Cysteine Proteinase Inhibitor Research Articles

Monomer-dimer equilibrium of human cystatin C during internalization into cancer cells.

Human cystatin C (hCC) is a physiologically important protein that serves as intra- and extracellular cysteine proteinase inhibitor in homeostasis. However, in pathological states it dimerizes and further oligomerizes accumulating into a toxic amyloid. HCC forms an active monomer in the extracellular space and becomes an inactive dimer when internalized in cellular organelles. However, hCC cell penetration and its oligomeric state during this process are not well understood. To determine if and how the oligomeric state influences hCC transmembrane migration, we investigated the internalization of the hCC wild type protein as well as three different mutants, which exclusively exist in the monomeric or multimeric state into HeLa cells via confocal fluorescence microscopy. Our results showed that the preferred pathway was endocytosis and that the oligomeric state did not significantly influence the internalization because both monomeric and dimeric hCC migrated into HeLa cells. Considering the differences of the active monomeric and the passive dimeric states of hCC, our findings contribute to a better understanding of the intra and extra cellular functions of hCC and their interaction with cysteine proteases.

Nicotinamide adenine dinucleotide treatment confers resistance to neonatal ischemia and hypoxia: effects on neurobehavioral phenotypes.

JOURNAL/nrgr/04.03/01300535-202412000-00031/figure1/v/2024-04-08T165401Z/r/image-tiff Neonatal hypoxic-ischemic brain injury is the main cause of hypoxic-ischemic encephalopathy and cerebral palsy. Currently, there are few effective clinical treatments for neonatal hypoxic-ischemic brain injury. Here, we investigated the neuroprotective and molecular mechanisms of exogenous nicotinamide adenine dinucleotide, which can protect against hypoxic injury in adulthood, in a mouse model of neonatal hypoxic-ischemic brain injury. In this study, nicotinamide adenine dinucleotide (5 mg/kg) was intraperitoneally administered 30 minutes before surgery and every 24 hours thereafter. The results showed that nicotinamide adenine dinucleotide treatment improved body weight, brain structure, adenosine triphosphate levels, oxidative damage, neurobehavioral test outcomes, and seizure threshold in experimental mice. Tandem mass tag proteomics revealed that numerous proteins were altered after nicotinamide adenine dinucleotide treatment in hypoxic-ischemic brain injury mice. Parallel reaction monitoring and western blotting confirmed changes in the expression levels of proteins including serine (or cysteine) peptidase inhibitor, clade A, member 3N, fibronectin 1, 5'-nucleotidase, cytosolic IA, microtubule associated protein 2, and complexin 2. Proteomics analyses showed that nicotinamide adenine dinucleotide ameliorated hypoxic-ischemic injury through inflammation-related signaling pathways (e.g., nuclear factor-kappa B, mitogen-activated protein kinase, and phosphatidylinositol 3 kinase/protein kinase B). These findings suggest that nicotinamide adenine dinucleotide treatment can improve neurobehavioral phenotypes in hypoxic-ischemic brain injury mice through inflammation-related pathways.

Inhibition of cysteine protease disturbs the topological relationship between bone resorption and formation in vitro

IntroductionOsteoporosis is a global health issue. Bisphosphonates that are commonly used to treat osteoporosis suppress both bone resorption and subsequent bone formation. Inhibition of cathepsin K, a cysteine proteinase secreted by osteoclasts, was reported to suppress bone resorption while preserving or increasing bone formation. Analyses of the different effects of antiresorptive reagents such as bisphosphonates and cysteine proteinase inhibitors will contribute to the understanding of the mechanisms underlying bone remodeling. Materials and MethodsOur team has developed an in vitro system in which bone remodeling can be temporally observed at the cellular level by 2-photon microscopy. We used this system in the present study to examine the effects of the cysteine proteinase inhibitor E-64 and those of zoledronic acid on bone remodeling. ResultsIn the control group, the amount of the reduction and the increase in the matrix were correlated in each region of interest, indicating the topological and quantitative coordination of bone resorption and formation. Parameters for osteoblasts, osteoclasts, and matrix resorption/formation were also correlated. E-64 disrupted the correlation between resorption and formation by potentially inhibiting the emergence of spherical osteoblasts, which are speculated to be reversal cells in the resorption sites.ConclusionThese new findings help clarify coupling mechanisms and will contribute to the development of new drugs for osteoporosis.

