Date of Defense
8-4-2025 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
Prof. Bassam R. Ali
Keywords
ER-associated protein degradation, ER stress; Familial hypercholesterolemia, Low-density lipoprotein (LDL); Low-density lipoprotein receptor (LDLR); Protein quality control; Receptor-mediated endocytosis; Very-low density lipoprotein receptor (VLDLR), CAMRQ, Reelin signaling.
Abstract
The Low-Density Lipoprotein Receptor (LDLR) and the Very-Low-Density Lipoprotein Receptor (VLDLR) are major members of the LDLR family of proteins, and they have been shown to be responsible for Familial hypercholesterolemia (FH) and Cerebellar ataxia, mental retardation, and dysequilibrium syndrome type 1 (CAMRQ1), respectively. FH is an autosomal dominant disorder characterized by increased LDL-cholesterol levels mainly caused by mutations in LDLR. Clinically, FH is characterized by increased levels of LDL-C, tendon xanthomas, corneal arcus, and premature coronary artery diseases (CAD) such as atherosclerosis, if left untreated. CAMRQ-related disorders are a group of rare, heterogeneous, autosomal recessive conditions characterized by cerebellar ataxia, intellectual disability, cerebellar hypoplasia, and delayed ambulation. CAMRQ1 is a subtype of these disorders caused by mutations in the VLDLR disrupting reelin signaling which results in delayed neuronal migration and abnormal brain architecture. The Endoplasmic Reticulum Associated protein degradation (ERAD) pathway has been previously associated with the pathogenesis of certain mutations causing both FH and CAMRQ1. This protein folding and assembly quality control system is residing within the Endoplasmic Reticulum (ER) network of all eukaryotic cells which is the site of synthesis and quality assurance of many proteins. Roughly one-third of all newly synthesized proteins undergo post-translational modifications in the ER and are subject to this stringent quality control system ensuring that proteins are properly folded and ready for trafficking to the Golgi apparatus for additional modifications before reaching their cellular final functional destinations. Misfolded secretory pathway targeted proteins are generally retained in the ER, recognized, and degraded via ERAD. However, the accumulation of misfolded proteins exerts stress on the ER, activating the Unfolded Protein Response (UPR). UPR alleviates ER stress by halting protein synthesis and increasing the expression of ERAD components to degrade misfolded proteins. In the first part of this thesis, I investigated the cellular trafficking and functional implications of ten LDLR missense variants (p.C167F, p.D178N, p.C243Y, p.E277K, p.G314R, p.H327Y, p.D477N, p.D622G, p.R744Q, and p.R814Q) reported in multiethnic suspected FH patients in UAE using biochemical and functional approaches. I also analyzed the functional impact of three other LDLR variants (p.D445E, p.D482H, and p.C677F), two of which were previously shown to be retained in the ER. The results show that p.D622G variant is largely retained in the ER, whereas p.R744Q is partially ER-retained, while the other variants were predominantly localized to the plasma membrane. LDL internalization assays in CHO-ldlA7 (LDLR deficient) cells indicated that p.D482H, p.C243Y, p.D622G, and p.C667F variants have quantitatively lost their ability to internalize Dil-LDL, with the others (p.C167F, p.D178N, p.G314R, p.H327Y, p.D445E, p.D477N, p.R744Q, and p.R814Q) showing significant losses except for p.E277K, which showed full activity. We also assessed the degradation routes of three LDLR variants, p.D482H, p.D622G, and p.R744Q, in HEK293 cells treated with a group of proteasomal and lysosomal inhibitors, which showed some accumulation when treated with the proteasomal inhibitor epoxomicin, suggesting that these variants are likely to be degraded via the ubiquitin-proteasome pathway. This was further confirmed in two HEK293 cell lines deficient in two major ERAD components: HRD1 and DERLIN1. A significant accumulation of these variants was observed in the two knock-out cell lines, confirming that these variants are largely degraded via the ubiquitin-proteasome pathway. The second part of the thesis presents the evaluation of several variants in the highly related protein VLDLR including a case study of CAMRQ1 caused by a novel missense variant, p.P565Q, in VLDLR. This variant was identified in a family from Senegal exhibiting clinical manifestations of CAMRQ1. In addition, I generated and evaluated several other VLDLR missense variants (p.N81S, p.I244M, p.Y378H, p.C419Y, p.G438D, p.V595G, p.R634H, and p.E654G) previously reported to cause hypobetalipoproteinemia, neurological, and neurodevelopmental disorders. The generated data show that VLDLR variants p.P565Q and p.V595G are largely ER-retained while p.N81S, p.C419Y, and R634H are partially ER-retained. The findings in this thesis conclusively consolidate the role of ERAD in the pathogenesis of FH and CAMRQ1 caused by variations in LDLR and VLDLR, respectively, which could pave the way for therapeutic interventions that could target ERAD to partially restore the functionality of LDLR or VLDLR and reduce their pathological impact. store cellular homeostasis.
