Identification and Characterization of HTLV-1 HBZ Post-Translational Modifications

Human T-cell leukemia virus type-1 (HTLV-1) is estimated to infect 15–25 million people worldwide, with several areas including southern Japan and the Caribbean basin being endemic. The virus is the etiological agent of debilitating and fatal diseases, for which there is currently no long-term cure. In the majority of cases of leukemia caused by HTLV-1, only a single viral gene, hbz, and its cognate protein, HBZ, are expressed and their importance is increasingly being recognized in the development of HTLV-1-associated disease. We hypothesized that HBZ, like other HTLV-1 proteins, has properties and functions regulated by post-translational modifications (PTMs) that affect specific signaling pathways important for disease development. To date, PTM of HBZ has not been described. We used an affinity-tagged protein and mass spectrometry method to identify seven modifications of HBZ for the first time. We examined how these PTMs affected the ability of HBZ to modulate several pathways, as measured using luciferase reporter assays. Herein, we report that none of the identified PTMs affected HBZ stability or its regulation of tested pathways.     

Biochemical and Genetic Characterization of Propionicin T1, a New Bacteriocin from Propionibacterium thoenii

A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii. This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii. The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA. The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.     

Purification and Characterization of a Neuropeptide-Degrading Aminopeptidase from Human Brain

The major aminopeptidase from human post-mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2-mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal-ion chelating agents, sulphydryl blocking agents, bestatin, and puromycin. A series of amino acyl-7-amido-4-methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 microM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5-enkephalin (Km 180 microM), cholecystokinin octapeptide, and Arg8-vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5-enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis.     

Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg²⁺, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K_m for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.     

Ph-Sensitive Liposomes: Structural Characterization and Possible Therapeutic Applications

Abstract

The definition of liposomal preparations where sensitivity to moderate drops of pH (i.e. from 7.4 to 6.8) can be induced by the presence of plasma itself has been investigated. Liposome stability was monitored using 5,6-carboxyfluorescein (CF). We used sulfatide as the pH sensitive molecule on the basis of our previous studies in which we demonstrated an enhanced anti-tumor activity against a transplantable metastatic tumor model for ADM entrapped liposomes containing sulfatide. The amount of CF released at pH 6.8 in the presence of 50% plasma from small unilamellar vesicles (SUV), composed of egg phosphatidylcholine (EPC) and bovine brain sulfatide (CS) (4:1 m/m), was 3-fold that at pH 7.4, whereas no significant differences were observed when the same liposomes were incubated in buffer at 7.4 and 6.8 respectively. The plasma dependent pH sensitivity of these liposomes seems to specifically depend on the presence of sulfatide in the bilayer since neither cholesterol 3 sulfate (Choi 3S) nor galactocerebroside, are able to induce pH sensitivity in EPC liposomes. Of all the plasma components considered, VLDL seemed preferentially involved in the pH sensitivity induced by CS since they promoted an almost complete release of CF from EPC-CS liposomes at pH 6.8.     

Purification and Characterization of Aminopeptidase from Euphausia superba

Krill aminopeptidase was purified about, 1,100-fold from an extract of Euphausia superba with DEAE-cellulose column chromatography, Toyopearl HW55, and hydroxyapatite column chromatography. The final preparation was electrophoretically homogeneous. The molecular weight was determined to be 140,000 by gel filtration and SDS-polyacrylamide disc gel electrophoresis. The optimum pH and optimum temperature were 8.4 and 45°C respectively. Krill aminopeptidase was inhibited by EDTA, Hg^{+ +} and amastatin, and partially by bestatin, and was activated by Co ^{+ +}. Alanyl-p-nitroanilide was hydrolyzed faster than leucyl-p-nitroanilide. Alanyl peptides (di-, tri-, tetra- and hexa-alanyl peptide) were hydrolyzed very fast.

These results suggest that krill aminopeptidase is an alanine aminopeptidase which is activated by cobaltous ion.     