Identification of Novel QTL for Mercury Accumulation in Maize Using an Enlarged SNP Panel.

Mercury (Hg) pollution not only poses a threat to the environment but also adversely affects the growth and development of plants, with potential repercussions for animals and humans through bioaccumulation in the food chain. Maize, a crucial source of food, industrial materials, and livestock feed, requires special attention in understanding the genetic factors influencing mercury accumulation. Developing maize varieties with low mercury accumulation is vital for both maize production and human health. In this study, a comprehensive genome-wide association study (GWAS) was conducted using an enlarged SNP panel comprising 1.25 million single nucleotide polymorphisms (SNPs) in 230 maize inbred lines across three environments. The analysis identified 111 significant SNPs within 78 quantitative trait loci (QTL), involving 169 candidate genes under the Q model. Compared to the previous study, the increased marker density and optimized statistical model led to the discovery of 74 additional QTL, demonstrating improved statistical power. Gene ontology (GO) enrichment analysis revealed that most genes participate in arsenate reduction and stress responses. Notably, GRMZM2G440968, which has been reported in previous studies, is associated with the significant SNP chr6.S_155668107 in axis tissue. It encodes a cysteine proteinase inhibitor, implying its potential role in mitigating mercury toxicity by inhibiting cysteine. Haplotype analyses provided further insights, indicating that lines carrying hap3 exhibited the lowest mercury content compared to other haplotypes. In summary, our study significantly enhances the statistical power of GWAS, identifying additional genes related to mercury accumulation and metabolism. These findings offer valuable insights into unraveling the genetic basis of mercury content in maize and contribute to the development of maize varieties with low mercury accumulation.

Different glycosylation profiles of cystatin F alter the cytotoxic potential of natural killer cells

Cystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell stage, called split-anergy. We show that N-glycosylation determines the localization and cellular function of cystatin F. Cystatin F mostly exhibited high-mannose glycosylation in U-937 cells, both high-mannose and complex glycosylation in NK-92 and primary NKs, and predominantly complex glycosylation in super-charged NKs. Manipulating N-glycosylation with kifunensine increased high-mannose glycosylation of cystatin F and lysosome localisation, which decreased cathepsin C activity and reduced NK cytotoxicity. Mannose-6-phosphate could significantly reduce the internalization of extracellular cystatin F. By comparing NK cells with different cytotoxic potentials, we found that high-mannose cystatin F was strongly associated with lysosomes and cathepsin C in NK-92 cell line. In contrast, in highly cytotoxic super-charged NKs, cystatin F with complex glycosylation was associated with the secretory pathway and less prone to inhibit cathepsin C. Modulating glycosylation to alter cystatin F localisation could increase the cytotoxicity of NK cells, thereby enhancing their therapeutic potential for treating cancer patients.Graphical abstract

A cysteine proteinase inhibitor ALLN alleviates bleomycin-induced skin and lung fibrosis

