| 1. |
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| 2. |
- Forsberg, Göte, et al.
(författare)
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Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease
- 2004
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Ingår i: American Journal of Gastroenterology. - : Ovid Technologies (Wolters Kluwer Health). - 0002-9270 .- 1572-0241. ; 99:5, s. 894-904
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Exposure to gliadin and related prolamins and appropriate HLA-DQ haplotype are necessary but not sufficient for contracting celiac disease (CD). Aberrant innate immune reactions could be contributing risk factors. Therefore, jejunal biopsies were screened for bacteria and the innate immune status of the epithelium investigated.METHODS: Children with untreated, treated, challenged CD, and controls were analyzed. Bacteria were identified by scanning electron microscopy. Glycocalyx composition and mucin and antimicrobial peptide production were studied by quantitative RT-PCR, antibody and lectin immunohistochemistry.RESULTS: Rod-shaped bacteria were frequently associated with the mucosa of CD patients, with both active and inactive disease, but not with controls. The lectin Ulex europaeus agglutinin I (UEAI) stained goblet cells in the mucosa of all CD patients but not of controls. The lectin peanut agglutinin (PNA) stained glycocalyx of controls but not of CD patients. mRNA levels of mucin-2 (MUC2), alpha-defensins HD-5 and HD-6, and lysozyme were significantly increased in active CD and returned to normal in treated CD. Their expression levels correlated to the interferon-gamma mRNA levels in intraepithelial lymphocytes. MUC2, HD-5, and lysozyme proteins were seen in absorptive epithelial cells. beta-defensins hBD-1 and hBD-2, carcinoembryonic antigen (CEA), CEA cell adhesion molecule-1a (CEACAM1a), and MUC3 were not affected.CONCLUSIONS: Unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients. These glycosylation differences could facilitate bacterial adhesion. Ectopic production of MUC2, HD-5, and lysozyme in active CD is compatible with goblet and Paneth cell metaplasia induced by high interferon-gamma production by intraepithelial lymphocytes.
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| 3. |
- Almstedt, Karin, 1980-, et al.
(författare)
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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
- 2004
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Ingår i: Journal of Molecular Biology. - Oxford : Elsevier. - 0022-2836 .- 1089-8638. ; 342:2, s. 619-633
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Andersson, D., et al.
(författare)
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Cofactor-induced refolding : Refolding of molten globule carbonic anhydrase induced by Zn(II) and Co(II)
- 2001
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 40:9, s. 2653-2661
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Tidskriftsartikel (refereegranskat)abstract
- The stability versus unfolding to the molten globule intermediate of bovine carbonic anhydrase II (BCA II) in guanidine hydrochloride (GuHCl) was found to depend on the metal ion cofactor [Zn(II) or Co(II)], and the apoenzyme was observed to be least stable. Therefore, it was possible to find a denaturant concentration (1.2 M GuHCl) at which refolding from the molten globule to the native state could be initiated merely by adding the metal ion to the apo molten globule. Thus, refolding could be performed without changing the concentration of the denaturant. The molten globule intermediate of BCA II could still bind the metal cofactor. Cofactor-effected refolding from the molten globule to the native state can be summarized as follows: (1) initially, the metal ion binds to the molten globule, (2) compaction of the metal-binding site region is then induced by the metal ion binding, (3) a functioning active center is formed, and (4) finally, the native tertiary structure is generated in the outer parts of the protein.
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| 5. |
- Grankvist, Hannah, 1976-, et al.
