Type I Cystatin Derived from Cysticercus pisiformis-Stefins, Suppresses LPS-Mediated Inflammatory Response in RAW264.7 Cells.

Cysticercus pisiformis is a kind of tapeworm larvae of Taenia pisiformis, which parasitizes the liver envelope, omentum, mesentery, and rectum of rodents such as rabbits. Cysteine protease inhibitors derived from helminth were immunoregulatory molecules of intermediate hosts and had an immunomodulatory function that regulates the production of inflammatory factors. Thus, in the present research, the recombinant Stefin of C. pisiformis was confirmed to have the potential to fight inflammation in LPS-Mediated RAW264.7 murine macrophages. CCK8 test showed that rCpStefin below 50 µg/mL concentration did not affect cellular viability. Moreover, the NO production level determined by the Griess test was decreased. In addition, the secretion levels of IL-1ß, IL-6, and TNF-α as measured by ELISA were decreased. Furthermore, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and proinflammatory mediators, including IL-1ß, IL-6, TNF-α, iNOS, and COX-2 at the gene transcription level, as measured by qRT-PCR. Therefore, Type I cystatin derived from C. pisiformis suppresses the LPS-Mediated inflammatory response of the intermediate host and is a potential candidate for the treatment of inflammatory diseases.

Stefin B alleviates the gouty arthritis in mice by inducing the M2 polarization of macrophages.

The present study aims to explore the therapeutic effect of Stefin B on gouty arthritis (GA) and the polarization of macrophages in mice. Stefin B-overexpressed or knockdown M0 macrophages were constructed. The GA model was established in mice by injecting 25 mg/mL MSU, followed by a single injecting of Stefin B-overexpressing adenovirus vector (GA model + Stefin B OE) or an empty vector (GA model + Stefin B OE NC). Stefin B was found lowly expressed in M1 macrophages. CD206 was markedly upregulated and IL-10 release was signally increased in Stefin B-overexpressed macrophages. In gouty arthritis mice, marked redness and swelling were observed in the ankle joint. Dramatical infiltration of inflammatory cells was observed in the GA model and GA model + Stefin B OE NC groups, which was suppressed in the Stefin B OE group. Increased proportion of F4/80+CD86+ cells observed in GA mice was markedly repressed by Stefin B overexpression, accompanied by the declined level of Caspase-1 and IL-17. Collectively, Stefin B alleviated the GA in mice by inducing the M2 polarization of macrophages.

The Roles of Cystatin B in the Brain and Pathophysiological Mechanisms of Progressive Myoclonic Epilepsy Type 1.

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disorder, also known as Unverricht-Lundborg disease (ULD). EPM1 patients suffer from photo-sensitive seizures, stimulus-sensitive myoclonus, nocturnal myoclonic seizures, ataxia and dysarthria. In addition, cerebral ataxia and impaired GABAergic inhibition are typically present. EPM1 is caused by mutations in the Cystatin B gene (CSTB). The CSTB protein functions as an intracellular thiol protease inhibitor and inhibits Cathepsin function. It also plays a crucial role in brain development and regulates various functions in neurons beyond maintaining cellular proteostasis. These include controlling cell proliferation and differentiation, synaptic functions and protection against oxidative stress, likely through regulation of mitochondrial function. Depending on the differentiation stage and status of neurons, the protein localizes either to the cytoplasm, nucleus, lysosomes or mitochondria. Further, CSTB can also be secreted to the extracellular matrix for interneuron rearrangement and migration. In this review, we will review the various functions of CSTB in the brain and discuss the putative pathophysiological mechanism underlying EPM1.

Stefin B Inhibits NLRP3 Inflammasome Activation via AMPK/mTOR Signalling.