BackgroundSystemic sclerosis (SSc) is a connective tissue disease that is characterized by fibrosis in the skin and internal organs, such as the lungs. Activated differentiation of progenitor cells, which are mainly resident fibroblasts, into myofibroblasts is considered a key mechanism underlying the overproduction of extracellular matrix and the resultant tissue fibrosis in SSc. Calpains are members of the Ca2+-dependent cysteine protease family, whose enzymatic activities participate in signal transduction and tissue remodeling, potentially contributing to fibrosis in various organs. However, the roles of calpain in the pathogenesis of SSc remain unknown. This study aimed to examine the anti-fibrotic properties of N-acetyl-Leu-Leu-norleucinal (ALLN), one of the cysteine proteinase inhibitors that primarily inhibit calpain, in vitro and in vivo, to optimally translate into the therapeutic utility in human SSc.MethodsNormal human dermal and lung fibroblasts pretreated with ALLN were stimulated with recombinant transforming growth factor beta 1 (TGF-β1), followed by assessment of TGF-β1/Smad signaling and fibrogenic molecules.ResultsALLN treatment significantly inhibited TGF-β1-induced phosphorylation and nuclear transport of Smad2/3 in skin and lung fibroblasts. TGF-β1-dependent increases in α-smooth muscle actin (αSMA), collagen type I, fibronectin 1, and some mesenchymal transcription markers were attenuated by ALLN. Moreover, our findings suggest that ALLN inhibits TGF-β1-induced mesenchymal transition in human lung epithelial cells. Consistent with these in vitro findings, administering ALLN (3 mg/kg/day) three times a week intraperitoneally remarkably suppressed the development of skin and lung fibrosis in a SSc mouse model induced by daily subcutaneous bleomycin injection. The number of skin- and lung-infiltrating CD3+ T cells decreased in ALLN-treated mice compared with that in control-treated mice. Phosphorylation of Smad3 and/or an increase in αSMA-positive myofibroblasts was significantly inhibited by ALLN treatment on the skin and lungs. However, no adverse effects were observed.ConclusionsOur results prove that calpains can be a novel therapeutic target for skin and lung fibrosis in SSc, considering its inhibitor ALLN.

Cystatins from the Human Liver Fluke Opisthorchis viverrini: Molecular Characterization and Functional Analysis.

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1-6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3-6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3-9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated.

Characterization and Differentiation of Grain Proteomes from Wild-Type Puroindoline and Variants in Wheat.

Premium wheat with a high end-use quality is generally lacking in China, especially high-quality hard and soft wheat. Pina-D1 and Pinb-D1 (puroindoline genes) influence wheat grain hardness (i.e., important wheat quality-related parameter) and are among the main targets in wheat breeding programs. However, the mechanism by which puroindoline genes control grain hardness remains unclear. In this study, three hard wheat puroindoline variants (MY26, GX3, and ZM1) were compared with a soft wheat variety (CM605) containing the wild-type puroindoline genotype. Specifically, proteomic methods were used to screen for differentially abundant proteins (DAPs). In total, 6253 proteins were identified and quantified via a high-throughput tandem mass tag quantitative proteomic analysis. Of the 208 DAPs, 115, 116, and 99 proteins were differentially expressed between MY26, GX3, and ZM1 (hard wheat varieties) and CM605, respectively. The cluster analysis of protein relative abundances divided the proteins into six clusters. Of these proteins, 67 and 41 proteins were, respectively, more and less abundant in CM605 than in MY26, GX3, and ZM1. Enrichment analyses detected six GO terms, five KEGG pathways, and five IPR terms that were shared by all three comparisons. Furthermore, 12 proteins associated with these terms or pathways were found to be differentially expressed in each comparison. These proteins, which included cysteine proteinase inhibitors, invertases, low-molecular-weight glutenin subunits, and alpha amylase inhibitors, may be involved in the regulation of grain hardness. The candidate genes identified in this study may be relevant for future analyses of the regulatory mechanism underlying grain hardness.

Maquiberry Cystatins: Recombinant Expression, Characterization, and Use to Protect Tooth Dentin and Enamel.

Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui (Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins' potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein (by One-way ANOVA and Tukey's Multiple Comparisons Test, p < 0.05), suggesting its potential usage in dental products.

SerpinA3N deficiency attenuates steatosis and enhances insulin signaling in male mice.

