A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Inserções de aminoácidos na protease têm sido raramente descritas em pacientes infectados pelo HIV. Uma destas inserções foi, recentemente, descrita no codon 35, embora seu impacto na resistência mantém-se pouco conhecido. Este trabalho apresenta um caso de uma variante viral com inserção no codon 35 da protease, descrita pela primeira vez em Bauru, São Paulo, Brasil, circulante em um homem, caucasiano, com 38 anos, o qual apresenta infecção assintomática pelo HIV desde 1997. A variante isolada mostrou uma inserção no codon 35 da protease de dois aminoácidos: uma treonina e um ácido aspártico, resultando na sequência de aminoácidos E35E_TD.

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion. Case presentation: We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine. Conclusions: A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

Molecular and functional characterizations of a Kunitz-type serine protease inhibitor FcKuSPI of the shrimp Fenneropenaeus chinensis

Serine proteinase inhibitors play important and diverse roles in biological processes such as coagulation, defense mechanisms, and immune responses. Here, we identified and characterized a Kunitz-type proteinase inhibitor, designated FcKuSPI, of the BPTI/Kunitz family of serine proteinase inhibitors from the hemocyte cDNA library of the shrimp Fenneropenaeus chinensis. The deduced amino acid sequence of FcKuSPI comprises 80 residues with a putative signal peptide of 15 amino acids. The predicted molecular weight of the mature peptide is 7.66 kDa and its predicted isoelectric point is 8.84. FcKuSPI includes a Kunitz domain containing six conserved cysteine residues that are predicted to form three disulfide bonds. FcKuSPI shares 44e53% homology with BPTI/Kunitz family members from other species. FcKuSPI mRNAwas expressed highly in the hemocytes and moderately in muscle in healthy shrimp. Recombinant FcKuSPI protein demonstrated anti-protease activity against trypsin and anticoagulant activity against citrated human plasma in a dose-dependent manner in in vitro assays.

Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

Jerdonase, a novel serine protease with kinin-releasing and fibrinogenolytic activity from Trimeresurus jerdonii venom

A novel kinin-releasing and fibrin (ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenymethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the Aalpha-chain of human fibrinogen with lower activity towards Bbeta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

A novel serine protease with clip domain from scallop Chlamys farreri

The serine proteases with clip domain are involved in various innate immune functions in invertebrate such as antimicrobial activity, cell adhesion, pattern recognition and regulation of the prophenoloxidase system. A serine protease with clip-domain cDNA (Cf SP) was obtained by Expressed sequence taggings (ESTs) method and rapid amplification of cDNA ends (RACE). The Cf SP full-length cDNA was of 1,152 bp, including a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 81 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 1,008 bp encoding a polypeptide of 336 amino acids with a putative signal peptide of 19 amino acids. The deduced amino acid sequence of Cf SP contained an amino-terminal clip domain with three disulfide bonds formed six conserved Cys residues, a carboxyl-terminal trypsin-like domain with the conserved His-Asp-Ser catalytic triad, and a low complexity linker sequence. The Cf SP was strongly expressed in hemocytes and the mRNA expression of Cf SP was up-regulated and increased 3.2-fold and 2.6-fold at 16 h after injection of Vibrio anguillarum and Micrococcus luteus. The results suggested that Cf SP gene might be involved in immune response of Gram-negative and Gram-positive microbial infection in scallop.

Neural Network and Bioinformatic Methods for Predicting HIV-1 Protease Inhibitor Resistance

This article presents a new method for predicting viral resistance to seven protease inhibitors from the HIV-1 genotype, and for identifying the positions in the protease gene at which the specific nature of the mutation affects resistance. The neural network Analog ARTMAP predicts protease inhibitor resistance from viral genotypes. A feature selection method detects genetic positions that contribute to resistance both alone and through interactions with other positions. This method has identified positions 35, 37, 62, and 77, where traditional feature selection methods have not detected a contribution to resistance. At several positions in the protease gene, mutations confer differing degress of resistance, depending on the specific amino acid to which the sequence has mutated. To find these positions, an Amino Acid Space is introduced to represent genes in a vector space that captures the functional similarity between amino acid pairs. Feature selection identifies several new positions, including 36, 37, and 43, with amino acid-specific contributions to resistance. Analog ARTMAP networks applied to inputs that represent specific amino acids at these positions perform better than networks that use only mutation locations.

A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods.

The coronavirus main protease, Mpro, is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compounds that was performed to identify potential Mpro inhibitors revealed etacrynic acid tert-butylamide (6a) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the phenyl ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-butyl moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (6b) as a promising lead for nonpeptidic active-site-directed Mpro inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compound 6b showed a Ki value of 35.3 M. Since the novel lead compound does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compound 6b.

Purification and properties of an alkaline protease of Aspergillus clavatus

An extracellular alkaline serine protease has been purified from a strain of Aspergillus clavatus, to apparent homogeneity, by ammonium sulfate precipitation and chromatography on Sephadex G-75. Its molar mass, estimated by SDS-PAGE, was 35 kDa. Maximum protease activity was observed at pH 9.5 and 40 degrees C. The enzyme was active between pH 6.0 and 11.0 and was found to be unstable up to 50 degrees C. Calcium at 5 mM increased its thermal stability. The protease was strongly inhibited by PMSF and chymostatin as well as by SDS, Tween 80 and carbonate ion. Substrate specificity was observed with N-p-Tos-Gly-Pro-Arg-p-nitroanilide and N-Suc-Ala-Ala-Ala-p-nitroanilide being active substates. Parts of the amino acid sequence were up to 81% homologous with those of several fungal alkaline serine proteases.

