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Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II
Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska högskolan.
Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
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2004 (engelsk)Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 342, nr 2, s. 619-633Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.

sted, utgiver, år, opplag, sider
Oxford: Elsevier , 2004. Vol. 342, nr 2, s. 619-633
Emneord [en]
Misfolding, loss-of-function, aggregation, molten globule, misfolding inhibitor
HSV kategori
Identifikatorer
URN: urn:nbn:se:liu:diva-21072DOI: 10.1016/j.jmb.2004.07.024OAI: oai:DiVA.org:liu-21072DiVA, id: diva2:240509
Tilgjengelig fra: 2009-09-28 Laget: 2009-09-28 Sist oppdatert: 2018-04-25bibliografisk kontrollert
Inngår i avhandling
1. Protein Misfolding in Human Diseases
Åpne denne publikasjonen i ny fane eller vindu >>Protein Misfolding in Human Diseases
2009 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories:

  1. Loss-of-function diseases
  2. Gain-of-toxic-function diseases
  3. Infectious misfolding diseases

 

Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.

 

The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease.

Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

sted, utgiver, år, opplag, sider
Linköping: Linköping University Electronic Press, 2009. s. 103
Serie
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1239
Emneord
Misfolding, carbonic anhydrase, prion protein, protein stability
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-21077 (URN)978-91-7393-698-9 (ISBN)
Disputas
2009-02-26, Planck, Campus Valla, Linköpings universitet, Linköping, 13:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2009-10-16 Laget: 2009-09-28 Sist oppdatert: 2018-04-25bibliografisk kontrollert

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