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BBA - Proteins and Proteomics (v.1814, #10)
Identification of amino acid residues responsible for taurocyamine binding in mitochondrial taurocyamine kinase from Arenicola brasiliensis by Kumiko Tanaka; Tamotsu Matsumoto; Tomohiko Suzuki (pp. 1219-1225).
In order to investigate the residues associated with binding of the substrate taurocyamine in Arenicola mitochondrial taurocyamine kinase (TK), we performed Ala-scanning of the amino acid sequence HTKTV at positions 67–71 on the GS loop, and determined apparent Km and Vmax (app Km and app Vmax, respectively) of the mutant forms for the substrates taurocyamine and glycocyamine. The app Km values for taurocyamine of the K69A, T70A and V71A mutants were significantly increased as compared with wild-type, suggesting that these residues are associated with taurocyamine binding. Of special interest is a property of V71A mutant: its catalytic efficiency for glycocyamine was twice that for taurocyamine, indicating that the V71A mutant acts like a glycocyamine kinase, rather than a TK. The role of the amino acid residue K95 of Arenicola MiTK was also examined. K95 was replaced with R, H, Y, I, A and E. K95R, K95H and K95I have a 3-fold higher affinity for taurocyamine, and activity was largely lost in K95E. On the other hand, the K95Y mutant showed a rather unique feature; namely, an increase in substrate concentration caused a decrease in initial velocity of the reaction (substrate inhibition). This is the first report on the key amino acid residues responsible for taurocyamine binding in mitochondrial TK.► First identification of residues for substrate binding in mitochondrial TK. ► Through Ala scanning, K67, T70 and V71 were shown to be associated with it. ► V71A caused a dramatic change, shifting the enzyme from TK to behaving like a GK. ► Typical substrate inhibition was observed for the K95Y mutant. ► The above inhibition was abolished in the V71A/K95Y double mutant.
Keywords: Abbreviations; TK; taurocyamine kinase; AK; arginine kinase; CK; creatine kinase; GK; glycocyamine kinase; LK; lombricine kinase; HTK; hypotaurocyamine kinase; MiTK; mitochondrial taurocyamine kinase; MiCK; mitochondrial creatine kinase; MiLK; mitochondrial lombricine kinaseTaurocyamine kinase; Glycocyamine kinase; Substrate inhibition; Arenicola; (polychaete)
Role of thiamine pyrophosphate in oligomerisation, functioning and import of peroxisomal 2-hydroxyacyl-CoA lyase by Patrizia Fraccascia; Minne Casteels; Evelyn De Schryver; Paul P. Van Veldhoven (pp. 1226-1233).
During peroxisomal α-oxidation, the CoA-esters of phytanic acid and 2-hydroxylated straight chain fatty acids are cleaved into a (n-1) fatty aldehyde and formyl-CoA by 2-hydroxyacyl-CoA lyase (HACL1). HACL1 is imported into peroxisomes via the PEX5/PTS1 pathway, and so far, it is the only known peroxisomal TPP-dependent enzyme in mammals. In this study, the effect of mutations in the TPP-binding domain of HACL1 on enzyme activity, subcellular localisation and oligomerisation was investigated. Mutations of the aspartate 455 and serine 456 residues within the TPP binding domain of the human HACL1 did not affect the targeting upon expression in transfected CHO cells, although enzyme activity was abolished. Gel filtration of native and mutated N-His6-fusions, expressed in yeast, revealed that the mutations did not influence the oligomerisation of the (apo)enzyme. Subcellular fractionation of yeast cells expressing HACL1 showed that the lyase activity sedimented at high density in a Nycodenz gradient. In these fractions TPP could be measured, but not when mutated HACL1 was expressed, although the recombinant enzyme was still targeted to peroxisomes. These findings indicate that the binding of TPP is not required for peroxisomal targeting and correct folding of HACL1, in contrast to other TPP-dependent enzymes, and suggest that transport of TPP into peroxisomes is dependent on HACL1 import, without requirement of a specific solute transporter.► In this study, the cofactor binding site of human 2-hydroxyacyl-CoA lyase (HACL1), an enzyme involved in alpha-oxidation was investigated. ► Mutational analysis revealed the importance of aspartate 455 and serine 456 for binding TPP and for enzymatic activity. ► Oligomerisation of HACL1 does not require TPP binding. ► Targeting of HACL1 to peroxisomes is not dependent on cofactor binding. ► Transport of TPP into peroxisomes is likely dependent on HACL1 import.
