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- Rafalik, M., et al.
(författare)
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The identification of discontinuous epitope in the human cystatin C – Monoclonal antibody HCC3 complex
- 2019
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 191, s. 58-67
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders. In this work we present epitope identification of the hCC-HCC3 complex using methods such as affinity chromatography, epitope excision and extraction MS approach, enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry (HDX MS). Comprehensive analysis of the obtained results allowed us to identify the epitope sequence with the key fragment covering hCC L1 loop and two potential epitopic fragments – α-helical part, hCC (17–28) and β4 strand in C-terminal part of hCC. The presence of the L1 loop in the epitope sequence accounts for the significant reduction of hCC dimer formation in the presence of HCC3 antibody. Significance of the study: Deciphering the mechanism of the cystatin C aggregation process and detailed analysis of the interactions between hCC, or its pathogenic variant, and monoclonal antibodies, potentially constituting aggregation inhibitors, might be of great value as there still is a complete lack of any kind of efficient therapy for young people with the pathogenic mutation of hCC.
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- Nilsson, M., et al.
(författare)
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Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C. Use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:23, s. 24236-24245
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Tidskriftsartikel (refereegranskat)abstract
- Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins. This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils. One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life. The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors. The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C. In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers. The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C. In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C. It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.
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- Rodziewicz-Motowidlo, S, et al.
(författare)
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Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: Why is the L68Q variant amyloidogenic?
- 2006
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1095-8657 .- 1047-8477. ; 154:1, s. 68-78
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Tidskriftsartikel (refereegranskat)abstract
- Human L68Q cystatin C is one of the known human amyloidogenic proteins. In its native state it is a monomer with alpha/beta structure. Experimental evidence suggests that L68Q variant associates into dimeric intermediates and that the dimers subsequently self-assemble to form amyloid deposits and insoluble fibrils. Details of the pathway of L68Q mutant amyloid formation are unclear; however, different experimental approaches with resolutions at molecular level have provided Some clues. Probably, the stability and flexibility of monomeric L68Q variant play essential roles in the early steps of amyloid formation; thus, it is necessary to characterize early conformational changes of L68Q cystatin C monomers. In this paper, we demonstrate the possibility that the differences between the monomeric forms of wild-type (wt) cystatin C and its L68Q variant are responsible for higher tendency of the L68Q cystatin C amyloidogenesis. We started our studies with the simulations of wt and L68Q cystatin C monomers. Nanosecond time scale molecular dynamics simulations at 308 K were performed using AMBER7.0 program, The results show that the structure of the L68Q monomer was changed, relative to the wt cystatin C structure. The results support earlier speculation that the L68Q point mutation would easily lead to dimer formation. (c) 2006 Published by Elsevier Inc.
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- Rodziewicz-Motowidlo, Sylwia, et al.
(författare)
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Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques
- 2007
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 87:1, s. 23-39
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Tidskriftsartikel (refereegranskat)abstract
- The beta-amyloid (A beta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the A beta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of A beta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish AP variants. The fragment was chosen because it been shown to be the most important for amyloid fibril formation detailed structure of both variants A beta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.
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- Stachowiak, K, et al.
(författare)
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Effect of antisense peptide binding on the dimerization of human cystatin C - gel electrophoresis and molecular modeling studies
- 2004
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Ingår i: Acta Biochimica Polonica. - 0001-527X. ; 51:1, s. 153-160
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C (HCC) shows a tendency to dimerize. This process is particularly easy in the case of the L68Q HCC mutant and might lead to formation of amyloid deposits in brain arteries of young adults. Our purpose was to find ligands of monomeric HCC that can prevent its dimerization. Eleven antisense peptide ligands of monomeric HCC were designed and synthesized. The influence of these ligands on HCC dimerization was studied using gel electrophoresis and molecular modeling methods. The results suggest that all the designed peptides interact with monomeric HCC facilitating its dimerization rather than preventing it.
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- Wieczerzak, E, et al.
(författare)
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Novel azapeptide inhibitors of cathepsins B and K. Structural background to increased specificity for cathepsin B
- 2005
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Ingår i: Journal of Peptide Research. - : Wiley. - 1397-002X .- 1399-3011. ; 66:S1, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- We have designed and synthesized a new series of azapeptides which act as potential inhibitors of cathepsin B and/or cathepsin K. Their structures are based upon the inhibitory sites of natural cysteine protease inhibitors, cystatins. For the synthesized azapeptides, the equilibrium constants for dissociation of inhibitor-enzyme complex, K-i, were determined. Comparison of these values indicated that all of the azainhibitors act much stronger toward cathepsin B. Z-Arg-Leu-His-Agly-Ile-Val-OMe (7) proved to be approximately 500 times more potent for cathepsin B than for cathepsin K. To be able to explain the obtained experimental values we used the molecular dynamics procedures to analyze the interactions between cathepsin B and compound 7. We also determined the structure of the most potent and selective cathepsin B azainhibitor by means of NMR studies and theoretical calculations. In this report, we describe SAR studies of azapeptide inhibitors indicating the influence of the conformational flexibility of the examined compounds on inhibition of cathepsins B and K.
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