Included in
ELUCIDATING THE PATHOGENIC MECHANISMS OF DISEASE-CAUSING VARIANTS IN THE LOW-DENSITY LIPOPROTEIN RECEPTOR (LDLR) AND THE VERY-LOW-DENSITY LIPOPROTEIN RECEPTOR (VLDLR)
Yanah Theatre
The Low-Density Lipoprotein Receptor (LDLR) and the Very-Low-Density Lipoprotein Receptor (VLDLR) are major members of the LDLR family of proteins, and they have been shown to be responsible for Familial hypercholesterolemia (FH) and Cerebellar ataxia, mental retardation, and dysequilibrium syndrome type 1 (CAMRQ1), respectively. FH is an autosomal dominant disorder characterized by increased LDL-cholesterol levels mainly caused by mutations in LDLR. Clinically, FH is characterized by increased levels of LDL-C, tendon xanthomas, corneal arcus, and premature coronary artery diseases (CAD) such as atherosclerosis, if left untreated. CAMRQ-related disorders are a group of rare, heterogeneous, autosomal recessive conditions characterized by cerebellar ataxia, intellectual disability, cerebellar hypoplasia, and delayed ambulation. CAMRQ1 is a subtype of these disorders caused by mutations in the VLDLR disrupting reelin signaling which results in delayed neuronal migration and abnormal brain architecture. The Endoplasmic Reticulum Associated protein degradation (ERAD) pathway has been previously associated with the pathogenesis of certain mutations causing both FH and CAMRQ1. This protein folding and assembly quality control system is residing within the Endoplasmic Reticulum (ER) network of all eukaryotic cells which is the site of synthesis and quality assurance of many proteins. Roughly one-third of all newly synthesized proteins undergo post-translational modifications in the ER and are subject to this stringent quality control system ensuring that proteins are properly folded and ready for trafficking to the Golgi apparatus for additional modifications before reaching their cellular final functional destinations. Misfolded secretory pathway targeted proteins are generally retained in the ER, recognized, and degraded via ERAD. However, the accumulation of misfolded proteins exerts stress on the ER, activating the Unfolded Protein Response (UPR). UPR alleviates ER stress by halting protein synthesis and increasing the expression of ERAD components to degrade misfolded proteins. In the first part of this thesis, I investigated the cellular trafficking and functional implications of ten LDLR missense variants (p.C167F, p.D178N, p.C243Y, p.E277K, p.G314R, p.H327Y, p.D477N, p.D622G, p.R744Q, and p.R814Q) reported in multiethnic suspected FH patients in UAE using biochemical and functional approaches. I also analyzed the functional impact of three other LDLR variants (p.D445E, p.D482H, and p.C677F), two of which were previously shown to be retained in the ER. The results show that p.D622G variant is largely retained in the ER, whereas p.R744Q is partially ER-retained, while the other variants were predominantly localized to the plasma membrane. LDL internalization assays in CHO-ldlA7 (LDLR deficient) cells indicated that p.D482H, p.C243Y, p.D622G, and p.C667F variants have quantitatively lost their ability to internalize Dil-LDL, with the others (p.C167F, p.D178N, p.G314R, p.H327Y, p.D445E, p.D477N, p.R744Q, and p.R814Q) showing significant losses except for p.E277K, which showed full activity. We also assessed the degradation routes of three LDLR variants, p.D482H, p.D622G, and p.R744Q, in HEK293 cells treated with a group of proteasomal and lysosomal inhibitors, which showed some accumulation when treated with the proteasomal inhibitor epoxomicin, suggesting that these variants are likely to be degraded via the ubiquitin-proteasome pathway. This was further confirmed in two HEK293 cell lines deficient in two major ERAD components: HRD1 and DERLIN1. A significant accumulation of these variants was observed in the two knock-out cell lines, confirming that these variants are largely degraded via the ubiquitin-proteasome pathway. The second part of the thesis presents the evaluation of several variants in the highly related protein VLDLR including a case study of CAMRQ1 caused by a novel missense variant, p.P565Q, in VLDLR. This variant was identified in a family from Senegal exhibiting clinical manifestations of CAMRQ1. In addition, I generated and evaluated several other VLDLR missense variants (p.N81S, p.I244M, p.Y378H, p.C419Y, p.G438D, p.V595G, p.R634H, and p.E654G) previously reported to cause hypobetalipoproteinemia, neurological, and neurodevelopmental disorders. The generated data show that VLDLR variants p.P565Q and p.V595G are largely ER-retained while p.N81S, p.C419Y, and R634H are partially ER-retained. The findings in this thesis conclusively consolidate the role of ERAD in the pathogenesis of FH and CAMRQ1 caused by variations in LDLR and VLDLR, respectively, which could pave the way for therapeutic interventions that could target ERAD to partially restore the functionality of LDLR or VLDLR and reduce their pathological impact. store cellular homeostasis.