Aberrant hepatic lipid metabolism is the major cause of non-alcoholic fatty liver disease (NAFLD) and is associated with insulin resistance and type 2 diabetes. Serine (or cysteine) peptidase inhibitor, clade A, member 3N (SerpinA3N) is highly expressed in the liver; however, its functional role in regulating NAFLD and associated metabolic disorders are not known. Male wildtype and hepatocyte Serpina3N knockout (HKO) mice were fed a control diet, methionine- and choline-deficient diet or high-fat high-sucrose diet to induce NAFLD and markers of lipid metabolism and glucose homeostasis were assessed. SerpinA3N protein was markedly induced in mice with fatty livers. Hepatic deletion of SerpinA3N attenuated steatosis which correlated with altered lipid metabolism genes, increased fatty acid oxidation activity and enhanced insulin signaling in mice with NAFLD. Additionally, SerpinA3N HKO mice had reduced epididymal white adipose tissue mass, leptin, and insulin levels, improved glucose tolerance, and enhanced insulin sensitivity which was associated with elevated insulin-like growth factor binding protein-1 (IGFBP1) and activation of the leptin receptor (LEPR)-STAT3 signaling pathway. Our findings provide a novel insight into the functional role of SerpinA3N in regulating NAFLD and glucose homeostasis.

An undefined cystatin CsCPI1 from tea plant Camellia sinensis harbors antithrombotic activity

Tea consumption has been linked to a decreased risk of cardiovascular disease (CVD) mortality, which imposes a heavy burden on the healthcare system; however, which components in tea cause this beneficial effect is not fully understood. Here we uncovered a cystatin (namely CsCPI1), which is a cysteine proteinase inhibitor (CPI) of the tea plant (Camellia sinensis) that promotes antithrombotic activity. Since thrombosis is a common pathogenesis of fatal CVDs, we investigated the effects of CsCPI1, which showed good therapeutic effects in mouse models of thrombotic disease and ischemic stroke. CsCPI1 significantly increases endothelial cell production of nitric oxide (NO) and inhibits platelet aggregation. Notably, CsCPI1 exhibited no cytotoxicity or resistance to pH and temperature changes, which indicates that CsCPI1 might be a potent antithrombotic agent that contributes to the therapeutic effects of tea consumption against CVD. Specifically, the antithrombotic effects of CsCPI1 are distinct from the classical function of plant cystatins against herbivorous insects. Therefore, our study proposes a new potential role of cystatins in CVD prevention and treatment, which requires further study.

Exploration of Microbially Derived Natural Compounds against Monkeypox Virus as Viral Core Cysteine Proteinase Inhibitors

Monkeypox virus (MPXV) is a member of the Orthopoxvirus genus and the Poxviridae family, which instigated a rising epidemic called monkeypox disease. Proteinases are majorly engaged in viral propagation by catalyzing the cleavage of precursor polyproteins. Therefore, proteinase is essential for monkeypox and a critical drug target. In this study, high-throughput virtual screening (HTVS) and molecular dynamics simulation were applied to detect the potential natural compounds against the proteinase of the monkeypox virus. Here, 32,552 natural products were screened, and the top five compounds were selected after implementing the HTVS and molecular docking protocols in series. Gallicynoic Acid F showed the minimum binding score of -10.56 kcal/mole in the extra precision scoring method, which reflected the highest binding with the protein. The top five compounds showed binding scores ≤-8.98 kcal/mole. These compound complexes were tested under 100 ns molecular dynamics simulation, and Vaccinol M showed the most stable and consistent RMSD trend in the range of 2 Å to 3 Å. Later, MM/GBSA binding free energy and principal component analysis were performed on the top five compounds to validate the stability of selected compound complexes. Moreover, the ligands Gallicynoic Acid F and H2-Erythro-Neopterin showed the lowest binding free energies of -61.42 kcal/mol and -61.09 kcal/mol, respectively. Compared to the native ligand TTP-6171 (ΔGBind = -53.86 kcal/mol), these two compounds showed preferable binding free energy, suggesting inhibitory application against MPXV proteinase. This study proposed natural molecules as a therapeutic solution to control monkeypox disease.

Trypanosoma brucei rhodesiense Inhibitor of Cysteine Peptidase (ICP) Is Required for Virulence in Mice and to Attenuate the Inflammatory Response.