Stefin B (cystatin B) is an inhibitor of lysosomal and nuclear cysteine cathepsins. The gene for stefin B is located on human chromosome 21 and its expression is upregulated in the brains of individuals with Down syndrome. Biallelic loss-of-function mutations in the stefin B gene lead to Unverricht-Lundborg disease-progressive myoclonus epilepsy type 1 (EPM1) in humans. In our past study, we demonstrated that mice lacking stefin B were significantly more sensitive to sepsis induced by lipopolysaccharide (LPS) and secreted higher levels of interleukin 1-ß (IL-1ß) due to increased inflammasome activation in bone marrow-derived macrophages. Here, we report lower interleukin 1-ß processing and caspase-11 expression in bone marrow-derived macrophages prepared from mice that have an additional copy of the stefin B gene. Increased expression of stefin B downregulated mitochondrial reactive oxygen species (ROS) generation and lowered the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in macrophages. We determined higher AMP-activated kinase phosphorylation and downregulation of mTOR activity in stefin B trisomic macrophages-macrophages with increased stefin B expression. Our study showed that increased stefin B expression downregulated mitochondrial ROS generation and increased autophagy. The present work contributes to a better understanding of the role of stefin B in regulation of autophagy and inflammasome activation in macrophages and could help to develop new treatments.

Amyloid Formation of Stefin B Protein Studied by Infrared Spectroscopy.

BACKGROUND: Stefin B, an established model protein for studying the stability and mechanism of protein folding, was used for monitoring protein aggregation and formation of amyloid structure by infrared spectroscopy. METHODS: The analyses of the integral intensities of the low frequency part of the Amide I band, which is directly connected to the appearance of the cross-ß structure reveals the temperature but not pH dependence of stefin B structure. RESULTS: We show that pH value has significant role in the monomer stability of stefin B. Protein is less stable in acidic environment and becomes more stable in neutral or basic conditions. While in the case of the Amide I band area analysis we apply only spectral regions characteristic for only part of the protein in cross-ß structure, the temperature study using multivariate curve resolution (MCR) analysis contains also information about the protein conformation states which do not correspond to native protein nor protein in cross-ß structure. CONCLUSIONS: These facts results in the slightly different shapes of fitted sigmoid functions fitted to the weighted amount of the second basic spectrum (sc2), which is the closed approximation of the protein spectra with cross-ß structure. Nevertheless, the applied method detects the initial change of the protein structure. Upon the analysis of infrared data a model for stefin B aggregation is proposed.

Type I Cystatin Derived from Fasciola gigantica Suppresses Macrophage-Mediated Inflammatory Responses.

There is an inverse relationship between the high incidence of helminth infection and the low incidence of inflammatory disease. Hence, it may be that helminth molecules have anti-inflammatory effects. Helminth cystatins are being extensively studied for anti-inflammatory potential. Therefore, in this study, the recombinant type I cystatin (stefin-1) of Fasciola gigantica (rFgCyst) was verified to have LPS-activated anti-inflammatory potential, including in human THP-1-derived macrophages and RAW 264.7 murine macrophages. The results from the MTT assay suggest that rFgCyst did not alter cell viability; moreover, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and mediators, including IL-1ß, IL-6, IL-8, TNF-α, iNOS, and COX-2 at the gene transcription and protein expression levels, as determined by qRT-PCR and Western blot analysis, respectively. Further, the secretion levels of IL-1ß, IL-6, and TNF-α determined by ELISA and the NO production level determined by the Griess test were decreased. Furthermore, in Western blot analysis, the anti-inflammatory effects involved the downregulation of pIKKα/ß, pIκBα, and pNF-κB in the NF-κB signaling pathway, hence reducing the translocation from the cytosol into the nucleus of pNF-κB, which subsequently turned on the gene of proinflammatory molecules. Therefore, cystatin type 1 of F. gigantica is a potential candidate for inflammatory disease treatment.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

In Silico, In Vitro, and Clinical Investigations of Cathepsin B and Stefin A mRNA Expression and a Correlation Analysis in Kidney Cancer.