(författare)
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Reshaping the folding energy landscape by chloride salt : Impact on molten-globule formation and aggregation behavior of carbonic anhydrase
- 2004
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 566:1-3, s. 95-99
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Tidskriftsartikel (refereegranskat)abstract
- During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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| 6. |
- Hammarström, Per, 1972-
(författare)
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Biophysical Studies of Protein Folding, Aggregation and Interactions with Molecular Chaperones
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The information for a protein to fold correctly into a well defined three-dimensional functional structure is embedded in the primary sequence. Much effort has been devoted to characterize the nature of the unfolded state, since this state represents the starting point of the folding reaction. Also the formation of protein aggregates has an important role in biotechnology and also causes numerous diseases. Nature has evolved molecular chaperones as a protection system to prevent protein aggregation. By studying the interactions between chaperones and their protein substrates many important clues about protein folding and misfolding can be found.It is very difficult to structurally investigate partially folded/unfolded proteins, because of their high intrinsic dynamics. Therefore, to obtain site specific information of the protein structure a cysteine labeling approach together with spectroscopic methods can be used. By this approach cysteines can be introduced at particular locations in the protein structure. The sulfhydryl groups of these Cys residues are either used as handles to which various spectroscopic labels can be attached or the chemical reactivity of the sulfhydryl group by itself constitute the probe function. Spin and fluorescent labels will provide dynamic information and report on changes in mobility and polarity. Communicating labels, such as pyrene-pyrene excimer fluorescence and fluorescence resonance energy transfer, FRET, can be used to monitor distances within proteins. In this thesis human carbonic anhydrase II, HCA II, has been used as a model protein for studies of protein folding, aggregation and interactions with the molecular chaperone GroEL.Residual structure in the unfolded state of HCA II: HCA II consists of 259 amino acids and folds into a mainly β-sheet protein. Ten β-strands span the central part of the protein and divide the molecule into two halves, one half containing the active site and the N-terminal subdomain, and the other half containing a large hydrophobic core. By utilization of a doubly labeled cysteine mutant (N67C/C206) labeled with pyrene fluorophores the unfolding process of the central part of HCA II could be monitored by the loss of pyrene excimer fluorescence separating the pyrenes. These measurements revealed an unfolding transition at a GuHC1 concentration significantly higher than that required to induce unfolding of the protein as monitored by circular dicrosim, CD. Utilizing fluorescence resonance energy transfer, FRET, between tryptophans and an inserted acceptor (AEDANS) at various positions (16, 54, 67, 79, 118, 142,146,244 and 245) in the protein revealed a continuous solvation of the central core of HCA II at very strong GuHC1 concentrations. In addition, the use of a single site (position 79) labeled with different probes in the periphery of the central hydrophobic core showed that a persistent cluster can form locally and be associated to the central core. These results provide a picture of stable structures within HCA II mostly composed of hydrophobic clusters that suggestively guide the folding process at the onset of folding.Aggregation of HCA II: Partial unfolding of HCA II results in a molten-globule intermediate, believed to retain native-like secondary structure but very little tertiary interactions. This molten-globule intermediate of HCA II was found to form aggregates. Local and long-range interactions in a protein sequence form a pattern of favorable interactions strongly dependent on each other, meaning that the protein folding process is highly cooperative. These intramolecular interactions are a prerequisite for correct folding. However, if such high affinity exists within a single protein chain, another protein molecule with the same exposed structural pattern can instead form intermolecular interactions with the first protein chain. This will result in the formation of protein oligomers and aggregates. During denaturation by 1-2 M GuHC1 HCA II forms aggregates that organize into an ensemble of soluble oligomers. During thermal denaturation HCA II forms aggregates that grow into micron sized precipitates. The aggregates are composed of partially folded proteins with a distribution of polar, dynamic as well as non-polar, compact regions. The intermolecular interactions involved in aggregate formation of HCA II were localized in a direct way by measuring pyreneexcimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. Another approach was the utilization of 9 site-specific AEDANS-labeled cysteine mutants for tryptophan-AEDANS FRET measurements. The data showed that the contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large β-sheet of the protein, within a limited region between the central β-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific β-sheet containing regions inaggregate formation.GroEL-HCA II interactions: Aggregation of HCA II is prevented by the presence of the chaperon in GroEL,and because GroEL and a molten-globule intermediate of HCA II form a complex at elevated temperatures the interactions between the two proteins can be studied. These interactions were mapped by site-directed spin-labeling and EPR (Electron Paramagnetic Resonance) measurements and by site-directed fluorescencelabeling and fluorescence shift and