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Elucidation of the Cellular and Molecular Mechanisms Of United Arab Emirates University Scholarworks@UAEU Dissertations Electronic Theses and Dissertations Winter 2-2015 ELUCIDATION OF THE CELLULAR AND MOLECULAR MECHANISMS OF MISSENSE MUTATIONS ASSOCIATED WITH FAMILIAL EXUDATIVE VITREORETINOPATHY AND CONGENITAL MYASTHENIC SYNDROME Reham Mahmoud Mohammed Milhem Follow this and additional works at: https://scholarworks.uaeu.ac.ae/all_dissertations Part of the Medical Pathology Commons Recommended Citation Milhem, Reham Mahmoud Mohammed, "ELUCIDATION OF THE CELLULAR AND MOLECULAR MECHANISMS OF MISSENSE MUTATIONS ASSOCIATED WITH FAMILIAL EXUDATIVE VITREORETINOPATHY AND CONGENITAL MYASTHENIC SYNDROME" (2015). Dissertations. 15. https://scholarworks.uaeu.ac.ae/all_dissertations/15 This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarworks@UAEU. It has been accepted for inclusion in Dissertations by an authorized administrator of Scholarworks@UAEU. For more information, please contact [email protected]. United Arab Emirates University College of Medicine and Health Sciences ELUCIDATION OF THE CELLULAR AND MOLECULAR MECHANISMS OF MISSENSE MUTATIONS ASSOCIATED WITH FAMILIAL EXUDATIVE VITREORETINOPATHY AND CONGENITAL MYASTHENIC SYNDROME Reham Mahmoud Mohammed Milhem This dissertation is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Under the Supervision of Professor Bassam R. Ali February 2015 ii Declaration of Original Work I, Reham Mahmoud Mohammed Milhem, the undersigned, a graduate student at the United Arab Emirates University (UAEU), and the author of this PhD dissertation, entitled “Elucidation of the cellular and molecular mechanisms of missense mutations associated with familial exudative vitreoretinopathy and congenital myasthenic syndrome”. I hereby solemnly declare that this dissertation is an original research work that has been done and prepared by me under the supervision of Professor Bassam R. Ali, in the College of Medicine and Health Sciences at the UAEU. This work has not been previously formed as the basis for the award of any academic degree, diploma or similar title at this or any other university. The materials borrowed from other sources and included in my dissertation have been properly cited and acknowledged. Student’s Signature ________________________ Date _________________ iii Copyright © 2015 Reham Mahmoud Mohammed Milhem All Rights Reserved iv Signatures This Doctorate Dissertation is approved by the following Examining Committee Members: 1) Advisor (Committee Chair): Prof. Bassam R. Ali Title: Professor Department of: Pathology College of: Medicine and Health Sciences Signature: _____________________________ Date: _________________ 2) Member: Prof. Abdul-kader Souid Title: Professor Department of: Pediatrics College of: Medicine and Health Sciences Signature: _____________________________ Date: _________________ 3) Member: Dr. Samir Attoub Title: Associate Professor Department of: Pharmacology and Therapeutics College of: Medicine and Health Sciences Signature: _____________________________ Date: _________________ 4) Member (External Examiner): Prof. Ineke Braakman Title: Professor of Cellular Protein Chemistry Department of: Bijvoet Center for Biomolecular Research Institution: Faculty of Science, Utrecht University, The Netherlands Signature: _____________________________ Date: _________________ v This Doctorate Dissertation is accepted by: Dean of the College of Medicine and Health Sciences: Professor Dennis Templeton Signature _________________________________ Date ____________________ Dean of the College of Graduate Studies: Professor Nagi Wakim Signature _________________________________ Date ____________________ Copy ____ of ____ vi Abstract The endoplasmic reticulum (ER), within eukaryotic cells, is a hub for protein folding and assembly. Misfolded proteins and unassembled subunits of protein complexes are retained in the ER and degraded by a process termed endoplasmic reticulum associated degradation (ERAD). Frizzled class receptor 4 (FZD4) and muscle, skeletal, receptor tyrosine kinase (MuSK) are Wnt receptors. These proteins contain the frizzled cysteine-rich domain (Fz-CRD) required for dimerization in the ER. Mutations in FZD4 and MuSK genes are known to cause familial exudative vitreoretinopathy (FEVR, an autosomal dominant disease) and congenital myasthenic syndrome (CMS, an autosomal recessive disease), respectively. It was hypothesized that missense mutations within Fz-CRD lead to misfolding of FZD4 and MuSK proteins and consequent ER-retention. Investigating the molecular mechanism of these mutations is important since misfolded protein and ER-targeted therapies are in development being developed. Wild-type and mutants of FZD4 and MuSK were