AT1, 9 of 18 Hartnup disease-associated variants led to endoplasmic reticulum retention, thereby affecting ACE2 trafficking. Notably, two mutations significantly impaired ACE2 plasma membrane targeting. This study deepens our understanding of ACE2 and B0AT1 biogenesis and their roles in disease, with potential implications for therapeutic development and biomarker discovery. In conclusion, this research confirms that while ACE2 is intolerant to loss of function, it is prone to aggregation with B0AT1. By identifying key variants and modulators, these findings contribute to potential targeted therapies. Future studies should further explore the clinical relevance of these insights and investigate additional molecular modulators to mitigate disease progression and improve patient outcomes.
Included in
THE CELLULAR TRAFFICKING AND TARGETING OF ANGIOTENSIN-CONVERTING-ENZYME-2 (ACE2) AND NEUTRAL-AMINO-ACID-TRANSPORTER (B0AT1) VARIANTS: IMPLICATIONS FOR THE PATHOGENESIS OF ASSOCIATED DISEASES AND THERAPY
Yanah Theatre
Angiotensin-converting enzyme 2 (ACE2) and the amino acid transporter B0AT1 are essential for blood pressure regulation, amino acid absorption, and viral entry. Their interaction is crucial in both normal physiology and disease, including hypertension, Hartnup disease, and SARS-CoV-2 infection. However, the effects of genetic variants on their biogenesis, trafficking, and function remain poorly understood. This PhD thesis investigates how specific ACE2 and B0AT1 variants influence their subcellular localization and interactions. A multidisciplinary approach, incorporating site-directed mutagenesis, confocal microscopy, Western blotting, and in silico modelling, was used to examine 39 ACE2 variants and 18 B0AT1 mutations. Results showed that wild-type ACE2 reaches the plasma membrane within 10 hours. While most genetic variants had no major impact on intracellular trafficking or membrane targeting, disruption of the signal peptide completely blocked trafficking. Drug screening revealed that ACE2 maturation is generally rapid and robust. Of 23 tested compounds, 8 significantly reduced ACE2 maturation levels, with 3 causing an approximate 20% decrease. Screening of trafficking inhibitors demonstrated strong effects from most molecular modulators, mild effects from proposed COVID-19 drugs, and no effects from statins. Given that altering ACE2 levels can be beneficial or detrimental depending on the context, therapeutic modulation requires careful evaluation. For B0AT1, 9 of 18 Hartnup disease-associated variants led to endoplasmic reticulum retention, thereby affecting ACE2 trafficking. Notably, two mutations significantly impaired ACE2 plasma membrane targeting. This study deepens our understanding of ACE2 and B0AT1 biogenesis and their roles in disease, with potential implications for therapeutic development and biomarker discovery. In conclusion, this research confirms that while ACE2 is intolerant to loss of function, it is prone to aggregation with B0AT1. By identifying key variants and modulators, these findings contribute to potential targeted therapies. Future studies should further explore the clinical relevance of these insights and investigate additional molecular modulators to mitigate disease progression and improve patient outcomes.