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Exploring small heat shock protein chaperones by crosslinking mass spectrometry

Lambert, Wietske LU (2012)
Abstract (Swedish)
Popular Abstract in Swedish

Byggstenarna av allt liv på denna planet är celler. Vår egen kropp till exempel består av ungefär 10 biljon (1013) celler. De flesta bakterier består av bara en cell. Inne i cellerna finns alla de biomolekyler som styr livet, såsom proteiner, sockerarter, lipider, och DNA. Celler är otroligt fullpackade, särskilt vad gäller proteiner, med protein koncentrationer av up till 300 mg/ml.

Proteiner är linjära molekyler som består av olika aminosyror. För att proteiner ska fungera, måste aminosyrakedjan vara veckad i en speciell tredimensionell form. Stressfaktorer, som till exempel värme, kan göra att proteinerna delvis förlorar denna veckning. Eftersom cellen är så fullpackad, så finns det... (More)
Popular Abstract in Swedish

Byggstenarna av allt liv på denna planet är celler. Vår egen kropp till exempel består av ungefär 10 biljon (1013) celler. De flesta bakterier består av bara en cell. Inne i cellerna finns alla de biomolekyler som styr livet, såsom proteiner, sockerarter, lipider, och DNA. Celler är otroligt fullpackade, särskilt vad gäller proteiner, med protein koncentrationer av up till 300 mg/ml.

Proteiner är linjära molekyler som består av olika aminosyror. För att proteiner ska fungera, måste aminosyrakedjan vara veckad i en speciell tredimensionell form. Stressfaktorer, som till exempel värme, kan göra att proteinerna delvis förlorar denna veckning. Eftersom cellen är så fullpackad, så finns det en stor risk att sådana delvis oveckade proteiner klistrar fast i varandra och börjar klumpa ihop sig. En liknande process sker när ett ägg utsätts för stress i form av värme (äggvita har en proteinkoncentration av ungefär 100 mg/ml): proteinerna veckas ut och klumpar ihop sig, och man ser att ägget stelnar när man steker eller kokar det. I cellen kan ihop-klumpade proteiner orsaka allvarliga problem, och många olika sjukdomar är konsekvensen av att proteiner klumpat ihop sig (Alzheimers och Parkinsons sjukdom, och grå starr, till exempel).

Lyckligtvis finns det en speciell klass av proteiner i cellen som heter chaperoner, vars funktion är att hindra andra proteiner från att klumpa ihop sig. Det är emellertid inte helt utrett, på en molekylär nivå, hur det går till när chaperonerna gör detta. Särskilt vad gäller en mycket viktig undergrupp av chaperoner, som paradoxalt nog kallas ‘small heat shock proteins’, finns det få undersökningar gjorda i jämförelse med andra chaperoner.

Fokus i denna avhandling är hur ett sådant ‘small heat shock protein’, kallat Hsp21, skyddar andra proteiner från att klumpa ihop sig. För att studera detta har en kombination av kemisk koppling och masspektrometri använts. Kemisk koppling innebär att ett speciellt kemiskt reagens används för att parvis koppla ihop aminosyror i de olika proteinerna. Efter det klyvs proteinerna i små bitar. Två bitar som kopplats ihop kan sedan detekteras med hjälp av masspektrometri. Genom att identifiera vilka bitar och vilka aminosyror av proteinerna som kopplats, kan man studera vilka delar av proteinerna som var nära varandra och interagerade med varandra precis när man tillsatte det kemiska reagenset. På det sättet, kan information utvinnas gällande interaktionen mellan Hsp21 och det proteinet som skyddas av Hsp21. Experiment som dessa bidrar till vår förståelse av hur chaperonerna hela tiden skyddar de viktiga proteinerna i alla våra celler. (Less)
Abstract
Together with other molecular chaperones, small heat shock proteins are key components of the protein quality control system, which is comprised of several hundred proteins and acts to maintain proteome homeostasis in the cell. Small heat shock proteins bind unfolding proteins at an early stage, to prevent these from further unfolding and aggregating. Partially unfolded proteins are being held in a refolding competent state, to be refolded by other chaperones or degraded by the degradation machinery. In the stress response, small heat shock proteins are among the most highly upregulated, preparing the cell to absorb large quantities of partially unfolded proteins. In this way, they form the first line of defence against the threat of... (More)
Together with other molecular chaperones, small heat shock proteins are key components of the protein quality control system, which is comprised of several hundred proteins and acts to maintain proteome homeostasis in the cell. Small heat shock proteins bind unfolding proteins at an early stage, to prevent these from further unfolding and aggregating. Partially unfolded proteins are being held in a refolding competent state, to be refolded by other chaperones or degraded by the degradation machinery. In the stress response, small heat shock proteins are among the most highly upregulated, preparing the cell to absorb large quantities of partially unfolded proteins. In this way, they form the first line of defence against the threat of protein aggregation under stress conditions. The polydispersity and dynamics of the large small heat shock protein oligomers have complicated their structural and functional characterization. In particular, the molecular mechanism of substrate protein protection remains poorly understood.