The protozoan Trypanosoma brucei rhodesiense causes Human African Trypanosomiasis, also known as sleeping sickness, and penetrates the central nervous system, leading to meningoencephalitis. The Cathepsin L-like cysteine peptidase of T. b. rhodesiense has been implicated in parasite penetration of the blood-brain barrier and its activity is modulated by the chagasin-family endogenous inhibitor of cysteine peptidases (ICP). To investigate the role of ICP in T. b. rhodesiense bloodstream form, ICP-null (Δicp) mutants were generated, and lines re-expressing ICP (Δicp:ICP). Lysates of Δicp displayed increased E-64-sensitive cysteine peptidase activity and the mutant parasites traversed human brain microvascular endothelial cell (HBMEC) monolayers in vitro more efficiently. Δicp induced E-selectin in HBMECs, leading to the adherence of higher numbers of human neutrophils. In C57BL/6 mice, no Δicp parasites could be detected in the blood after 6 days, while mice infected with wild-type (WT) or Δicp:ICP displayed high parasitemia, peaking at day 12. In mice infected with Δicp, there was increased recruitment of monocytes to the site of inoculation and higher levels of IFN-γ in the spleen. At day 14, mice infected with Δicp exhibited higher preservation of the CD4+, CD8+, and CD19+ populations in the spleen, accompanied by sustained high IFN-γ, while NK1.1+ populations receded nearly to the levels of uninfected controls. We propose that ICP helps to downregulate inflammatory responses that contribute to the control of infection.

Role of cystatin C in urogenital malignancy.

Urogenital malignancy accounts for one of the major causes of cancer-related deaths globally. Numerous studies have investigated novel molecular markers in the blood circulation, tumor tissue, or urine in order to assist in the clinical identification of tumors at early stages, predict the response of therapeutic strategies, and give accurate prognosis assessment. As an endogenous inhibitor of lysosomal cysteine proteinases, cystatin C plays an integral role in diverse processes. A substantial number of studies have indicated that it may be such a potential promising biomarker. Therefore, this review was intended to provide a detailed overview of the role of cystatin C in urogenital malignancy.

Transcriptomic analysis of bakanae disease resistant and susceptible rice genotypes in response to infection by Fusarium fujikuroi.

Fusarium fujikuroi causing bakanae is one of the most significant pathogens of rice and much responsible for yield losses thereby emerging as a major risk to food security. In the present study transcriptomic analysis was conducted between two contrasting resistant (C101A51) and susceptible (Rasi) genotypes of rice with the combinations of C101A51 control (CC) vs. C101A51 inoculated (CI); Rasi control (RC) vs. Rasi inoculated (RI) and C101A51 inoculated (CI) vs. Rasi inoculated (RI). In CC vs. CI commonly expressed genes were 12,764. Out of them 567 (4%) were significantly upregulated and 1399 (9%) genes were downregulated. For the RC vs. RI 14, 333 (79%) genes were commonly expressed. For CI vs. RI 13,662 (72%) genes were commonly expressed. Genes related to cysteine proteinase inhibitor 10, disease resistance protein TAO1-like, oleosin 16kDa-like, pathogenesis-related protein (PR1), (PR4), BTB/POZ and MATH domain-containing protein 5-like, alpha-amylase isozyme were upregulated in resistant genotype C101A51. Whereas, genes related to GDSL esterase/lipase, serine glyoxylate aminotransferase, CASP-like protein 2C1, WAT1-related protein, Cytoplasmic linker associated proteins, xyloglucan endotransglucosylase/hydrolase protein and β-D xylosidase 7 were upregulated in susceptible genotype Rasi. Gene ontology analysis showed functions related to defence response (GO:0006952), regulation of plant hypersensitive type response (GO:0010363), Potassium ion transmembrane activity (GO:0015079), chloroplast (GO:0009507), response to wounding (GO:0009611), xylan biosynthetic process (GO:0045492) were upregulated in resistant genotype C101A51 under inoculated conditions. Real time PCR based validation of the selected DEGs showed that the qRT-PCR was consistent with the RNA-Seq results. This is the first transcriptomic study against bakanae disease of rice in Indian genotypes. Further, functional studies on identified genes and their utilization through different methodology will be helpful for the development of bakanae disease management strategies.

