Interaction between oligomers of stefin B and amyloid-beta in vitro and in cells.
ABSTRACT: To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-beta-(1-40) peptide (Abeta). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Abeta is oligomer specific. The dimers and tetramers of stefin B, which bind Abeta, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Abeta fibril formation. When expressed in cultured cells, stefin B co-localizes with Abeta intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Abeta epitope. Thus, stefin B is another APP/Abeta-binding protein in vitro and likely in cells.
Project description:Stefin B (cystatin B) is an intracellular inhibitor of cysteine cathepsins and mutations in the stefin B gene, resulting in the development of Unverricht-Lundborg disease, which is a form of myoclonic epilepsy. It was suggested that a key mechanism behind stefin B-mediated disease progression was impaired redox homeostasis. Stefin B-deficient mice were found more sensitive to lipopolysaccharide (LPS)-induced sepsis as a consequence of increased expression of caspase-11 and Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing (NLRP nflammasome activation and higher levels of mitochondrial reactive oxygen species (ROS). In the present study, we investigated if LPS-triggered oxidative stress affected the protein levels and redox status of redox sensitive proteins-thioredoxin, peroxiredoxins, and superoxide dismutases in macrophages and spleens of LPS-injected mice. LPS challenge was found to result in a marked elevation in mitochondrial peroxiredoxin 3 (Prx3), sulfiredoxin, and superoxide dismutase 2 (Sod2) in stefin B-deficient macrophages and spleens. We determined that sulfiredoxin is targeted to mitochondria after LPS challenge. In conclusion, the upregulation of mitochondrial redox-sensitive proteins Prx3 and Sod2 in stefin B-deficient cells implies a protective role of stefin B in mitochondrial function.
Project description:The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins (G, QxVxG) was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain (G, QxVxG, PW), a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn ?-helix, a five stranded ?-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.
Project description:A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallographic R factor is 0.19. Like cystatin, the stefin molecule consists of a five stranded beta-sheet wrapped around a five turn alpha-helix, but with an additional carboxy terminal strand running along the convex side of the sheet. Topological equivalence of stefin and cystatin reveal the previous sequence alignment to be incorrect in part, through deletion of the intermediate helix. The conserved residues form a tripartite wedge, which slots into the papain active site as proposed through consideration of the tertiary structures of the individual components (Bode et al., 1988). The main interactions are provided by the amino terminal 'trunk' (occupying the 'unprimed' subsites of the enzyme), and by the first hairpin loop, containing the highly conserved QVVAG sequence, with minor contributions from the second hairpin loop. The carboxyl terminus of stefin provides an additional interaction region with respect to cystatin. The interaction is dominated by hydrophobic contacts. Inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serine proteinase inhibitors.
Project description:The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.
Project description:Unlike a number of amyloid-forming proteins, stefins, and in particular stefin B (cystatin B) form amyloids under conditions where the native state predominates. In order to trigger oligomerization processes, the stability of the protein needs to be compromised, favoring structural re-arrangement however, accelerating fibril formation is not a simple function of protein stability. We report here on how optimal conditions for amyloid formation lead to the destabilization of dimeric and tetrameric states of the protein in favor of the monomer. Small, highly localized structural changes can be mapped out that allow us to visualize directly areas of the protein which eventually become responsible for triggering amyloid formation. These regions of the protein overlap with the Cu (II)-binding sites which we identify here for the first time. We hypothesize that in vivo modulators of amyloid formation may act similarly to painstakingly optimized solvent conditions developed in vitro. We discuss these data in the light of current structural models of stefin B amyloid fibrils based on H-exchange data, where the detachment of the helical part and the extension of loops were observed.
Project description:Parasite inhibitors of cysteine peptidases are known to influence a vast range of processes linked to a degradation of either the parasites' own proteins or proteins native to their hosts. We characterise a novel type I cystatin (stefin) found in a sanguinivorous fish parasite Eudiplozoon nipponicum (Platyhelminthes: Monogenea). We have identified a transcript of its coding gene in the transcriptome of adult worms. Its amino acid sequence is similar to other stefins except for containing a legumain-binding domain, which is in this type of cystatins rather unusual. As expected, the recombinant form of E. nipponicum stefin (rEnStef) produced in Escherichia coli inhibits clan CA peptidases - cathepsins L and B of the worm - via the standard papain-binding domain. It also blocks haemoglobinolysis by cysteine peptidases in the worm's excretory-secretory products and soluble extracts. Furthermore, we had confirmed its ability to inhibit clan CD asparaginyl endopeptidase (legumain). The presence of a native EnStef in the excretory-secretory products of adult worms, detected by mass spectrometry, suggests that this protein has an important biological function at the host-parasite interface. We discuss the inhibitor's possible role in the regulation of blood digestion, modulation of antigen presentation, and in the regeneration of host tissues.
