A Chloroplast Localized Small Heat Shock Protein, Hsp21. Importance of Hsp21 in stress protection of transgenic Arabidopsis thaliana, recombinant expression and characterization of oxidation-dependent conformational changes.
(2000)- Abstract
- A specific group of heat shock proteins, the so called small heat shock proteins (sHsps), exists in all organisms but are especially abundant in plants. The sHsps prevent aggregation of other proteins (substrate proteins) during transient heat and oxidative stress. Molten globule forms of the substrate proteins are kept in a re-folding competent state by binding onto the outer surface of the sHsp. Members of the superfamily of sHsps and a-crystallins share a conserved C-terminal domain, the a-crystallin domain. This thesis is concerned with a chloroplast localized sHsp, Hsp21. In addition to the a-crystallin domain Hsp21 contains a conserved N-terminal domain predicted to form an amphipathic a-helix with highly conserved methionine... (More)
- A specific group of heat shock proteins, the so called small heat shock proteins (sHsps), exists in all organisms but are especially abundant in plants. The sHsps prevent aggregation of other proteins (substrate proteins) during transient heat and oxidative stress. Molten globule forms of the substrate proteins are kept in a re-folding competent state by binding onto the outer surface of the sHsp. Members of the superfamily of sHsps and a-crystallins share a conserved C-terminal domain, the a-crystallin domain. This thesis is concerned with a chloroplast localized sHsp, Hsp21. In addition to the a-crystallin domain Hsp21 contains a conserved N-terminal domain predicted to form an amphipathic a-helix with highly conserved methionine residues on the hydrophobic side. Using transgenic Arabidopsis thaliana plants that overexpress Hsp21 I found that Hsp21 improves the plant performance during concomitant heat and light stress and that Hsp21 undergoes conformational changes during stress. To characterize the different conformations, recombinant Hsp21 protein was used and different biochemical and biophysical methods as circular dichroism, fluorescence spectroscopy, mass spectrometry and site-directed mutagenesis. A similar conformational change could be induced in the recombinant Hsp21 protein by oxidation with hydrogen peroxide. In the oxidized Hsp21 protein the highly conserved methionine residues was found to be in methionine sulfoxide form. The oxidized Hsp21 showed loss in a-helical secondary structure and chaperone activity. An enzyme, peptide methionine sulfoxide reductase, was able to reduce the methionine sulfoxides and recover the chaperone activity. The present data indicate that the methionine-rich amphipathic a-helix, which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone activity very dependent on the chloroplast redox state. (Less)
- Abstract (Swedish)
- Popular Abstract in Swedish
När en organism utsätts för stress, t. ex. vid hög temperatur, starkt solljus, torka eller köld, produceras särskilda hjälpar-proteiner, s. k. ¨stress-proteiner¨. Syntesen av cellens normala proteiner stängs av och istället produceras bara stress-proteiner. Dessa stress-proteiner är till för att skydda andra protein som skadas under stress. De s. k. ¨värme-stress¨ proteinerna bildar en familj av proteiner som först upptäcktes då organismer utsattes för värme-stress. Man har senare upptäckt att många värme-stress proteiner också produceras vid andra typer av stress, som t. ex. oxidativ stress.
Gemensamt för alla värme-stress proteiner är att de känner igen ytor på protein som... (More) - Popular Abstract in Swedish
När en organism utsätts för stress, t. ex. vid hög temperatur, starkt solljus, torka eller köld, produceras särskilda hjälpar-proteiner, s. k. ¨stress-proteiner¨. Syntesen av cellens normala proteiner stängs av och istället produceras bara stress-proteiner. Dessa stress-proteiner är till för att skydda andra protein som skadas under stress. De s. k. ¨värme-stress¨ proteinerna bildar en familj av proteiner som först upptäcktes då organismer utsattes för värme-stress. Man har senare upptäckt att många värme-stress proteiner också produceras vid andra typer av stress, som t. ex. oxidativ stress.
Gemensamt för alla värme-stress proteiner är att de känner igen ytor på protein som exponeras under stress, s. k. hydrofoba ytor. Hydrofoba ytor är klistriga ytor som lätt klistrar ihop med hydrofoba ytor i andra protein. Då proteiner med hydrofoba ytor klistras ihop bildas stora aggregat som kan vara dödliga för cellen. Värme-stress proteiner binder till dessa hydrofoba ytor och hindrar därigenom proteiner från att aggregera, på så sätt hjälper värme-stress proteinerna cellen att överleva. Aggregering av protein är inblandat vid många sjukdomar, t. ex. vid neuro-degenerativa sjukdomar som Alzheimers och galna-ko sjukan.
