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.
We previously reported that cystatin B (CSTB) is a progression marker of human ovarian cancer (OC); however, the regulatory mechanism of CSTB and its function in OC remain unclear. The present study ...aimed to explore the mechanism underlying transforming growth factor-β (TGF-β) 1-mediated CSTB regulation, and to examine the function of CSTB on OC cell proliferation and apoptosis. Using the online program, miRWalk, a microRNA (miR)-143-3p was detected, which contains a homologous sequence of the potential binding site to the 3'-untranslated region (3'-UTR) of CSTB. A dual-luciferase reporter assay confirmed the interaction between miR-143-3p and CSTB 3'-UTR. Treating OC cells with miR-143-3p mimics or inhibitors resulted in a decrease or an increase of CSTB expression at mRNA and protein levels, respectively. Additionally, CSTB was significantly overexpressed, whereas miR-143-3p was downregulated in human OC tissues compared with normal ovarian tissues. A negative correlation between miR-143-3p and CSTB mRNA expression was observed in ovarian malignant tumors. The levels of primary and mature miR-143-3p expression were upregulated in OC cells after TGF-β1 treatment; the action of TGF-β1 was abolished in the presence of an inhibitor of TGF-β type I receptor. These results indicated an axis between TGF-β, miR-143-3p and CSTB in OC cells. Furthermore, high levels of CSTB expression were associated with the poor overall survival of patients with OC. Knockdown of CSTB resulted in a decrease in OC cell proliferation and arrested cells in G2/M phase. In addition, suppression of CSTB induced cell apoptosis. In conclusion, CSTB was overexpressed and miR-143-3p was downregulated in ovarian malignant tumors. Mature miR-143-3p directly bound CSTB 3'-UTR, leading to a decrease in CSTB expression in OC cells, which was regulated by TGF-β1. Our findings suggest the potential therapeutic application of targeting the TGF-β/miR-143-3p/CSTB axis for treating patients with OC.
Glutamate dehydrogenase has been recently identified as a tissue‐specific histone H3‐specific clipping enzyme. We have previously shown that it cleaves free as well as chromatin‐bound histone H3. ...However, the physiological significance of this enzyme is still not clear. The present study aimed to improve our understanding of its significance in vivo. Using biochemical and cell biological approaches, we show that glutamate dehydrogenase is primarily associated with euchromatin, and it re‐localizes from the nuclear periphery to the nucleolus upon DNA damage. The cysteine protease inhibitor stefin B regulates the H3 clipping activity of the enzyme. Chromatin structure and certain histone modifications influence H3 clipping activity. Interestingly, we also observed that an in vivo truncated form of H3 lacks H3K56 acetylation, which is a code for the DNA damage response. Together, these results suggest that glutamate dehydrogenase is a euchromatin‐associated enzyme, and its H3 clipping activity is regulated by chromatin structure, histone modifications and an in vivo inhibitor. In response to DNA damage, it re‐localizes to the nuclei, and hence may be involved in regulation of gene expression in vivo.
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.
Menadione (MD) treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some 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. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases.
Epilepsy is a disorder of the brain characterized by an enduring predisposition to generate epileptic seizures. In the last two decades, numerous gene defects underlying different forms of epilepsy ...have been identified with most of these genes encoding ion channel proteins. Despite these developments, the etiology of majority of non-familial epilepsies has no known associated genetic mutations and cannot be explained by defects in identified ion channels alone. We hypothesize that
formation of ion channels by naturally unfolded proteins (NUPs) increases neuronal excitability. Altered ionic homeostasis may initiate/contribute to cellular cascades related to epileptogenesis in susceptible individuals. Here, we consider two small proteins, namely, α-synuclein and stefin B, as prototypical candidates to illustrate the underlying mechanism(s). Previous work points to an association between epilepsy and α-synuclein or stefin B, but the mechanism(s) underlying such association remains elusive. We review the evidence to link the structure-function of these proteins with disease processes. Epigenetic mechanisms unrelated to altered DNA sequence(s) that may affect epileptogenesis include transcriptional or posttranscriptional regulation. Such epigenetic mechanisms or their combination(s) enhance the levels of these proteins and as a result the ability to form annular structures, which upon incorporation into membrane form novel ion channels and disturb intracellular ion homeostasis. Alternative epigenetic mechanisms may change amyloidogenic proteins by posttranslational modifications, thereby increasing their propensity to form channels. Further research elucidating the details about the formation of ion channels through these mechanisms and their role in epileptogenesis may define new molecular targets and guide the development of new drug targets.
Cystatins are a large family of the proteins that function as reversible and tight-binding inhibitors of cysteine proteases, which consequently regulate multiple physiological activities including ...apoptosis and innate immunity. In the present study, we cloned a gene from Crassostrea gigas encoding cystatin, which is related to cystatin A superfamily. CgCytA was comprised of a cystatin-like domain with two conserved glycine residues (GG) near the N-terminal and a highly conserved glutamine-valine-glycine (Q-X-V-X-G) motif in the form of QVVAG loop. Transcription analysis of CgCytA indicated its constitutive expression in all tissues including mantle, gill, digestive tract, hemocytes, heart, adductor muscle, and gonads. Immune challenge with Vibrio alginolyticus, resulted in significant down-regulation of CgCytA expression at the initial stages of infection (till 12 h post infection) and the expression of cystatin increased 48 h post infection. Protease assay demonstrated the concentration of cystatin needed to inhibit half of the maximum biological response of cysteine protease is 14.4 μg/L (IC50). Furthermore, RNAi of CgCytA resulted in increase of apoptotic cell population in hemocytes of C. gigas, suggesting protection role of CgCytA from hemocytes apoptosis. Unexpectedly, knockdown of CgCytA leaded to enhancement of bacterial clearance in vivo, implying that CgCytA may negatively regulate immune defense by suppressing endogenous cysteine protease. Therefore, CgCytA plays dual roles in protection of host hemocytes from apoptosis and control of bacterial clearance, which may server as one of key endogenous balancer between apoptosis and innate immunity in oyster.
•One homolog of cystatin was identified from Crassostrea gigas.•CgCytA mRNA fluctuated after bacterial challenge.•Recombinant CgCytA could effectively inhibit the activity of cysteine proteases.•Knockdown of CgCytA leaded to increase of hemoctyes apoptosis and bacterial clearance in vivo.
Conformational diseases constitute a group of heterologous disorders in which a constituent host protein undergoes changes in conformation, leading to aggregation and deposition. To understand the ...molecular mechanisms of the process of amyloid fibril formation, numerous in vitro and in vivo studies, including model and pathologically relevant proteins, have been performed. Understanding the molecular details of these processes is of major importance to understand neurodegenerative diseases and could contribute to more effective therapies. Many models have been proposed to describe the mechanism by which proteins undergo ordered aggregation into amyloid fibrils. We classify these as: (a) templating and nucleation; (b) linear, colloid-like assembly of spherical oligomers; and (c) domain-swapping. In this review, we stress the role of domain-swapping and discuss the role of proline switches.
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.
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-β structure reveals the temperature but not pH dependence of stefin B structure.
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-β 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-β structure.
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-β 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.
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