Structure and function of mitochondrial small heat shock protein 22 in Drosophila melanogaster

Authors: Dabbaghizadeh, Afrooz
Advisor: Tanguay, Robert M.
Abstract: The small heat shock proteins (sHsps) were first discovered in Drosophila. Members of this family are molecular chaperones and are present in most eukaryotic and prokaryotic. Although, they are induced in response to most of the stressors including heat shock, they are also expressed in absence of stress. SHsps for mdynamic structures that assemble into oligomers which are essential during stress conditions by preventing aggregation of denatured proteins and promoting their folding by ATP dependent molecular chaperones. Drosophila melanogaster genome encodes 12 sHsps, that have developmental expression patterns, diverse intracellular localizations and distinct substrate specificities. DmHsp22 is up to now the only sHsp localized in mitochondria before and after heat shock. It is preferentially regulated during ageing and in response to heat and oxidative stresses. Over-expression of DmHsp22 increases lifespan and resistance to stress and its down-regulation is detrimental. It is an efficient chaperone and could be involved in the mitochondrial unfolding protein response (UPRMT). However, the exact mechanism of its action is poorly understood. Structurally, DmHsp22 forms one population of oligomers similar to the many metazoan sHsps but DmHsp27. Sequence alignment of DmHsp22 with sHsps in Drosophilaand other organisms at the alpha crystalline domain (ACD) region demonstrated the presence of three highly conserved arginine residues in this domain. Strong conservation of these residues suggest their possible involvement in structure and function of DmHsp22. Substitution of highly conserved arginine residues in mammalian sHsps is associated with some pathogenesis and triggers protein conformational changes as well as intracellular protein aggregation. Mutation of arginine to glycine at three highly conserved residues of ACD in DmHsp22 (R105, R109, R110) results in one oligomeric population as well which in the case of R110G disrupts the structure and causes formation of smaller oligomers. Although DmHsp22 as well as mutants have been characterized as effective in vitro chaperones, the exact mechanism(s) of action in mitochondria and information about protective behavior requires defining of in vivoprotein interacting network. We have used immunoaffinity conjugation (IAC) technique to recover 60 proteins that specifically interact with DmHsp22 in vivo during normal and heat treatment using cell extract of mammalian cells expressing DmHsp22. The IAC performed on mitochondrial fraction identified 39 proteins that specifically interact with DmHsp22. Combination of IAC with mass spectroscopy analysis of mitochondria of HeLa cells transfected with DmHsp22 resulted in identification of DmHsp22-binding partners under normal andunder heat shock conditions. Interaction between DmHsp22 and two other mitochondrial chaperones was validated by immunoblotting. Our approach showed that HeLa cells expressing DmHsp22 increase maximal mitochondrial oxygen consumption and ATP contents which provides a new mechanistic role for DmHsp22 in mitochondria. Further more, exogenous luciferase activity slightly increased in HeLa cells expressing DmHsp22 after the enzyme activity reduced as a result of exposure to heat. In summary, this project has characterized the oligomeric structure of DmHsp22 and a number of mutants inthe alpha crystalline domain while providing a potential mechanistic role in mitochondrial homeostasis. Determining mitochondrial network of DmHsp22 suggest its importance in this organelle not only as a molecular chaperone but also as a protein involved in several significant cellular functions.
Document Type: Thèse de doctorat
Issue Date: 2018
Open Access Date: 18 April 2019
Permalink: http://hdl.handle.net/20.500.11794/34491
Grantor: Université Laval
Collection:Thèses et mémoires

Files in this item:
Description SizeFormat 
34474.pdf4.94 MBAdobe PDFThumbnail
View/Open
All documents in CorpusUL are protected by Copyright Act of Canada.