Semiquantitative assessment of 99mTc-MIBI uptake in parathyroids of secondary hyperparathyroidism patients with chronic renal failure.

ObjectiveTo explore the valuably influential factors and improve the diagnostic accuracy and efficiency of 99mTc-methoxyisobutylisonitrile (MIBI) uptake in parathyroids of secondary hyperparathyroidism (SHPT) patients with chronic renal failure (CRF).MethodsThe correlation analysis was performed between clinical indices related to CRF and 99mTc-MIBI uptake intensity TBR (the gray value mean ratio between the parathyroid target and the bilateral neck background, semiquantitatively calculated with ImageJ software). All clinical indices and TBRs were compared by a three- or two-level grouping method of MIBI uptake, which was visually qualitatively assessed. The three-level grouping method comprised slight, medium, and high groups with little, faint, and distinct MIBI concentration in parathyroids, respectively. The two-level grouping method comprised insignificant and significant groups with TBR greater than or less than 0.49–0.71, respectively.ResultsMIBI uptake was significantly positively related to patient age, CRF course, hemodialysis vintage, serum parathyroid hormone (PTH), and alkaline phosphatase (AKP) but was significantly negatively related to serum uric acid (UA). MIBI washout was significantly positively related to patient age but was significantly negatively related to serum phosphorus (P) and calcium (Ca) × P. Oral administration of calcitriol and calcium could significantly reduce the MIBI uptake. MIBI uptake tendency might alter. Such seven indices, namely the MIBI uptake, CRF course, hemodialysis vintage, serum AKP, calcium, cysteine proteinase inhibitor C, and PTH, were comparable between the slight and medium groups but were significantly different between the slight and high groups or between the medium and high groups. The above seven indices plus blood urea nitrogen/creatinine were all significantly different between the insignificant and significant groups. All above significances were with P < 0.05.ConclusionsPatient age, CRF course, hemodialysis vintage, serum PTH, AKP, UA, phosphorus, Ca × P, oral administration of calcitriol and calcium, and parathyroids themselves can significantly influence MIBI uptake in parathyroids of SHPT patients with CRF. The two-level grouping method of MIBI intensity should be adopted to qualitatively diagnose the MIBI uptake.

Identification of Proteins Responsible for High Activity of Cysteine Proteinase Inhibitor in the Blood of Nile Tilapia Oreochromis niloticus

Cysteine proteinase inhibitors (CPIs) protect tissues and organs against cysteine proteinases in animal blood and have attracted much attention for use in food processing and medical sciences for humans and animals. Several CPI proteins, which include stefins, cystatins, kininogens, histidine-rich glycoproteins (HRG) and fetuins, have been identified and characterized in mammals. Fish blood also contains high CPI activity, but the identity of the major protein responsible for this activity has not been clarified. This study was conducted to screen CPI activity by examining papain inhibitory activity from various different tissues in Nile tilapia Oreochromis niloticus and to identify major proteins for the activity in the blood. CPI activity was highest in the serum among the tissues screened in this study (at least fourfold higher than in other tissues)(P &lt; 0.05). Major proteins for CPI activity in serum were purified using a CNBr-activated sepharose 4B column, gel filtration and an ion exchange FPLC column. From these purifications, two proteins with strong CPI activity were isolated and partially sequenced. Based on their molecular weights and partial amino sequences, the two major proteins with CPI activity from the blood in this species were found to be fetuin B (60 kDa) and kininogen (54 kDa).

Apple vacuolar processing enzyme 4 is regulated by cysteine protease inhibitor and modulates fruit disease resistance.

