Proteases from the fungus Metarhizium anisopliae toxic for Galleria mellonella larvae

The high molecular fraction of the extract from Metarhizium anisopliae grown on wheat bran contains proteolytic enzymes which are toxic for Galleria mellonella larvae. The complex of proteases was fractionated using precipitation with ammonium sulfate, gel filtration, and electrofocusing. Two components have been found: one with the optimum of activity on hemoglobin at pH 6.5, and the second with the optimum around pH 9. The prevailing protease acting at pH 6.5 was inhibited by phenylmethylsulfonyl fluoride and the inhibition was followed by decrease of toxicity. The molecular weights of the enzymes are 35 × 10³ and 71 × 10³.     

Cadmium causes the oxidative modification of proteins in pea plants

In pea (Pisum sativum L.) leaves from plants grown in the presence of 50 µm CdCl₂ the oxidative production of carbonyl groups in proteins, the rate of protein degradation and the proteolytic activity were investigated. In leaf extracts the content of carbonyl groups measured by derivatization with 2,4‐dinitrophenylhydrazine (DNPH), was two‐fold higher in plants treated with Cd than in control plants. The identification of oxidized proteins was carried out by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of proteins derivatized with DNPH and immunochemical detection with an antibody against DNPH. The intensity of the reactive bands was higher in plants exposed to Cd than in controls. By using different antibodies some of the oxidized proteins were identified as Rubisco, glutathione reductase, manganese superoxide dismutase, and catalase. The incubation of leaf crude extracts with increasing H₂O₂ concentrations showed a progressive enhancement in carbonyl content and the pattern of oxidized proteins was similar to that found in Cd‐treated plants. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased 20% due to the metal treatment. In peroxisomes purified from pea leaves a rise in the carbonyl content similar to that obtained in crude extracts from Cd‐treated plants was observed, but the functionality of the peroxisomal membrane was not apparently affected by Cd. Results obtained demonstrate the participation of both oxidative stress, probably mediated by H₂O₂, and proteolytic degradation in the mechanism of Cd toxicity in leaves of pea plants, and they appear to be involved in the Cd‐induced senescence previously reported in these plants.     

PGAM5: A crucial role in mitochondrial dynamics and programmed cell death

In response to mitochondrial damage, mitochondria activate mitochondrial dynamics to maintain normal functions, and an imbalance in mitochondrial dynamics triggers multiple programmed cell death processes. Recent studies have shown that phosphoglycerate mutase 5 (PGAM5) is associated with mitochondrial damage. PGAM5 activates mitochondrial biogenesis and mitophagy to promote a cellular compensatory response when mitochondria are mildly damaged, whereas severe damage to mitochondria leads to PGAM5 inducing excessive mitochondria fission, disruption to mitochondrial movement, and amplification of apoptosis, necroptosis and mitophagic death signals, which eventually evoke cell death. PGAM5 functions mainly through protein-protein interactions and specific Ser/Thr/His protein phosphatase activity. PGAM5 is also regulated by mitochondrial proteases. Detection of PGAM5 and its interacting protein partners should enable a more accurate evaluation of mitochondrial damage and a more precise method for the diagnosis and treatment of diseases.     

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (K_b) values between 10.23?×?10³ and 6.52?×?10³ M^?1. The binding reactions were enthalpy driven with ΔH_b values between ?50.99 and ?46.07 kJ mol^?1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.     

Ligand design,synthesis and biological anti-HCV evaluations for genotypes 1b and 4a of certain 4-(3- & 4-[3-(3,5-dibromo-4-hydroxyphenyl)-propylamino]phenyl) butyric acids and 3-(3,5-dibromo-4-hydroxyphenyl)-propylamino-acetamidobenzoic acid esters

4-(4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylamino]phenyl)-4-oxo-butyric acid (V), 4-(3- & 4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylaminophenyl]-2-aryl-4-oxo-butyric acids (Xa–e) and 4-(2-alkyl-2-[N-3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-propylamino]acetamido) benzoate esters (XVa–e) were designed, synthesized and biologically evaluated as anti-HCV for genotypes 1b and 4a. The design was based on their docking scores with HCV NS3/4A protease-binding site of the genotype 1b (1W3C), which is conserved in the genotype 4a structure. The docking scores predicted that most of these molecules have higher affinity to the HCV NS3/4A enzyme more than Indoline lead. These compounds were synthesized and evaluated for their cytopathic inhibitory activity against RAW HCV cell cultures of genotype 4a and also examined against Huh 5–2 HCV cell culture of genotype 1b, utilizing Luciferase and MTS assays. Compounds Xa and Xb have 95 and 80% of the activity of Ribavirin against genotype 4a and compounds XVa, XVb and XVd exerted high percentage inhibitory activity against genotype 1b equal 87.7, 84.3 and 82.8%, respectively, with low EC₅₀ doses.     

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.     

CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases

Background

CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles.

Methods and results

In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at ²⁹⁶SNLD²⁹⁹ and ³⁴⁸TCPD³⁵¹, and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified ³²⁰EPRT³²³ as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at ³²⁰EPRT³²³. Additionally, the cleavage catalyzed by the ³²⁰EPRT³²³ protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment.

Conclusion

CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the “³²⁰EPRT³²³ protease(s).” Furthermore, ³²⁰EPRT³²³ cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage.

General significance

CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

Relevance of signaling molecules for apoptosis induction on influenza A virus replication

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.