Keywords: Abbreviations; 2-OH-FA; 2-hydroxylated fatty acid; HACL1; 2-hydroxyacyl-CoA lyase 1; HPLC; high performance liquid chromatography; PNS; post nuclear supernatant; PTS; peroxisomal targeting signal; TPP; thiamine pyrophosphateα-oxidation; Peroxisomes; Phytanic acid; Pristanal; Solute transporter; Vitamin B; 1
Aggregation and structural changes of αS1-, β- and κ-caseins induced by homocysteinylation by Yulia Y. Stroylova; Jaroslaw Zimny; Reza Yousefi; Jean-Marc Chobert; Hieronim Jakubowski; Vladimir I. Muronetz; Haertle Thomas Haertlé (pp. 1234-1245).
Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. αS1-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, αS1-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1–3μm. Homocysteinylation of αS1- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400–800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features.► We report unknown previously modifications of bovine caseins potentially relevant in their structural transformations. ► We consider that this report could help in the identification of one of possible biological roles of N-Hcy lactone as inducer of amyloid formation.
Keywords: Abbreviations; ANS; 1-anilino-8-naphthalene sulfonate; beta; -ME; beta-mercaptoethanol; BSA; bovine serum albumin; CD; circular dichroism; CN; casein; DLS; dynamic light scattering; DTNB; 5,5′-dithio-bis (2-nitrobenzoic acid); Hcy; homocysteine; DTT; dithiothreitol; IUP; intrinsically unstructured proteins; MOPS; 3-(N-morpholino) propane sulfonic acid; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ThT; thioflavin TCasein; Aggregation; Homocysteinylation; Amyloid structures; Intrinsically unstructured proteins
The presence of Nε-(Carboxymethyl) lysine in the human epidermis by Keigo Kawabata; Harumi Yoshikawa; Keishi Saruwatari; Yumiko Akazawa; Takafumi Inoue; Tetsuya Kuze; Tetsuya Sayo; Noriko Uchida; Yoshinori Sugiyama (pp. 1246-1252).
It is well known that advanced glycation end products (AGEs) are formed in long-lived dermal proteins such as collagen, and that their formation is related to skin aging. To examine the distribution of AGEs in skin tissue, we performed immunofluorescence studies on the human skin using an anti-AGEs antibody. Interestingly, AGEs signals were observed not only in the dermis but also in the epidermis. The objectives of this study were to confirm the presence of Nε-(Carboxymethyl) lysine (CML), an AGE structure, in the epidermis and to characterize the CML-modified proteins. The presence of CML in the stratum corneum (SC) was examined using liquid chromatography–electrospray ionization time-of-flight mass spectrometry. Concordance between the retention times of a compound in the SC hydrolysate and authentic CML, as well as with the specific mass transition of CML, was detected. This result showed that CML is present in the epidermis. In order to characterize the CML-modified proteins in the epidermis, protein samples extracted from the SC were analyzed using two-dimensional electrophoresis followed by an amino acid sequence analysis. The clarified peptide sequences covered approximately 27% of the amino acid sequences of cytokeratin 10 (K10). In the immunoblotting experiment following the two-dimensional electrophoresis, where protein samples extracted from whole epidermis were used, the position of the major CML-positive spots corresponded to those of K10. Taken together these results showed that CML is present in the human epidermis, and suggest that K10 is one of the target molecules for CML modification in the epidermis.► CML, an AGEs structure, was detected in the human epidermis by immunofluorescence. ► The presence of CML in the epidermis was confirmed using LC-ESI-TOF-MS. ► Cytokeratin10 (K10) was one of the target molecules for CML modification. ► CML-modified K10 may relate to the functional impairment of an aged epidermis.
Keywords: AGEs; CML; LC-ESI-TOF-MS; Epidermis; Cytokeratin 10
Vertebrate slow skeletal muscle actin — Conservation, distribution and conformational flexibility by Robert C.C. Mercer; Wasana A.K.A. Mudalige; Tolulope O. Ige; David H. Heeley (pp. 1253-1260).