The cysteine protease Cathepsin B (CtsB) plays a critical role in multiple signaling pathways, intracellular protein degradation, and processing. Endogenous inhibitors regulate its enzymatic activity, including stefins and other cystatins. Recent data proved that CtsB is implicated in tumor extracellular matrix remodeling, cell invasion, and metastasis: a misbalance between cathepsins and their natural inhibitors is often considered a sign of disease progression. In the present study, we investigated CtsB and stefin A (StfA) expression in renal cell carcinoma (RCC). mRNA analysis unveiled a significant CTSB and STFA increase in RCC tissues compared to adjacent non-cancerogenic tissues and a higher CtsB expression in malignant tumors than in benign renal neoplasms. Further analysis highlighted a positive correlation between CtsB and StfA expression as a function of patient sex, age, tumor size, grade, lymph node invasion, metastasis occurrence, and survival. Alternative overexpression and silencing of CtsB and StfA confirmed the correlation expression between these proteins in human RCC-derived cells through protein analysis and fluorescent microscopy. Finally, the ectopic expression of CtsB and StfA increased RCC cell proliferation. Our data strongly indicated that CtsB and StfA expression play an important role in RCC development by mutually stimulating their expression in RCC progression.

Targeting Secreted Protease/Anti-Protease Balance as a Vaccine Strategy against the Helminth Fasciola hepatica.

The liver fluke Fasciola hepatica is an economically important global pathogen of humans and their livestock. To facilitate host invasion and migration, F. hepatica secretes an abundance of cathepsin peptidases but prevents excessive damage to both parasite and host tissues by co-secreting regulatory peptidase inhibitors, cystatins/stefins and Kunitz-type inhibitors. Here, we report a vaccine strategy aimed at disrupting the parasite's protease/anti-protease balance by targeting these key inhibitors. Our vaccine cocktail containing three recombinant stefins (rFhStf-1, rFhStf-2, rFhStf-3) and a Kunitz-type inhibitor (rFhKT1) formulated in adjuvant Montanide 61VG was assessed in two independent sheep trials. While fluke burden was not reduced in either trial, in Trial 1 the vaccinated animals showed significantly greater weight gain (p < 0.05) relative to the non-vaccinated control group. In both trials we observed a significant reduction in egg viability (36-42%). Multivariate regression analyses showed vaccination and increased levels of IgG2 antibodies specific for the F. hepatica peptidase inhibitors were positive indicators for increased weight gain and levels of haemoglobin within the normal range at 16 weeks post-infection (wpi; p < 0.05). These studies point to the potential of targeting peptidase inhibitors as vaccine cocktails for fasciolosis control in sheep.

Synergy of the Inhibitory Action of Polyphenols Plus Vitamin C on Amyloid Fibril Formation: Case Study of Human Stefin B.

In order to study how polyphenols and vitamin C (vitC) together affect protein aggregation to amyloid fibrils, we performed similar in vitro studies as before using stefin B as a model and a potentially amyloid-forming protein (it aggregates upon overexpression, under stressful conditions and some progressive myoclonus epilepsy of tape 1-EPM1-missense mutations). In addition to the chosen polyphenol, this time, we added a proven antioxidant concentration of 0.5 mM vitC into the fibrillation mixture and varied concentrations of resveratrol, quercetin, and curcumin. Synergy with vitC was observed with curcumin and quercetin.

Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians.

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.

Proteases as Therapeutic Targets Against the Parasitic Cnidarian Ceratonova shasta: Characterization of Molecules Key to Parasite Virulence In Salmonid Hosts.

Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss, to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets.

Grass carp (Ctenopharyngodon idella) stefin A: Systematic research on its cloning, expression, characterization and tissue distribution.

To fully understand the properties of piscine stefins (family I cystatins), the 294-bp stefinA gene from grass carp (Ctenopharyngodon idella, Ci) was cloned and expressed in E. coli BL21 (DE3). After purification by Ni2+-NTA agarose affinity chromatography, the CiStefin A protein was tested to have a molecular weight of 11.48 kDa and an isoelectric point of 8.7. The typical motif of the cystatins superfamily was characterized from CiStefin A (QVVQG). CiStefin A specifically inhibited the activity of papain and cathepsin B/L. The Ki value of CiStefin A against papain was 6.5 × 10-11 M. CiStefin A showed excellent heat and acid-base tolerance. StefinA gene transcription occurred in all tested tissues of grass carp, with the highest level in the hepatopancreas. Immunolocalization staining with an anti-CiStefinA antibody revealed the CiStefinA protein distribution in all tested tissues at various levels. Overall, these results clarified the physical and biochemical properties of grass carp stefin A.

