Identification and characterization of antibodies elicited by human cystatin C fragment

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.     

Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C

Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.     

Identification of the epitope for anti-cystatin C antibody

Human cystatin C (hCC), like many other amyloidogenic proteins, has been shown to form dimers by exchange of subdomains of the monomeric protein. Considering the model of hCC fibrillogenesis by propagated domain swapping, it seems possible that inhibition of this process should also suppress the entire process of dimerization and fibrillogenesis which leads to specific amyloidosis (hereditary cystatin C amyloid angiopathy (HCCAA)). It was reported that exogenous agents like monoclonal antibody against cystatin C are able to suppress formation of cystatin C dimers. In the effort to find a way of controlling the cystatin fibrillization process, the interactions between monoclonal antibody Cyst-13 and cystatin C were studied in detail. The present work describes the determination of the epitope of hCC to a monoclonal antibody raised against cystatin C, Cyst-13, by MALDI mass spectrometry, using proteolytic excision of the immune complex. The shortest epitope sequence was determined as hCC(107-114). Affinity studies of synthetic peptides revealed that the octapeptide with epitope sequence does not have binding ability to Cyst-13, whereas its longer counterpart, hCC(105-114), binds the studied antibody. The secondary structure of the peptides with epitope sequence was studied using circular dichroism and NMR spectroscopy.     

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: why is the L68Q variant amyloidogenic?

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 308K 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.     

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

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.     

Thermal and guanidine hydrochloride-induced denaturation of human cystatin C

Wild-type human cystatin C is directly involved in pathological fibrils formation, leading to hemorrhage, dementia and eventually death of people suffering from cerebral amyloid angiopathy. Some studies on cystatin C oligomerization have been already done but some points are still unclear. In order to learn more about this important process, we have investigated thermal and chemical (guanidine hydrochloride-induced) denaturation of human cystatin C. Studies performed using tryptophan fluorescence, calorimetry, circular dichroism and Fourier transform infrared spectroscopy demonstrate that neither chemical nor thermal denaturation of hCC are simple two-state events. One recognized intermediate form was dimeric cystatin C, whose appearance was preceded mainly by changes in the L2 binding loop. The other form occurred only in the chemical denaturation process and was characterized by partially recovered interactions maintaining the protein tertiary structure. Our studies also strongly indicate that the -structural motif of cystatin C is directly implicated in formation of temperature-induced aggregates.Abbreviations Gdn.HCl guanidine hydrochloride - hCC human cystatin C     

Structural studies of the C‐terminal 19‐peptide of serum amyloid A and its Pro→Ala variants interacting with human cystatin C

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.     

Characteristics of C‐terminal, β‐amyloid peptide binding fragment of neuroprotective protease inhibitor,cystatin C

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.     

Pressure as a denaturing agent in studies of single-point mutants of an amyloidogenic protein human cystatin c

Recently, we presented a convenient method combining a deuterium‐hydrogen exchange and electrospray mass spectrometry for studying high‐pressure denaturation of proteins (Stefanowicz et al., Biosci Rep 2009; 30:91–99). Here, we present results of pressure‐induced denaturation studies of an amyloidogenic protein—the wild‐type human cystatin C (hCC) and its single‐point mutants, in which Val57 residue from the hinge region was substituted by Asn, Asp or Pro, respectively. The place of mutation and the substituting residues were chosen mainly on a basis of theoretical calculations. Observation of H/D isotopic exchange proceeding during pressure induced unfolding and subsequent refolding allowed us to detect differences in the proteins stability and folding dynamics. On the basis of the obtained results we can conclude that proline residue at the hinge region makes cystatin C structure more flexible and dynamic, what probably facilitates the dimerization process of this hCC variant. Polar asparagine does not influence stability of hCC conformation significantly, whereas charged aspartic acid in 57 position makes the protein structure slightly more prone to unfolding. Our experiments also point out pressure denaturation as a valuable supplementary method in denaturation studies of mutated proteins. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

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.     