expressed at 37 °C in HeLa, COS-7, and HEK293 cells and their subcellular localizations were investigated using confocal microscopy imaging and glycosidase treatments. Abnormal trafficking was demonstrated in 10 of 21 mutants studied; nine mutants were within Fz-CRD and one was distant from Fz-CRD. These ER-retained mutants were improperly N-glycosylated confirming ER-localization. They were tagged with polyubiquitin chains confirming targeting for proteasomal degradation. The half-lives of wild-type MuSK and P344R-MuSK were 45 and 37 minutes, respectively; the latter half-life improved on incubation with proteasomal inhibitor MG132. The P344R-MuSK kinase mutant showed around 50% of its in vivo autophosphorylation activity. Trafficking defects in three of the 10 mutants (M105T- FZD4, C204Y-FZD4, and P344R-MuSK) were rescued by expression at 27 °C and by chemical chaperones (2.5-7.5% glycerol, 0.1-1% dimethyl sulfoxide, 10 µM thapsigargin, or 1 µM curcumin). Trafficking of wild-type FZD4 was not affected by vii co-expression with any of the nine ER-retained mutants, suggesting haploinsufficiency as the mechanism of disease. Thus, all nine Fz-CRD mutants of FEVR and CMS studied resulted in misfolded proteins. In contrast, only one of the 12 mutants outside Fz-CRD resulted in ER-retention. These findings demonstrate a common mechanism for diseases associated with Fz-CRD missense mutations. Disorders of Fz-CRD may be receptive to novel therapies that alleviate protein misfolding. Keywords: Familial exudative vitreoretinopathy; congenital myasthenic syndrome; endoplasmic reticulum associated degradation; frizzled class receptor 4; muscle, skeletal, receptor tyrosine kinase; frizzled cysteine-rich domain. viii Title and Abstract in Arabic توضيح اﻵلية الخلوية والجزيئية للطفرات المغلطة المرتبطة بإعتﻻل الشبكية الزجاجي الوراثي )FEVR( ومتﻻزمة الوهن العضلي الخلقية )CMS( الملخص تعتبر الشبكة اﻹندوبﻻزمية (endoplasmic reticulum (ER في الخﻻيا حقيقية النواة محور لطي البروتين وتجميعه. البروتينات ذات الطي الخاطئ وكذلك الوحدات الجزئية غير المجمعة من مجمعات البروتين تستقر في الشبكة اﻹندوبﻻزمية (ER) و تتدهور من خﻻل عملية تسمى التدهور المترابط في الشبكة اﻹندوبﻻزمية (Endoplasmic reticulum associated degradation (ERAD. البروتين المستقبل فريزلد (FZD4) والبروتين المستقبل مسك (MuSK) يعتبران من مستقبﻻت وينت )Wnt(. تحتوي هذه البروتينات على السستين (frizzled cysteine-rich domain (Fz-CRD الضروري لعمليات ربط جزيئات البروتينات )dimerization( في الشبكة اﻹندوبﻻزمية )ER(. الطفرات في جين فرزلد )FZD4( وجين مسك )MuSK( هي المسببة لمرض (familial exudative vitreoretinopathy (FEVR ولمرض congenital myasthenic (syndrome (CMS على التوالي. لقد تم اﻹفتراض بأن الطفرات المغلطة في المجاﻻت الغنية بفرزلد- السستين )Fz-CRD( تؤدي إلى الطي الخاطئ في بروتينات فرزلد )FZD4( ومسك )MuSK( وإلى اﻹستبقاء في الشبكة اﻹندوبﻻزمية )ER(. البحث في اﻵلية الجزيئية لهذه الطفرات مهم كون والعﻻجات المستهدفة للطي الخاطئ للبروتين و للشبكة اﻹندوبﻻزمية في تطور. تم إختبارالبروتين السليم والطفرات لكل من فرزلد )FZD4( و مسك )MuSK( عند درجة حرارة 37 سلسيوس في خﻻيا )HeLa وCOS -7 و HEK293( كما تم البحث في التمركز التحت خلوي )subcellular localization( لهما بواسطة التصوير المجهري متحد البؤر)confocal microscopy( وعﻻجات غليكوزيداز )glycosidase treatments(. ظهرت عمليات نقل غير طبيعية في عشرة طفرات من الطفرات اﻹحدى والعشرين التي تمت دراستها، حيث كانت تسع من هذة الطفرات ضمن المجاﻻت الغنية بفرزلد السستين )Fz-CRD( وواحدة بعيدة عنها. هذة الطفرات المستقرة في الشبكة اﻹندوبﻻزمية )ER( كانت غير صحيحة اﻹرتباط الغليكوزيلي )N-glycosylated( مما يؤكد تمركزها في الشبكة اﻹندوبﻻزمية و كانت موسومة بسلسلة البوليوبيكويتين )polyubiquitin chains( مما جعلها هدف للتدهور البروتيزوملي )proteasomal degradation(. كانت أعمار النصف للنوع الطبيعي من مسك )MuSK( و النوع المطفرن )P344R- MuSK( هي 45 و 37 دقيقة على التوالي، وقد تحسن نصف عمر اﻷخير خﻻل فترة الحضانة بواسطة استخدام المانع البروتيزوملي )proteasomal inhibitor MG132(. أظهرت الطفرة مسك )P344R-MuSK( نحو 50% من نشاط الفسفرة اﻵلي في الجسم الحي )in vivo autophosphorylation(. لقد تم تصحيح عيوب عملية النقل في ثﻻث طفرات من العشر وهي )M105T-FZD4 و C204Y-FZD4 و P344R-MuSK( من خﻻل زراعة الخﻻيا المختبرة عند درجة حرارة 27 سلسيوس واستخدام المواد الكيميائية ) Chemical chaperones( الجلسرين و ثنائي ميثيل السلفوكسيد و ميكرومتر الثابسيجارجن و الكركمين ) 2.5-7.5% glycerol, 0.1-1% dimethyl sulfoxide, 10 µM thapsigargin, 1 µM curcumin(. لم تتأثر عملية نقل البروتين السليم فرزلد )FZD4( بأي من طفرات فرزلد )FZD4( التسع عندما تم احتضانها بشكل ثنائي مع كل منها مما يدل على أن عدم كفاية الهابلو (haploinsufficiency) هي اﻵلية المسببة لهذا المرض. لذا فإن كافة الطفرات التسع الموجودة في )FZ- CRD( والمسببة لمرض (FEVR) ولمرض (CMS) تنتج عن الطي الخاطئ للبروتين. وعلى العكس من ذلك فإن واحدة فقط من الطفرات اﻹثنا عشر الموجودة خارج )FZ- CRD( أدت الى اﻹستقرار في الشبكة اﻹندوبﻻزمية )ER(. أظهرت هذه النتائج
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