Structural and Biochemical Characterization of a Halophilic Archaeal Alkaline Phosphatase

Phosphate is an essential component of all cells that must be taken up from the environment. Prokaryotes commonly secrete alkaline phosphatases (APs) to recruit phosphate from organic compounds by hydrolysis. In this study, the AP from Halobacterium salinarum, an archaeon that lives in a saturated salt environment, has been functionally and structurally characterized. The core fold and the active-site architecture of the H. salinarum enzyme are similar to other AP structures. These generally form dimers composed of dominant β-sheet structures sandwiched by α-helices and have well-accessible active sites. The surface of the enzyme is predicted to be highly negatively charged, like other proteins of extreme halophiles. In addition to the conserved core, most APs contain a crown domain that strongly varies within species. In the H. salinarum AP, the crown domain is made of an acyl-carrier-protein-like fold. Different from other APs, it is not involved in dimer formation. We compare the archaeal AP with its bacterial and eukaryotic counterparts, and we focus on the role of crown domains in enhancing protein stability, regulating enzyme function, and guiding phosphoesters into the active-site funnel.     

Biochemical Characterization and Structural Analysis of a Bifunctional Cellulase/Xylanase from Clostridium thermocellum

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.     

Structural and Biochemical Characterization of Free Methionine-R-sulfoxide Reductase from Neisseria meningitidis

A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys¹¹⁸ is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.     

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1’) with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.     

Molecular and Genetic Characterization of a Novel Nisin Variant Produced by Streptococcus uberis

Streptococcus uberis is one of the principal causative agents of bovine mastitis. In this study, we report that S. uberis strain 42 produces a lantibiotic, nisin U, which is 78% identical (82% similar) to nisin A from Lactococcus lactis. The 15.6-kb nisin U locus comprises 11 open reading frames, similar in putative functionality but differing in arrangement from that of the nisin A biosynthetic cluster. The nisin U producer strain exhibits specific resistance (immunity) to nisin U and cross-resistance to nisin A, a finding consistent with the 55% sequence similarity of their respective immunity peptides. Homologues of the nisin U structural gene were identified in several additional S. uberis strains, and in each case cross-protective immunity was expressed to nisin A and to the other producers of nisin U and its variants. To our knowledge, this is the first report both of characterization of a bacteriocin by S. uberis, as well as of a member of the nisin family of peptides in a species other than L. lactis.     

PTMs in Conversation: Activity and Function of Deubiquitinating Enzymes Regulated via Post-Translational Modifications

Deubiquitinating enzymes (DUBs) constitute a diverse protein family and their impact on numerous biological and pathological processes has now been widely appreciated. Many DUB functions have to be tightly controlled within the cell, and this can be achieved in several ways, such as substrate-induced conformational changes, binding to adaptor proteins, proteolytic cleavage, and post-translational modifications (PTMs). This review is focused on the role of PTMs including monoubiquitination, sumoylation, acetylation, and phosphorylation as characterized and putative regulative factors of DUB function. Although this aspect of DUB functionality has not been yet thoroughly studied, PTMs represent a versatile and reversible method of controlling the role of DUBs in biological processes. In several cases PTMs might constitute a feedback mechanism insuring proper functioning of the ubiquitin proteasome system and other DUB-related pathways.     

一种新的人CNA1基因转录本的克隆和功能鉴定

钙调神经磷酸酶（calcineurin,CN）是唯一依赖于Ca2+和钙调蛋白（calmodulin,CaM）的丝氨酸/苏氨酸型蛋白磷酸酶,由1个催化亚基CNA和1个调节亚基CNB组成. CNA 有3种亚型,最常见的是由CNA1基因编码的α亚型（CNAα）. 在克隆CNA1基因cDNA的过程中,发现了1种新的人CNA1转录本-CNAα4. 与CNA1基因的其它转录本相比,CNAα4缺失第2外显子,其编码蛋白质由454个氨基酸组成,具有比其它3种CNAα亚型更短的磷酸酶催化结构域. CNAα4具有与CNAα1相似的CaM亲和力,但是其激活活化T细胞核因子（nuclear factor of activated T cells,NFAT）的活性明显强于CNAα1,提示CNAα4所缺失的氨基酸序列（Ala20 Thr86）并非CNA催化结构域所必需,相反,Ala²⁰-Thr⁸⁶缺失可能有助于其酶活性中心与NFAT的结合并发挥作用.     