The existence of a unique sarcomeric actin is demonstrated in teleosts that possess substantial amounts of slow skeletal muscle in the trunk. The slow skeletal isotype is conserved. There is one amino acid substitution between Atlantic herring slow skeletal actin and the equivalent in salmonids. Conversely, the intra-species variation is considerable; 13 substitutions between different herring skeletal isotypes (slow versus fast). The isomorphisms (non-conservative underlined: residues, 2, 3,103,155, 160, 165,278,281,310, 329, 358,360 and 363) are restricted to sub-domains 1 and 3 and include the substitution Asp-360 in ‘slow’ to Gln in ‘fast’ which results in an electrophoretic shift at alkaline pH. The musculature of the trunk facilitates the preparation of isoactins for biochemical study. Herring slow skeletal G-actin (Ca.ATP) is more susceptible to thermal, and urea, -induced denaturation and subtilisin cleavage than that in fast skeletal, but more stable than the counterpart in salmonids (one substitution, Gln354Ala) highlighting the critical nature of actin's carboxyl-terminal insert. Fluorescent spectra of G-actin isoforms containing the isomorphism Ser155Ala in complexation with 2′-deoxy 3′ O-(N′-Methylanthraniloyl) ATP infer similar polarity of the nucleotide binding cleft. An electrophoretic survey detected two skeletal actins in some (smelt and mackerel) but not all teleosts. One skeletal muscle actin was detected in frog and bird.► Slow skeletal actin is absent in higher vertebrates. ► Slow skeletal actin is absent in higher vertebrates. ► Skeletal actins from Atlantic herring share 13 replacements including Gln360Asp. ► Slow skeletal G-actin (Ca-ATP) is more flexible than fast skeletal actin. ► The isomorphism Ala354Gln alters the Tm by 5 °C.
Keywords: Actin; Slow skeletal muscle; Isomorphism; Conformational flexibility; Cold-adaptation
Steric factors moderate conformational fluidity and contribute to the high proton sensitivity of Root effect hemoglobins by Celia Bonaventura; Robert Henkens; Joel Friedman; Claire J. Parker Siburt; Daniel Kraiter; Alvin L. Crumbliss (pp. 1261-1268).
The structural basis of the extreme pH dependence of oxygen binding to Root effect Hbs is a long-standing puzzle in the field of protein chemistry. A previously unappreciated role of steric factors in the Root effect was revealed by a comparison of pH effects on oxygenation and oxidation processes in human Hb relative to Spot ( Leiostomus xanthurus) and Carp ( Cyprinodon carpio) Hbs. The Root effect confers five-fold increased pH sensitivity to oxygenation of Spot and Carp Hbs relative to Hb A0 in the absence of anionic effectors, and even larger relative elevations of pH sensitivity of oxygenation in the presence of 0.2M phosphate. Remarkably, the Root effect was not evident in the oxidation of the Root effect Hbs. This finding rules out pH-dependent alterations in the thermodynamic properties of the heme iron, measured in the anaerobic oxidation reaction, as the basis of the Root effect. The alternative explanation supported by these results is that the elevated pH sensitivity of oxygenation of Root effect Hbs is attributable to globin-dependent steric effects that alter oxygen affinity by constraining conformational fluidity, but which have little influence on electron exchange via the heme edge. This elegant mode of allosteric control can regulate oxygen affinity within a given quaternary state, in addition to modifying the T–R equilibrium. Evolution of Hb sequences that result in proton-linked steric barriers to heme oxygenation could provide a general mechanism to account for the appearance of the Root effect in the structurally diverse Hbs of many species.► We sought insight into the allosteric controls of Hb oxygenation and oxidation. ► Human and Root effect Hbs differ in pH-sensitivity of oxygenation, not of oxidation. ► Conformational fluidity of key residues affects oxygenation more than oxidation. ► Conformational fluidity moderates O2 binding within T-states of human and fish Hbs. ► Allosteric controls of conformational fluidity underlie Root effect Hb adaptations.
Keywords: Hemoglobin; Root effect; Conformational fluidity; Redox potential; Allostery; Steric controls
The isolated major homology region of the HIV capsid protein is mainly unfolded in solution and binds to the intact protein by Domenech Rosa Doménech; Rebeca Bocanegra; Velazquez-Campoy Adrián Velázquez-Campoy; José L. Neira (pp. 1269-1278).
Assembly of the mature human immunodeficiency virus type 1 (HIV-1) capsid involves the oligomerization of the capsid protein, CA. During retroviral maturation, the CA protein undergoes structural changes and forms exclusive intermolecular interfaces in the mature capsid shell, different from those in the immature precursor. The most conserved region of CA, the major homology region (MHR), is located in the C-terminal domain of CA (CTD). The MHR is involved in both immature and mature virus assembly; however, its exact function during both assembly stages is unknown. To test its conformational preferences and to provide clues on its role during CA assembly, we have used a minimalist approach by designing a peptide comprising the whole MHR (MHRpep, residues Asp152 to Ala174). Isolated MHRpep is mainly unfolded in aqueous solution, with residual structure at its C terminus. MHRpep binds to monomeric CTD with an affinity of ~30μM (as shown by fluorescence and ITC); the CTD binding region comprises residues belonging to α-helices 10 and 11. In the immature virus capsid, the MHR and α-helix 11 regions of two CTD dimers also interact [Briggs JAG, Riches JD, Glass B, Baratonova V, Zanetti G and Kräusslich H-G (2009) Proc. Natl. Acad. Sci. USA 106, 11090–11095]. These results can be considered a proof-of-concept that the conformational preferences and binding features of isolated peptides derived from virus proteins could be used to mimic early stages of virus assembly.Display Omitted► The isolated MHR from HIV-1 in aqueous solution is mainly disordered. ► MHR binds to the C-terminal domain (CTD) of the capsid protein with KL ~30μM. ► The CTD region involved in MHR binding is formed by α-helices 10 and 11. ► In the immature capsid, α-helix 11 and the MHR of two CTD molecules interact. ► Peptide binding could be a model of the early stages of viral assembly.