Fibrillogenic oligomers of human cystatin C are formed by propagated domain swapping

Cystatin C and the prion protein have been shown to form dimers via three-dimensional domain swapping, and this process has also been hypothesized to be involved in amyloidogenesis. Production of oligomers of other amyloidogenic proteins has been reported to precede fibril formation, suggesting oligomers as intermediates in fibrillogenesis. A variant of cystatin C, with a Leu68-->Gln substitution, is highly amyloidogenic, and carriers of this mutation suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adulthood. This work describes doughnut-shaped oligomers formed by wild type and L68Q cystatin C upon incubation of the monomeric proteins. Purified oligomers of cystatin C are shown to fibrillize faster and at a lower concentration than the monomeric protein, indicating a role of the oligomers as fibril-assembly intermediates. Moreover, the present work demonstrates that three-dimensional domain swapping is involved in the formation of the oligomers, because variants of monomeric cystatin C, stabilized against three-dimensional domain swapping by engineered disulfide bonds, do not produce oligomers upon incubation under non-reducing conditions. Redox experiments using wild type and stabilized cystatin C strongly suggest that the oligomers, and thus probably the fibrils as well, are formed by propagated domain swapping rather than by assembly of domain-swapped cystatin C dimers.     

Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.     

Physico-chemical properties of the N-terminally truncated L68Q cystatin C found in amyloid deposits of brain haemorrhage patients

Cystatin C, a major extracellular cysteine proteinase inhibitor, is deposited as amyloid in brain haemorrhage patients with hereditary cystatin C amyloid angiopathy (HCCAA). A disease-causing mutation on the genetic level results in the substitution Leu68-->Gln (L68Q) in cystatin C, which causes protein instability. Besides carrying the L68Q substitution, cystatin C in amyloid deposits isolated from patients is N-terminally truncated by 10 amino acids. To elucidate the role of the N-terminal truncation for protein stability and aggregation properties, (delta1-10,L68Q)-cystatin C was produced in an Escherichia coli expression system and characterised. Unlike wild-type cystatin C, this variant rapidly dimerised under physiological conditions. Two unfolding intermediates of (delta1-10,L68Q)-cystatin C were identified, under the same pH and ionic strength conditions as required to form intermediates of full-length L68Q cystatin C. No evidence was found that the N-terminal truncation per se alters protein stability and leads to higher forms of aggregation. Monomeric as well as dimeric L68Q cystatin C incubated with neutrophil elastase was truncated as in HCCAA patients' amyloid. A protein variant with a thrombin cleavage site placed in front of residue Gly11 in L68Q cystatin C was constructed and used to confirm that the N-terminal segment is similarly accessible to proteinases in the monomeric and dimeric states of L68Q cystatin C. Thus, the N-terminal segment of L68Q cystatin C is exposed to proteolytic attack and does not seem to be involved in intramolecular contacts leading to dimerisation or higher-order aggregation. We conclude that the N-terminal truncation likely is an event secondary to amyloid formation, and of no relevance for the development of HCCAA.     

Interaction of serum amyloid A with human cystatin C—identification of binding sites

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.     

The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three‐dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the β2 and β3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the β2‐L1‐β3 (His43‐Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the β2‐L1‐β3 peptide, in which the Val57 residue was replaced by residues promoting β‐turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the β‐hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 373–383, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The emerging role of cystatins in Alzheimer's disease

Recently opposing effects of cysteine protease inhibitors, the human cystatins, on neurodegeneration have been reported. Human cystatin C is a risk factor for late‐onset Alzheimer's disease (AD), whereas human stefin B (cystatin B) has no direct involvement in AD. Conflicting data show that their target protease, cathepsin B, might be anti‐amyloidogenic, helping in amyloid‐beta (Aβ) clearance or, instead, might be involved in Aβ production. Some reports claim that cystatin C binds soluble Aβ, making transgenic animals healthier, others, in contrast, that deleting cystatins genes may contribute to amyloid pathology in animal models of AD.     