Project description:Protein activity is often regulated by altering the oligomerization state. One mechanism of multimerization involves domain swapping, wherein proteins exchange parts of their structures and thereby form long-lived dimers or multimers. Domain swapping has been specifically observed in amyloidogenic proteins, for example the cystatin superfamily of cysteine protease inhibitors. Cystatins are twin-headed inhibitors, simultaneously targeting the lysosomal cathepsins and legumain, with important roles in cancer progression and Alzheimer's disease. Although cystatin E is the most potent legumain inhibitor identified so far, nothing is known about its propensity to oligomerize. In this study, we show that conformational destabilization of cystatin E leads to the formation of a domain-swapped dimer with increased conformational stability. This dimer was active as a legumain inhibitor by forming a trimeric complex. By contrast, the binding sites toward papain-like proteases were buried within the cystatin E dimer. We also showed that the dimers could further convert to amyloid fibrils. Unexpectedly, cystatin E amyloid fibrils contained functional protein, which inhibited both legumain and papain-like enzymes. Fibril formation was further regulated by glycosylation. We speculate that cystatin amyloid fibrils might serve as a binding platform to stabilize the pH-sensitive legumain and cathepsins in the extracellular environment, contributing to their physiological and pathological functions.
Project description:At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.
Project description:Cystatins are reversible, tightly binding inhibitors of cysteine proteases. Filarial cystatins have been ascribed immunomodulatory properties and have been implicated in protective immunity. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cystatin gene locus of A. viteae. The gene is composed of 4 exons separated by 3 introns and spans approximately 2 kb of genomic DNA. The upstream genomic sequence contains transcriptional factor binding sites such as AP-1 and NF-Y, an inverted CCAAT sequence, and a TATA box. To investigate sites of cystatin expression, Caenorhabditis elegans worms were transformed by microinjection with the putative promoter region and the first exon of the A. viteae cystatin gene fused to the reporter GFP. In transgenic worms fluorescence was observed in the pharyngeal and rectal gland cells suggesting that cystatin is secreted. Additionally, A. viteae cystatin was expressed in C. elegans to explore its potential as an expression system for filarial genes.
Project description:Recent evidence suggests that high molecular weight soluble oligomeric Abeta (oAbeta) assemblies (also known as Abeta-derived diffusible ligands, or ADDLs) may represent a primary neurotoxic basis for cognitive failure in Alzheimer disease (AD). To date, most in vivo studies of oAbeta/ADDLs have involved injection of assemblies purified from the cerebrospinal fluid of human subjects with AD or from the conditioned media of Abeta-secreting cells into experimental animals. We sought to study the bioactivities of endogenously formed oAbeta/ADDLs generated in situ from the physiological processing of human amyloid precursor protein (APP) and presenitin1 (PS1) transgenes.We produced and histologically characterized single transgenic mice overexpressing APP(E693Q) or APP(E693Q) X PS1DeltaE9 bigenic mice. APP(E693Q) mice were studied in the Morris water maze (MWM) task at 6 and 12 months of age. Following the second MWM evaluation, mice were sacrificed, and brains were assayed for Abetatotal, Abeta40, Abeta42, and oAbeta/ADDLs by enzyme-linked immunosorbent assay (ELISA) and were also histologically examined. Based on results from the oAbeta/ADDL ELISA, we assigned individual APP(E693Q) mice to either an undetectable oAbeta/ADDLs group or a readily detectable oAbeta/ADDLs group. A days to criterion (DTC) analysis was used to determine delays in acquisition of the MWM task.Both single transgenic and bigenic mice developed intraneuronal accumulation of APP/Abeta, although only APP(E693Q) X PS1Delta9 bigenic mice developed amyloid plaques. The APP(E693Q) mice did not develop amyloid plaques at any age studied, up to 30 months. APP(E693Q) mice were tested for spatial learning and memory, and only 12-month-old APP(E693Q) mice with readily detectable oAbeta/ADDLs displayed a significant delay in acquisition of the MWM task when compared to nontransgenic littermates.These data suggest that cerebral oAbeta/ADDL assemblies generated in brain in situ from human APP transgenes may be associated with cognitive impairment. We propose that a DTC analysis may be a sensitive method for assessing the cognitive impact in mice of endogenously generated oligomeric human Abeta assemblies. ANN NEUROL 2010.