Jag har studerat ett värme-stress protein som finns i växters kloroplaster och som betecknas Hsp21 (heat shock protein 21). Jag fann att om man manipulerar växter så att de ökar sin produktion av Hsp21 ökar växtens stresstålighet då de utsätts för värme-stress och starkt solljus. Jag fann även att Hsp21 ändrar form under själva stressen men då temperaturen sänktes återfick Hsp21 sin ursprungliga form. För att studera formförändringen använde jag rent Hsp21 protein och fann att om man oxiderade proteinet i ett provrör uppstod samma formförändring som uppstod under stress i växten. Det visade sig att den oxiderade formen inte kunde förhindra aggregering av värme-stressade proteiner. Oxidering av Hsp21 förstör alltså den skyddande funktionen Hsp21 har.
Nyligen har man upptäckt ett enzym i växters kloroplaster som kan reducera oxiderat Hsp21. Då Hsp21 först oxiderades och sedan reducerades med detta enzym återfick Hsp21 sin skyddande funktion. Hsp21's skyddande funktion kan alltså regleras genom oxidering/reducering i ett provrör.
Exakt hur värme-stress proteinerna fungerar i cellen vet man inte. Genom att använda rent Hsp21 protein och studera Hsp21´s skyddande funktion utanför cellen har vi fått ledtrådar om hur Hsp21 kan fungera i cellen. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/40788
- author
- Härndahl, Ulrika LU
- supervisor
- opponent
-
- Professor Arrigo, André-Patrick, Equipe Stress oxydant, Chaperons et Apoptose, Centre de Génétique Moléculaire et Cellulaire, Université Claude Bernard, Lyon 1, Villeurbanne Cedex, France
- organization
- publishing date
- 2000
- type
- Thesis
- publication status
- published
- subject
- keywords
- a-crystallin, sHsp, chaperone, amphipathic a-helix, oligomer, redox-regulated, methionine sulfoxidation, oxidative stress, regulation, protein structure, circular dichroism, mass spectrometry, fluorescence spectroscopy, Biochemistry, Metabolism, Biokemi
- pages
- 158 pages
- publisher
- Department of Biochemistry, Lund University
- defense location
- Nils Alwalls room, Ideon Gamma, Sölvegatan 41, Lund
- defense date
- 2000-10-06 10:15:00
- external identifiers
-
- other:LUNKDL/(NKBL-1065)/1-190/2000
- ISBN
- 91-7874-087-8
- language
- English
- LU publication?
- yes
- id
- f36b0b38-0665-4de3-94cc-92497ef1f02a (old id 40788)
- date added to LUP
- 2016-04-04 12:23:49
- date last changed
- 2018-11-21 21:10:43
@phdthesis{f36b0b38-0665-4de3-94cc-92497ef1f02a,
abstract = {{A specific group of heat shock proteins, the so called small heat shock proteins (sHsps), exists in all organisms but are especially abundant in plants. The sHsps prevent aggregation of other proteins (substrate proteins) during transient heat and oxidative stress. Molten globule forms of the substrate proteins are kept in a re-folding competent state by binding onto the outer surface of the sHsp. Members of the superfamily of sHsps and a-crystallins share a conserved C-terminal domain, the a-crystallin domain. This thesis is concerned with a chloroplast localized sHsp, Hsp21. In addition to the a-crystallin domain Hsp21 contains a conserved N-terminal domain predicted to form an amphipathic a-helix with highly conserved methionine residues on the hydrophobic side. Using transgenic Arabidopsis thaliana plants that overexpress Hsp21 I found that Hsp21 improves the plant performance during concomitant heat and light stress and that Hsp21 undergoes conformational changes during stress. To characterize the different conformations, recombinant Hsp21 protein was used and different biochemical and biophysical methods as circular dichroism, fluorescence spectroscopy, mass spectrometry and site-directed mutagenesis. A similar conformational change could be induced in the recombinant Hsp21 protein by oxidation with hydrogen peroxide. In the oxidized Hsp21 protein the highly conserved methionine residues was found to be in methionine sulfoxide form. The oxidized Hsp21 showed loss in a-helical secondary structure and chaperone activity. An enzyme, peptide methionine sulfoxide reductase, was able to reduce the methionine sulfoxides and recover the chaperone activity. The present data indicate that the methionine-rich amphipathic a-helix, which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone activity very dependent on the chloroplast redox state.}},
author = {{Härndahl, Ulrika}},
isbn = {{91-7874-087-8}},
keywords = {{a-crystallin; sHsp; chaperone; amphipathic a-helix; oligomer; redox-regulated; methionine sulfoxidation; oxidative stress; regulation; protein structure; circular dichroism; mass spectrometry; fluorescence spectroscopy; Biochemistry; Metabolism; Biokemi}},
language = {{eng}},
publisher = {{Department of Biochemistry, Lund University}},
school = {{Lund University}},
title = {{A Chloroplast Localized Small Heat Shock Protein, Hsp21. Importance of Hsp21 in stress protection of transgenic Arabidopsis thaliana, recombinant expression and characterization of oxidation-dependent conformational changes.}},
year = {{2000}},
}