Ring rot is a destructive apple disease caused by Botryosphaeria dothidea. The resistance mechanism of apple plants to B. dothidea remains unclear. Here, we show that APPLE VACUOLAR PROCESSING ENZYME 4 (MdVPE4) is involved in resistance to B. dothidea. MdVPE4 silencing reduced fruit disease resistance, whereas its overexpression improved resistance. Gene expression analysis revealed that MdVPE4 influenced the expression of fruit disease resistance-related genes, such as APPLE POLYGALACTURONASE 1 (MdPG1), APPLE POLYGALACTURONASE INHIBITOR PROTEIN 1 (MdPGIP1), APPLE ENDOCHITINASE 1 (MdCHI1), and APPLE THAUMATIN-LIKE PROTEIN 1 (MdTHA1). The expression of the four genes responding to B. dothidea infection decreased in MdVPE4-silenced fruits. Further analysis demonstrated that B. dothidea infection induced MdVPE4 expression and enzyme activation in apple fruits. Moreover, MdVPE4 activity was modulated by apple cysteine proteinase inhibitor 1 (MdCPI1), which also contributed to resistance towards B. dothidea, as revealed by gene overexpression and silencing analysis. MdCPI1 interacted with MdVPE4 and inhibited its activity. However, MdCPI1 expression was decreased by B. dothidea infection. Taken together, our findings indicate that the interaction between MdVPE4 and MdCPI1 plays an important role in modulating fruit disease resistance to B. dothidea.

The fungal Clitocybe nebularis lectin binds distinct cell surface glycoprotein receptors to induce cell death selectively in Jurkat cells.

Clitocybe nebularis lectin (CNL) is a GalNAcβ1-4GlcNAc-binding lectin that exhibits an antiproliferative effect exclusively on the Jurkat leukemic T cell line by provoking homotypic aggregation and dose-dependent cell death. Cell death of Jurkat cells exhibited typical features of early apoptosis, but lacked the activation of initiating and executing caspases. None of the features of CNL-induced cell death were effectively blocked with the pan-caspase inhibitor or different cysteine peptidase inhibitors. Furthermore, CNL binding induced Jurkat cells to release the endogenous damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1). A plant lectin with similar glycan-binding specificity, Wisteria floribunda agglutinin (WFA) showed less selective toxicity and induced cell death in Jurkat, Tall-104, and Hut-87 cell lines. HMGB1 release was also detected when Jurkat cells were treated with WFA. We identified the CD45 and CD43 cell surface glycoproteins on Jurkat cells as the main targets for CNL binding. However, the blockade of CD45 phosphatase activity failed to block either CNL-induced homotypic agglutination or cell death. Overall, our results indicate that CNL triggers atypical cell death selectively on Jurkat cells, suggesting the potential applicability of CNL in novel strategies for treating and/or detecting acute T cell leukemia.

Vacuolar Processing Enzymes Modulating Susceptibility Response to Fusarium oxysporum f. sp. cubense Tropical Race 4 Infections in Banana.

Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) is a destructive necrotrophic fungal pathogen afflicting global banana production. Infection process involves the activation of programmed cell death (PCD). In this study, seven Musa acuminata vacuolar processing enzyme (MaVPE1–MaVPE7) genes associated with PCD were successfully identified. Phylogenetic analysis and tissue-specific expression categorized these MaVPEs into the seed and vegetative types. FocTR4 infection induced the majority of MaVPE expressions in the susceptible cultivar “Berangan” as compared to the resistant cultivar “Jari Buaya.” Consistently, upon FocTR4 infection, high caspase-1 activity was detected in the susceptible cultivar, while low level of caspase-1 activity was recorded in the resistant cultivar. Furthermore, inhibition of MaVPE activities via caspase-1 inhibitor in the susceptible cultivar reduced tonoplast rupture, decreased lesion formation, and enhanced stress tolerance against FocTR4 infection. Additionally, the Arabidopsis VPE-null mutant exhibited higher tolerance to FocTR4 infection, indicated by reduced sporulation rate, low levels of H2O2 content, and high levels of cell viability. Comparative proteomic profiling analysis revealed increase in the abundance of cysteine proteinase in the inoculated susceptible cultivar, as opposed to cysteine proteinase inhibitors in the resistant cultivar. In conclusion, the increase in vacuolar processing enzyme (VPE)-mediated PCD played a crucial role in modulating susceptibility response during compatible interaction, which facilitated FocTR4 colonization in the host.