Protease production by different thermophilic fungi

A comparative study was carried out to evaluate protease production in solid-state fermentation (SSF) and submerged fermentation (SmF) by nine different thermophilic fungi - Thermoascus aurantiacus Miehe, Thermomyces lanuginosus, T. lanuginosus TO.03, Aspergillus flavus 1.2, Aspergillus sp. 13.33, Aspergillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36 and Rhizomucor sp. 13.37 - using substrates containing proteins to induce enzyme secretion. Soybean extract (soybean milk), soybean flour, milk powder, rice, and wheat bran were tested. The most satisfactory results were obtained when using wheat bran in SSF. The fungi that stood out in SSF were T. lanuginosus, T. lanuginosus TO.03, Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37, and those in SmF were T. aurantiacus, T. lanuginosus TO.03, and 13.37. In both fermentation systems, A. flavus 1.2 and R. pusillus 13.36 presented the lowest levels of proteolytic activity.

Purification, characterization, and specificity determination of a new serine protease secreted by penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl2, KCl, and BaCl, and partially inhibited by CuCl2, CoCl2 and totally inhibited by AlCl3 and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k cat/K m of 10,666 mM-1 s-1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k cat/K m of 7,500 mM -1 s-1. Basic and acidic side chain-containing amino acids performed best at subsite S1. Subsites S2, S3, S′ 2, and S′ 1, S ′ 3 showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k cat/K m were observed for the subsites S2, S3, and S′ 2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level. © 2012 Springer Science+Business Media New York.

Characterization, subsite mapping and N-terminal sequence of miliin, a serine-protease isolated from the latex of Euphorbia milii

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mannose-binding lectin and MBL-associated serine protease-2 gene polymorphisms in a Brazilian population from Rio de Janeiro

Mannose-binding lectin (MBL) is a protein able to bind to carbohydrate patterns on pathogen membranes; upon MBL binding, its associated serine protease MBL-associated serine protease type 2 (MASP2) is autoactivated, promoting the activation of complement via the lectin pathway. For both MBL2 and MASP2 genes, the frequencies of polymorphisms are extremely variable between different ethnicities, and this aspect has to be carefully considered when performing genetic studies. While polymorphisms in the MBL-encoding gene (MBL2) have been associated, depending upon ethnicity, with several diseases in different populations, little is known about the distribution of MASP2 gene polymorphisms in human populations. The aim of our study was thus to determine the frequencies of MBL2 (exon 1 and promoter) and MASP2 (p.D371Y) polymorphisms in a Brazilian population from Rio de Janeiro. A total of 294 blood donor samples were genotyped for 27 polymorphisms in the MBL2 gene by direct sequencing of a region spanning from the promoter polymorphism H/L rs11003125 to the rs1800451 polymorphism (at codon 57 in the first exon of the gene). Genotyping for MASP2 p.D371Y was carried out using fluorogenic probes. To our knowledge, this is the first study reporting the prevalence of the MASP2 p.D371Y polymorphism in a Brazilian population. The C allele frequency 39% is something intermediate between the reported 14% in Europeans and 90% in Sub-Saharan Africans. MBL2 polymorphisms frequencies were quite comparable to those previously reported for admixed Brazilians. Both MBL2 and MASP2 polymorphisms frequencies reported in our study for the admixed Brazilian population are somehow intermediate between those reported in Europeans and Africans, reflecting the ethnic composition of the southern Brazilian population, estimated to derive from an admixture of Caucasian (31%), African (34%) and Native American (33%) populations. In conclusion, our population genetic study describes the frequencies of MBL2 and MASP2 functional SNPs in a population from Rio de Janeiro, with the aim of adding new information concerning the distribution of these SNPs in a previously unanalysed Brazilian population, thus providing a new genetic tool for the evaluation of the association of MBL2 and MASP2 functional SNPs with diseases in Brazil, with particular emphasis on the state of Rio de Janeiro.

Prognosis in pediatric hematologic malignancies is associated with serum concentration of mannose-binding lectin-associated serine protease-2 (MASP-2)

BACKGROUND: Mannose-binding lectin (MBL) and MBL-associated serine protease-2 (MASP-2) are key components of the lectin pathway of complement activation. Their serum concentrations show a wide interindividual variability. This study investigated whether the concentration of MBL and MASP-2 is associated with prognosis in pediatric patients with cancer. METHODS: In this retrospective multicenter study, MBL and MASP-2 were measured by commercially available ELISA in frozen remnants of serum taken at diagnosis. Associations of overall survival (OS) and event-free survival (EFS) with MBL and MASP-2 were assessed by multivariate Cox regression accounting for prognostically relevant clinical variables. RESULTS: In the 372 patients studied, median serum concentration of MBL was 2,808 microg/L (range, 2-10,060) and 391 microg/L (46-2,771) for MASP-2. The estimated 4-year EFS was 0.60 (OS, 0.78). In the entire, heterogeneous sample, MBL and MASP-2 were not significantly associated with OS or EFS. In patients with hematologic malignancies, however, higher MASP-2 was associated with better EFS in a significant and clinically relevant way (hazard ratio per tenfold increase (HR), 0.22; 95% CI, 0.09-0.54; P = 0.001). This was due to patients with lymphoma (HR, 0.11; 95% CI, 0.03-0.47; P = 0.003), but less for those with acute leukemia (HR, 0.35; 95% CI, 0.11-1.15; P = 0.083). CONCLUSION: In this study, higher MASP-2 was associated with better EFS in pediatric patients with hematologic malignancies, especially lymphoma. Whether MASP-2 is an independent prognostic factor affecting risk stratification and anticancer therapy needs to be assessed in prospective, disease-specific studies.