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

Rosmarinic acid prevents fibrillization and diminishes vibrational modes associated to β sheet in tau protein linked to Alzheimer’s disease

Alzheimer’s disease is a common tauopathy where fibril formation and aggregates are the hallmark of the disease. Efforts targeting amyloid-β plaques have succeeded to remove plaques but failed in clinical trials to improve cognition; thus, the current therapeutic strategy is at preventing tau aggregation. Here, we demonstrated that four phenolic diterpenoids and rosmarinic acid inhibit fibrillization. Since, rosmarinic acid was the most active compound, we observe morphological changes in atomic force microscopy images after treatment. Hence, rosmarinic acid leads to a decrease in amide regions I and III, indicating that rosmarinic acid prevents β-sheet assembly. Molecular docking study inside the steric zipper model of the hexapeptide ³⁰⁶VQIVYK³¹¹ involved in fibrillization and β sheet formation, suggests that rosmarinic acid binds to the steric zipper with similar chemical interactions with respect to those observed for orange G, a known pharmacofore for amyloid.     

Fertility Defects in Mice Expressing the L68Q Variant of Human Cystatin C: A ROLE FOR AMYLOID IN MALE INFERTILITY*

Hereditary cystatin C amyloid angiopathy is an autosomal dominant disorder in which a variant form of cystatin C (L68Q) readily forms amyloid deposits in cerebral arteries in affected individuals resulting in early death. L68Q protein deposits in human cystatin C amyloid angiopathy patients have also been found in tissues outside of the brain including the testis, suggesting possible effects on fertility. Heterozygous transgenic mice (L68Q) that express the human L68Q variant of cystatin C under the control of the mouse cystatin C promoter were unable to generate offspring, suggesting the presence of L68Q cystatin C amyloid affected sperm function. In vitro studies showed that epididymal spermatozoa from L68Q mice were unable to fertilize oocytes and exhibited poor sperm motility. Furthermore, spermatozoa from L68Q mice exhibited reduced cell viability compared with wild type (WT) spermatozoa and often were detected in large agglutinated clumps. Examination of the epididymal fluid and spermatozoa from L68Q mice showed increased levels and distinct forms of cystatin C amyloid that were not present in WT mice. The addition of epididymal fluid from L68Q mice to WT spermatozoa resulted in a recapitulation of the L68Q phenotype in that WT spermatozoa showed reduced cell viability and motility compared with WT spermatozoa incubated in epididymal fluid from WT mice. L68Q epididymal fluid that was depleted of cystatin C amyloids, however, did not impair the motility of WT spermatozoa. Taken together these studies suggest that amyloids in the epididymal fluid can be cytotoxic to the maturing spermatozoa resulting in male infertility.     

New Human Islet Amyloid Polypeptide Fragments Susceptible to Aggregation

Human Islet Amyloid Polypeptide (hIAPP) plays a key role in the pathogenesis of type II diabetes. The aim of this research was to search for new amyloidogenic fragments of hIAPP. An initial attempt to predict the amyloidogenic cores of polypeptides/proteins using five different computer programs did not provide conclusive results. Therefore, we synthesized hIAPP fragments covering the entire hormone. The fragments were assessed for their aggregation ability, using recommended methods to search for the amyloidogenic fragments of the polypeptides/proteins. It was found that fragments (18–22) H‐HSSNN‐OH and (33–37) H‐GSNTY‐NH₂ aggregate and form stable amyloid‐like structures. Both of these fragments have a much higher antiproliferative activity relative to the RIN‐5F cell compared to the (23–27) H‐FGAIL‐OH fragment widely regarded as the amyloidogenic core of amylin. The analog of (33–37) H‐GSNTY‐NH₂ containing a free carboxy group on the C‐terminal amino acid (H‐GSNTY‐OH) does not have amyloidogenic properties and can therefore be considered as a potential inhibitor of amylin aggregation. Research on the use of non‐aggregating amylin fragments as potential hormone aggregation inhibitors is ongoing.