AEDANS FRET measurements. The interaction with GroEL was shown to include interactions with outer parts of the HCA II molecule, such as peripheral β-strands and the N-terminal domain, which have previously been shown to be rather unstable. As a result of the interaction, the rigid and compact hydrophobic core was shown to exhibit higher flexibility. FRET measurements showed that the volume of HCA II expanded significantly compared to the molten-globule intermediate as a result of the interaction with GroEL. This suggests an active role of GroEL when exerting its chaperone action and the unfolding action is likely to facilitate rearrangements of misfolded structure in the substrate protein. How does GroEL exert this unfoldase activity? By the use of site-specific labeling of cysteines in GroEL it was possible to observe conformational changes induced by HCA II binding. i) Using iodoacetate to measure cysteine reactivity showed that the cysteines became more accesible during HCA II binding. ii)Determining reactivity with spin labels and thereafter performing EPR measurements demonstrated that the cysteines in GroEL were buried and became accessible upon binding of HCA II. iii) In GroEL that was spinlabeled at room temperature and subsequently used in HCA II binding experiments, the labels showed greater mobility in the presence of HCA II. iv) Fluoresceine-labeled GroEL displayed decreased fluorescence anisotropy, indicating higher flexibility during HCA II binding. This is qualitatively similar to results obtained from GroES binding, which has previously been shown to cause GroEL to expand. v) The quantum yield of AEDANS-labeled GroEL was increased in the presence of HCA II. All data indicated a more flexible and open structure of GroEL as a cause of binding HCA II, that can be utilized to further unfold the protein substrate. GroEL was, however, not capable to dissolve aggregates that were preformed from a molten-globule intermediate. Thus, it seems that the surface that is actively affected by GroEL must be exposed, and not hidden as in the HCA II aggregates, to allow GroEL to exert its chaperone activity. This suggests that surfaces responsible for off-pathway aggregation are overlapping with the surfaces involved in the interaction with chaperones.
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| 7. |
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| 8. |
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| 9. |
- Hammarström, Per, et al.
(författare)
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D18G transthyretin is monomeric, aggregation prone, and not detectable in plasma and cerebrospinal fluid : A prescription for central nervous system amyloidosis?
- 2003
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 42:22, s. 6656-6663
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Tidskriftsartikel (refereegranskat)abstract
- Over 70 transthyretin (TTR) mutations facilitate amyloidosis in tissues other than the central nervous system (CNS). In contrast, the D18G TTR mutation in individuals of Hungarian descent leads to CNS amyloidosis. D18G forms inclusion bodies in Escherichia coli, unlike the other disease-associated TTR variants overexpressed to date. Denaturation and reconstitution of D18G from inclusion bodies afford a folded monomer that is destabilized by 3.1 kcal/mol relative to an engineered monomeric version of WT TTR. Since TTR tetramer dissociation is typically rate limiting for amyloid formation, the monomeric nature of D18G renders its amyloid formation rate 1000-fold faster than WT. It is perplexing that D18G does not lead to severe early onset systemic amyloidosis, given that it is the most destabilized TTR variant characterized to date, more so than variants exhibiting onset in the second decade. Instead, CNS impairment is observed in the fifth decade as the sole pathological manifestation, however, benign systemic deposition is also observed. Analysis of heterozygote D18G patient's serum and cerebrospinal fluid (CSF) detects only WT TTR, indicating that D18G is either rapidly degraded postsecretion or degraded within the cell prior to secretion, consistent with its inability to form hybrid tetramers with WT TTR. The nondetectable levels of D18G TTR in human plasma explain the absence of an early onset systemic disease. CNS disease may result owing to the sensitivity of the CNS to lower levels of D18G aggregate. Alternatively, or in addition, we speculate that a fraction of D18G made by the choroid plexus can be transiently tetramerized by the locally high thyroxine (T4) concentration, chaperoning it out into the CSF where it undergoes dissociation and amyloidogenesis due to the low T4 CSF concentration. Selected small molecule tetramer stabilizers can transform D18G from a monomeric aggregation-prone state to a nonamyloidogenic tetramer, which may prove to be a useful therapeutic strategy against TTR-associated CNS amyloidosis.
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| 10. |
- Hammarström, Per, et al.
(författare)
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High-resolution probing of local conformational changes in proteins by the use of multiple labeling : Unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes
- 2001
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 80:6, s. 2867-2885
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Tidskriftsartikel (refereegranskat)abstract
- Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2.2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)ethyl)amino)naphtalene-1 -sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation, HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 Angstrom from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 Angstrom from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At similar to1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 Angstrom in diameter depending on the experimental conditions and spectroscopic technique used.
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