Characterization of Aminopeptidase in the Free-living Nematode Panagrellus redivivus: Subcellular Distribution and Possible Role in Neuropeptide Metabolism

Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide. Subcellular distribution of activity was 80% soluble and 20% membrane-associated. Aminopeptidases in the two fractions differed in affinity for Ala-4-NA, with Km''s of 0.65 mM (soluble) and 2.90 mM (membrane). Specific activities (units/mg) at pH 7.8, 27°C were 9.10 (soluble) and 14.30 (membrane). Each enzyme was competitively inhibited by amastatin (90% at 100 μM inhibitor, IC₅₀ = 3.7 μM) and inhibited by puromycin (30% at 500 μM) and 1,10-phenanthroline (IC_50''s:; 148 μM, soluble; 89 μM, membrane). Activity was restored by Zn⁺⁺, with maximum recoveries of 50% (soluble) and 90% (membrane), each at 23 μM ZnCl₂. Estimated molecular masses for each were ∼150 kDa. FMRFamide-like neuropeptides behaved as competitive inhibitors. Modification of the N-terminal F of FMRFamide weakened inhibition by 95%, suggesting that the N-terminus is essential for binding to the enzyme. Two nematode FMRFamides, APKPFIRFa and RNKFEFIRFa, were the most potent tested. This is the first biochemical characterization of aminopeptidase in a free-living nematode other than Caenorhabditis elegans and demonstrates the high selectivity of the P. redivivus enzymes for neuropeptide substrates.     

An Intragenic Suppressor of a Calmodulin Mutation in Paramecium: Genetic and Biochemical Characterization

We describe a suppressor of the calmodulin mutant cam1 in Paramecium tetraurelia. The cam1 mutant, which has a SER----PHE change at residue 101 of the third calcium-binding domain, inhibits the activity of the Ca(2+)-dependent K+ current and causes exaggerated behavioral responses to most stimuli. An enrichment scheme, based on an increased sensitivity to Ba2+ in cam1 cells, was used to isolate suppressors. One such suppressor, designated cam101, restores both the activity of the Ca(2+)-dependent K+ current and behavioral responses of the cells. We show that the cam101 mutant is an intragenic suppressor of cam1, based on genetic and microinjection data. The cam101 calmodulin is shown to be similar to wild-type calmodulin in terms of its ability to stimulate calmodulin-dependent phosphodiesterase at low concentrations of free calcium. However, the cam101 calmodulin has a reduced affinity for a monoclonal antibody to wild-type Paramecium calmodulin, as does the parental cam1 calmodulin, and a different mobility on acid-urea gels relative to both wild-type and cam1 calmodulin. We have been able to demonstrate that the isolation of intragenic suppressors of a calmodulin mutation is possible, which allows for the further genetic analysis of structure-function relationships in the calmodulin molecule.     

Purification and Partial Characterization of an Aminopeptidase from Lactobacillus lactis

A surface-bound aminopeptidase of Lactobacillus lactis cells was solubilized with lysozyme, and the extract was subjected to streptomycin sulfate precipitation, ammonium sulfate fractionation, chromatography on Sephadex G-100 and diethylaminoethyl-Sephadex A-50, and preparative polyacrylamide gel electrophoresis. The purified enzyme was homogeneous in disc electrophoretic analysis and consisted of a single polypeptide chain with a molecular weight of 78,000 to 81,000. The optimal pH and optimal temperature for enzyme activity were 6.2 to 7.2 and 47.5 degrees C, respectively, for l-lysine-4-nitroanilide as the substrate. The enzyme was activated by Co and Zn ions and inhibited by Cu, Hg, and Fe ions and by the metal-complexing reagents ethylenediaminetetraacetic acid, 1,10-phenanthroline, and alpha,alpha'-dipyridyl. Higher concentrations of substrate and hydrolysis products also inhibited the activity of the enzyme. The aminopeptidase had broad substrate specificity and hydrolyzed many amino acid arylamides and many peptides with unsubstituted NH(2)-terminal amino acids.     

Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K_m values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.