Molecular communication between the monogenea and fish immune system.

Monogeneans parasitise mainly the outer structures of fish, such as the gills, fins, and skin, that is, tissues covered with a mucous layer. While attached by sclerotised structures to host's surface, monogeneans feed on its blood or epidermal cells and mucus. Besides being a rich source of nutrients, these tissues also contain humoral immune factors and immune cells, which are ready to launch defence mechanisms against the tegument or gastrointestinal tract of these invaders. The exploitation of hosts' resources by the Monogenea must, therefore, be accompanied by suppressive and immunomodulatory mechanisms which protect the parasites against attacks by host immune system. Elimination of hosts' cytotoxic molecules and evasion of host immune response is often mediated by proteins secreted by the parasites. The aim of this review is to summarise existing knowledge on fish immune responses against monogeneans. Results gleaned from experimental infections illustrate the various interactions between parasites and the innate and adaptive immune system of the fish. The involvement of monogenean molecules (mainly inhibitors of peptidases) in molecular communication with host immune system is discussed.

Site-1 protease ablation in the osterix-lineage in mice results in bone marrow neutrophilia and hematopoietic stem cell alterations.

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells, via single-cell RNA-Seq and protein mass spectrometry, demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro, recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells, with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells, S1P ablation results in an increased production of granulocyte-macrophage progenitors, the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche.

Interplay of ERα binding and DNA methylation in the intron-2 determines the expression and estrogen regulation of cystatin A in breast cancer cells.

Despite advances in early detection and treatment, invasion and metastasis of breast tumors remains a major hurdle. Cystatin A (CSTA, also called stefin A), an estrogen-regulated gene in breast cancer cells, is an inhibitor of cysteine cathepsins, and a purported tumor suppressor. Loss of CSTA expression in breast tumors evidently shifts the balance in favor of cysteine cathepsins, thereby promoting extracellular matrix remodeling, tumor invasion and metastasis. However, the underlying mechanism behind the loss of CSTA expression in breast tumors is not known. Here, we have analyzed CSTA expression, and methylation of upstream and intron-2 CpG sites within the CSTA locus in human breast cancer cell lines and breast tumors of the TCGA cohort. Results showed an inverse relationship between expression and methylation. Sequence analysis revealed a potential estrogen response element (ERE) in the intron-2. Analysis of ChIP-seq data (ERP000380) and our own ChIP experiments showed that 17ß-estradiol (E2) enhanced ERα binding to this ERE in MCF-7 cells. This ERE was located amidst the differentially methylated intron-2 CpG sites, which provoked us to examine the possible conflict between estrogen-regulation of CSTA and DNA methylation in the intron-2. We analyzed the expression of CSTA and its regulation by E2 in MDA-MB-231 and T47D cells subjected to global demethylation by 5-azacytidine (5-aza). 5-aza significantly demethylated intron-2 CpGs, and enhanced estrogen-induced ERα occupancy at the intron-2 ERE, leading to restoration of estrogen-regulation. Taken together, our results indicate that DNA methylation-dependent silencing could play a significant role in the loss of CSTA expression in breast tumors. The potential of DNA methylation as an indicator of CSTA expression or as a marker of tumor progression can be explored in future investigations. Furthermore, our results indicate the convergence of ERα-mediated estrogen regulation and DNA methylation in the intron-2, thereby offering a novel context to understand the role of estrogen-ERα signaling axis in breast tumor invasion and metastasis.

Menadione-induced apoptosis in U937 cells involves Bid cleavage and stefin B degradation.

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumor Necrosis Factor-α Induced Apoptosis in U937 Cells Promotes Cathepsin D-Independent Stefin B Degradation.

Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc.

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation.

Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to ß2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders.