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Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. One of rather atypical, occurring at a young age amyloidosis is hereditary cystatin C amyloid angiopathy (HCCAA) related to aggregation of L68Q variant of human cystatin C (hCC). Human cystatin C plays a very important role in many aspects of human health; however, its amyloidogenic properties manifested in HCCAA present a real, lethal threat to some populations and any work on factors that can affect possible influencing hCC aggregation is not to overestimate. It was proved that interaction of hCC with monoclonal antibodies suppresses significantly hCC dimerization process. Therefore, immunotherapy seems to be the right approach toward possible HCCAA treatment. In this work, the hCC fragment encompassing residue 60-70 (in 2 variants: linear peptide and multiple antigenic peptide) was used as an immunogen in rabbit immunization. As a result, specific anti-hCC antibodies were found in both rabbit sera. Surprisingly, rabbit antibodies were obtained after immunization with only a short peptide. The obtained antibodies were characterized, and their influence on the aggregation propensity of the hCC molecules was evaluated. The antibodies turned out not to have any significant influence on the cystatin C dimerization process. Nevertheless, we hope that antibodies elicited in rabbits by other hCC fragments could lead to elaboration of effective treatment against HCCAA., (Copyright © 2017 John Wiley & Sons, Ltd.)

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aβ and the hCC-induced inhibition of Aβ fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C-terminal fragment hCC(101-117) was identified as the Aβ-binding region. The fragment of Aβ peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aβ(17-24). Structures and affinities of both Aβ and hCC binding sites were characterized by the enzyme-linked immunosorbent assay-like assay, by surface plasmon resonance, and by nano-ESI-FTICR MS of the hCC-Aβ-binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101-117), revealed the highest relative inhibitory effect toward Aβ-fibril formation. Herein, we present further studies on molecular details of the hCC-Aβ complex. With Ala substitution, affinity experiments, and enzyme-linked immunosorbent assay-like assays for the Aβ-binding fragment, hCC(101-117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101-117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Serum amyloid A (SAA) is a multifunctional acute-phase protein whose concentration in serum increases markedly following a number of chronic inflammatory and neoplastic diseases. Prolonged high SAA level may give rise to reactive systemic amyloid A (AA) amyloidosis, where the N-terminal segment of SAA is deposited as amyloid fibrils. Besides, recently, well-documented association of SAA with high-density lipoprotein or glycosaminoglycans, in particular heparin/heparin sulfate (HS), and specific interaction between SAA and human cystatin C (hCC), the ubiquitous inhibitor of cysteine proteases, was proved. Using a combination of selective proteolytic excision and high-resolution mass spectrometry, a hCC binding site in the SAA sequence was determined as SAA(86-104). The role of this SAA C-terminal fragment as a ligand-binding locus is still not clear. It was postulated important in native SAA folding and in pathogenesis of AA amyloidosis. In the search of conformational details of this SAA fragment, we did its structure and affinity studies, including its selected double/triple Pro → Ala variants. Our results clearly show that the SAA(86-104) 19-peptide has rather unordered structure with bends in its C-terminal part, which is consistent with the previous results relating to the whole protein. The results of affinity chromatography, fluorescent ELISA-like test, CD and NMR studies point to an importance of proline residues on structure of SAA(86-104). Conformational details of SAA fragment, responsible for hCC binding, may help to understand the objective of hCC-SAA complex formation and its importance for pathogenesis of reactive amyloid A amyloidosis., (Copyright © 2015 John Wiley & Sons, Ltd.)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life-threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute-phase-reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases--human cystatin C (hCC)--has been proved. Using a combination of selective proteolytic excision and high-resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86-104) and hCC(96-102), respectively. Here, we report further details concerning the hCC-SAA interaction. With the use of affinity tests and florescent ELISA-like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser-98 and Tyr-102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC-SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins., (Copyright © 2013 John Wiley & Sons, Ltd.)

Serum amyloid A (SAA) is a multifunctional acute-phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N-terminal 76-amino acid residue-long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases--human cystatin C (hCC)--has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA-hCC complex, using a combination of selective proteolytic excision and high-resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86-104), whereas the binding site in hCC sequence was identified as hCC(96-102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA-hCC complex., (Copyright © 2012 John Wiley & Sons, Ltd.)