The work described in this thesis aims to characterize the molecular interactions between the plant small heat shock protein Hsp21 and model substrate proteins by crosslinking mass spectrometry. The model substrate proteins citrate synthase and malate dehydrogenase, both especially vulnerable to temperature-induced aggregation, were protected from aggregation by Hsp21 and therefore used to investigate the Hsp21-substrate interactions that confer protection. To be able to study the transient Hsp21-substrate interaction by crosslinking mass spectrometry, a workflow was developed based on isotope-labelled lysine-specific crosslinking, nano-LC MALDI-TOF/TOF mass spectrometry, and data analysis with the specialized software FINDX. During the development of this workflow, interactions within Hsp21 itself were characterized as a way to evaluate the method and to learn more about the conformation of Hsp21 in absence of substrate. The interpretation of the identified Hsp21-Hsp21 crosslinks required structural information on the Hsp21 oligomer, which was obtained by single particle negative stain electron microscopy. The combination of these data with native mass spectrometry and homology modelling, led to a structure model of the Hsp21 dodecamer. The in-depth analysis of Hsp21-Hsp21 crosslinks provided a framework for further application of the crosslinking mass spectrometry workflow to the Hsp21-substrate interactions. Finally, Hsp21-substrate crosslinks were identified that support the view that unfolding substrate proteins interact with the intrinsically disordered N-terminal region of the small heat shock protein Hsp21. (Less)
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author
supervisor
opponent
  • Dr. Benesch, Justin L.P., University of Oxford
organization
publishing date
type
Thesis
publication status
published
subject
keywords
cross-linking, mass spectrometry, protein-protein interactions, Hsp21, BS3, DTSSP, MALDI-TOF/TOF, client protein, substrate, aggregation, unfolded protein, chaperone, Small heat shock protein, crosslinking
pages
168 pages
publisher
Department of Chemistry, Lund University
defense location
sal K:B, Kemicentrum, Getingevägen 60, Lund
defense date
2012-06-01 13:15
ISBN
978-91-7422-299-9
language
English
LU publication?
yes
id
8fb5873c-df61-4f35-bd04-77eae9e2915c (old id 2534872)
date added to LUP
2012-05-07 12:26:31
date last changed
2016-09-19 08:45:02
@misc{8fb5873c-df61-4f35-bd04-77eae9e2915c,
  abstract     = {Together with other molecular chaperones, small heat shock proteins are key components of the protein quality control system, which is comprised of several hundred proteins and acts to maintain proteome homeostasis in the cell. Small heat shock proteins bind unfolding proteins at an early stage, to prevent these from further unfolding and aggregating. Partially unfolded proteins are being held in a refolding competent state, to be refolded by other chaperones or degraded by the degradation machinery. In the stress response, small heat shock proteins are among the most highly upregulated, preparing the cell to absorb large quantities of partially unfolded proteins. In this way, they form the first line of defence against the threat of protein aggregation under stress conditions. The polydispersity and dynamics of the large small heat shock protein oligomers have complicated their structural and functional characterization. In particular, the molecular mechanism of substrate protein protection remains poorly understood.<br/><br>
The work described in this thesis aims to characterize the molecular interactions between the plant small heat shock protein Hsp21 and model substrate proteins by crosslinking mass spectrometry. The model substrate proteins citrate synthase and malate dehydrogenase, both especially vulnerable to temperature-induced aggregation, were protected from aggregation by Hsp21 and therefore used to investigate the Hsp21-substrate interactions that confer protection. To be able to study the transient Hsp21-substrate interaction by crosslinking mass spectrometry, a workflow was developed based on isotope-labelled lysine-specific crosslinking, nano-LC MALDI-TOF/TOF mass spectrometry, and data analysis with the specialized software FINDX. During the development of this workflow, interactions within Hsp21 itself were characterized as a way to evaluate the method and to learn more about the conformation of Hsp21 in absence of substrate. The interpretation of the identified Hsp21-Hsp21 crosslinks required structural information on the Hsp21 oligomer, which was obtained by single particle negative stain electron microscopy. The combination of these data with native mass spectrometry and homology modelling, led to a structure model of the Hsp21 dodecamer. The in-depth analysis of Hsp21-Hsp21 crosslinks provided a framework for further application of the crosslinking mass spectrometry workflow to the Hsp21-substrate interactions. Finally, Hsp21-substrate crosslinks were identified that support the view that unfolding substrate proteins interact with the intrinsically disordered N-terminal region of the small heat shock protein Hsp21.},
  author       = {Lambert, Wietske},
  isbn         = {978-91-7422-299-9},
  keyword      = {cross-linking,mass spectrometry,protein-protein interactions,Hsp21,BS3,DTSSP,MALDI-TOF/TOF,client protein,substrate,aggregation,unfolded protein,chaperone,Small heat shock protein,crosslinking},
  language     = {eng},
  pages        = {168},
  publisher    = {ARRAY(0x81648d8)},
  title        = {Exploring small heat shock protein chaperones by crosslinking mass spectrometry},
  year         = {2012},
}