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.     

Serum uromodulin predicts graft failure in renal transplant recipients

Objective and methods: Test the ability of serum uromodulin concentrations 1–3 months after renal transplantation to predict all-cause mortality (ACM) and graft loss (GL) in 91 patients.

Results: uromodulin predicted GL equivalently to the other markers studied: the risk for GL was reduced by 0.21 per one standard deviation (SD) increase (cystatin C: hazard ratio [HR] 4.57, creatinine: HR 4.53, blood-urea-nitrogen [BUN]: HR 2.50, estimated glomerular filtration rate [eGFR]: HR 0.10). In receiver-operating-characteristic (ROC) analysis, uromodulin predicted GL with an area-under-the curve of 0.782 at an optimal cut-off (OCO) of 24.0?ng/ml with a sensitivity of 90.0% and a specificity of 70.2%.

Conclusion: Serum uromodulin predicted GL equivalently compared to conventional biomarkers of glomerular filtration.     

Nuclear localization of cystatin B, the cathepsin inhibitor implicated in myoclonus epilepsy (EPM1)

Cystatin B is an anti-protease implicated in myoclonus epilepsy, a degenerative disease of the central nervous system. In vitro, cystatin B interacts with and inhibits proteases of the cathepsin family. Confocal microscopy analysis of the subcellular localization of cystatin B and cathepsin B shows that, in vivo, the two proteins are concentrated in different cell compartments. In fact, cystatin B is found mainly in the nucleus of proliferating cells and both in the nucleus and in the cytoplasm of differentiated cells, while cathepsin B, in either case, is essentially cytoplasmic. However, colocalization of cystatin and cathepsin B is observed in the isolated cell matrix and in the nuclear scaffold of differentiated neuroblastoma cells but not of proliferating cells. This suggests that at least a fraction of cystatin B is bound to the protease in differentiated cells. The electron microscopy analysis of the cell matrix confirms the observation made with confocal microscopy. The cellular activity of cathepsin B was analyzed with a fluorogenic cytochemical assay. A fluorescent signal is observed in the cytoplasm of proliferating cells but is undetectable in the cytoplasm of differentiated cells, suggesting that cathepsin B is active mainly during the cell cycle. This result is consistent with the separate compartimentalization of cystatin B and cathepsin B that we have observed in growing cells.     

Investigation of candidate genes for meat quality in dry-cured ham production: the porcine cathepsin B (CTSB) and cystatin B (CSTB) genes

Excessive softness is a serious defect of dry cured hams which seems related to high activity of lysosomal cysteine proteinases, such as cathepsin B, in fresh pork muscles a few days after slaughtering. As it has been shown that cathepsin B activity has a moderate heritability in Italian Large White pigs we started a candidate gene approach to identify the gene(s) that affect(s) this parameter. Here, we studied two candidate genes: cathepsin B (CTSB) and cystatin B (CSTB). We amplified and sequenced porcine DNA fragments for these two genes that were used to identify polymorphisms by SSCP and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Four and two alleles were detected at the CTSB and CSTB loci, respectively. Sequencing of the CSTB alleles showed a missense mutation that changes a codon for aspartic acid into a codon for asparagine in exon 3 of the gene. Allele frequencies for the two loci differed among the pig breeds studied (Large White, Landrace, Duroc, Belgian Landrace, Hampshire, Piétrain, Meishan, Cinta Senese, Casertana, Calabrese and Nero di Sicilia). Linkage, somatic cell hybrid panel and radiation hybrid panel analyses assigned CTSB to porcine chromosome (Sscr) 14 and CSTB to Sscr 13. The markers identified at the CTSB and CSTB loci were used in association studies with several traits of economic importance including parameters that may indicate the suitability of pig meat to produce dry-cured hams. Significant associations were observed between CTSB and back-fat thickness and between CSTB and average daily gain. In this study, cathepsin B activity was not associated with the polymorphisms identified at the CTSB and CSTB loci.     

3D domain swapping: as domains continue to swap

Three-dimensional (3D) domain swapping creates a bond between two or more protein molecules as they exchange their identical domains. Since the term '3D domain swapping' was first used to describe the dimeric structure of diphtheria toxin, the database of domain-swapped proteins has greatly expanded. Analyses of the now about 40 structurally characterized cases of domain-swapped proteins reveal that most swapped domains are at either the N or C terminus and that the swapped domains are diverse in their primary and secondary structures. In addition to tabulating domain-swapped proteins, we describe in detail several examples of 3D domain swapping which show the swapping of more than one domain in a protein, the structural evidence for 3D domain swapping in amyloid proteins, and the flexibility of hinge loops. We also discuss the physiological relevance of 3D domain swapping and a possible mechanism for 3D domain swapping. The present state of knowledge leads us to suggest that 3D domain swapping can occur under appropriate conditions in any protein with an unconstrained terminus. As domains continue to swap, this review attempts not only a summary of the known domain-swapped proteins, but also a framework for understanding future findings of 3D domain swapping.     

Different propensity to form amyloid fibrils by two homologous proteins-Human stefins A and B: searching for an explanation

By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.     

Differential effects of anti-cancer and anti-hepatitis drugs on liver cystatin

The drug–protein interaction has been the subject of increasing interest over the decades. In the present communication, the interaction of liver cystatin with anti-cancer (adriamycin) and anti-hepatitis (adevofir dipivoxil) drugs was studied by thiol-protease inhibitory assay, UV absorption, fluorescence spectroscopy and circular dichroism (CD). A static type of quenching was observed between the protein and the drug molecules. Binding constant (Ka) of adriamycin to liver cystatin (LC) was found to be 1.08 × 10⁶ M⁻¹. Moreover, binding site number was found to be 2. Importantly, cystatin loses its activity in the presence of adriamycin. However, intrinsic fluorescence studies in the presence of adevofir dipivoxil showed enhancement in the fluorescence intensity suggesting that binding of adevofir to LC caused unfolding of the protein. The unfolding of the test protein was also accompanied by significant loss of inhibitory activity. CD spectroscopy result showed, both adriamycin and adevofir dipivoxil caused perturbation in the secondary structure of liver cystatin. The possible implications of these results will help in combating drug induced off target effects.Abbreviations: LC, liver cystatin; ADR, adriamycin; CD, circular dichroism; Ka, binding constant; HBV, human hepatitis B virus