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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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    1688 • The Journal of Neuroscience, February 13, 2008 • 28(7):1688–1696 Cellular/Molecular Muscle-Specific Receptor Tyrosine Kinase Endocytosis in Acetylcholine Receptor Clustering in Response to Agrin Dan Zhu,1 Zhihua Yang,1 Zhenge Luo,2 Shiwen Luo,1 Wen C. Xiong,1 and Lin Mei1 1Program of Developmental Neurobiology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, and 2Institute of Neuroscience and Key Laboratory of Neurobiology, Shanghai Institutes for Biological Scineces, Chinese Academy of Sciences, Shanghai 200031, China Agrin, a factor used by motoneurons to direct acetylcholine receptor (AChR) clustering at the neuromuscular junction, initiates signal transduction by activating the muscle-specific receptor tyrosine kinase (MuSK). However, the underlying mechanisms remain poorly defined. Here, we demonstrated that MuSK became rapidly internalized in response to agrin, which appeared to be required for induced AChR clustering. Moreover, we provided evidence for a role of N-ethylmaleimide sensitive factor (NSF) in regulating MuSK endocytosis and subsequent signaling in response to agrin stimulation. NSF interacts directly with MuSK with nanomolar affinity, and treatment of muscle cells with the NSF inhibitor N-ethylmaleimide, mutation of NSF, or suppression of NSF expression all inhibited agrin-induced AChR clustering. Furthermore, suppression of NSF expression and NSF mutation attenuate MuSK downstream signaling. Our study reveals a potentially novel mechanism that regulates agrin/MuSK signaling cascade. Key words: neuromuscular junction; nicotinic acetylcholine receptors; agrin; endocytosis; MuSK; NSF Introduction dependent manner (Okada et al., 2006; Jones et al., 2007). Agrin/ The vertebrate neuromuscular junction (NMJ) is a synapse MuSK signaling is mediated or regulated by several interacting formed between motoneurons and skeletal muscle fibers.
  • Viral Mediated Tethering to SEL1L Facilitates ER-Associated Degradation of IRE1

    Viral Mediated Tethering to SEL1L Facilitates ER-Associated Degradation of IRE1

    bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.330779; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 Viral mediated tethering to SEL1L facilitates ER-associated degradation of IRE1 4 5 6 Florian Hinte, Jendrik Müller, and Wolfram Brune* 7 8 Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany 9 10 * corresponding author. [email protected] 11 12 Running head: MCMV M50 promotes ER-associated degradation of IRE1 13 14 15 Word count 16 Abstract: 249 17 Text: 3275 (w/o references) 18 Figures: 7 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.330779; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 19 Abstract 20 The unfolded protein response (UPR) and endoplasmic reticulum (ER)-associated 21 degradation (ERAD) are two essential components of the quality control system for proteins 22 in the secretory pathway. When unfolded proteins accumulate in the ER, UPR sensors such 23 as IRE1 induce the expression of ERAD genes, thereby increasing protein export from the ER 24 to the cytosol and subsequent degradation by the proteasome. Conversely, IRE1 itself is an 25 ERAD substrate, indicating that the UPR and ERAD regulate each other.
  • View of All NF-Κb Post-Translational Modifications See Review by Perkins [179]

    View of All NF-Κb Post-Translational Modifications See Review by Perkins [179]

    UNIVERSITY OF CINCINNATI Date: 8-May-2010 I, Michael Wilhide , hereby submit this original work as part of the requirements for the degree of: Master of Science in Molecular, Cellular & Biochemical Pharmacology It is entitled: Student Signature: Michael Wilhide This work and its defense approved by: Committee Chair: Walter Jones, PhD Walter Jones, PhD Mohammed Matlib, PhD Mohammed Matlib, PhD Basilia Zingarelli, MD, PhD Basilia Zingarelli, MD, PhD Jo El Schultz, PhD Jo El Schultz, PhD Muhammad Ashraf, PhD Muhammad Ashraf, PhD 5/8/2010 646 Hsp70.1 contributes to the NF-κΒ paradox after myocardial ischemic insults A thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirement for the degree of Master of Science (M.S.) in the Department of Pharmacology and Biophysics of the College of Medicine by Michael E. Wilhide B.S. College of Mount St. Joseph 2002 Committee Chair: W. Keith Jones, Ph.D. Abstract One of the leading causes of death globally is cardiovascular disease, with most of these deaths related to myocardial ischemia. Myocardial ischemia and reperfusion causes several biochemical and metabolic changes that result in the activation of transcription factors that are involved in cell survival and cell death. The transcription factor Nuclear Factor-Kappa B (NF-κB) is associated with cardioprotection (e.g. after permanent coronary occlusion, PO) and cell injury (e.g. after ischemia/reperfusion, I/R). However, there is a lack of knowledge regarding how NF- κB mediates cell survival vs. cell death after ischemic insults, preventing the identification of novel therapeutic targets for enhanced cardioprotection and decreased injurious effects.