Keywords: Abbreviations; CA; capsid protein of HIV-1; CD; circular dichroism; CTD; C-terminal domain of CA; CTDW184A; monomeric mutant of CTD with an alanine at the position 184; EM; electron microscopy; HSQC; heteronuclear single quantum coherence; ITC; isothermal titration calorimetry; MA; matrix protein; MD; molecular dynamics; MHR; major homology region; MHRpep; the peptide comprising residues Asp152 to Ala174 of CA; NC; nucleocapsid protein; NMR; nuclear magnetic resonance; NTD; N-terminal domain of CA; T; m; thermal-denaturation midpoint; TFE; 2,2,2-trifluoroethanol; TSP; sodium trimethylsilyl-[2,2,3,3-; 2; H; 4; ]-propionate; UV; ultravioletPeptide; Folding; Structure; Fluorescence; ITC; Binding
The kinetics of molecular oxygen migration in the isolated α chains of human hemoglobin as revealed by molecular dynamics simulations and laser kinetic spectroscopy by Sergei V. Lepeshkevich; Sergey A. Biziuk; Alexander M. Lemeza; Boris M. Dzhagarov (pp. 1279-1288).
Bimolecular and geminate molecular oxygen (O2) rebinding to isolated α chains of human adult hemoglobin in solutions is analyzed. Multiple extended molecular dynamics (MD) simulations of the O2 migration within the protein after dissociation are described. Computational modeling is exploited to identify hydrophobic pockets within the αchains and internal O2 migration pathways associated with the experimentally observed ligand rebinding kinetics. To initiate dissociation, trajectories of the liganded protein are interrupted, the iron–dioxygen bond is broken, and the parameters of the iron–nitrogen bonds are simultaneously altered to produce a deoxyheme conformation. MD simulations provide 140 essentially independent trajectories (up to 25-ns long) of the O2 migration in the protein. The time dependence of cavities occupancy, obtained by the MD simulations, and the kinetics of O2 rebinding, measured by flash-photolysis techniques, allow us to obtain the kinetics of the entire O2 migration process within the nanosecond time range and construct an explicit kinetic model of the O2 migration and rebinding process. The amino acids that have the most pronounced effect on the ligand migration within the α chain matrix are predicted.Display Omitted► Bimolecular and geminate O2 rebinding to α chains of human hemoglobin in solutions is analyzed. ► Multiple extended molecular dynamics simulations of the O2 migration within the protein after dissociation are described. ► The kinetics of the entire O2 migration process within the nanosecond time range were obtained. ► An explicit kinetic model of the O2 migration and rebinding process was constructed.
Keywords: Abbreviations; HbA; human hemoglobin; O; 2; molecular oxygen; Mb; myoglobin; CO; carbon monoxide; MD; molecular dynamics; MEM; maximum entropy method; BR; bimolecular rebinding; GR; geminate rebinding; RMSD; root mean square deviationHuman hemoglobin; Photodissociation; Geminate and bimolecular recombination; Molecular dynamic simulation
Studies on the parameters controlling the stability of the TET peptidase superstructure from Pyrococcus horikoshii revealed a crucial role of pH and catalytic metals in the oligomerization process by Eva Rosenbaum; Mylène Ferruit; Dura M. Asunción Durá; Bruno Franzetti (pp. 1289-1294).
The TET proteases from Pyrococcus horikoshii are metallopeptidases that form large dodecameric particles with high thermal stability. The influence of various physico-chemical parameters on PhTET3 quaternary structure was investigated. Analytical ultracentrifugation and biochemical analyses showed that the PhTET3 quaternary structure and enzymatic activity are maintained in high salt and that the complex is stable under extreme acidic conditions. Under basic pH conditions the complex disassembled into a low molecular weight species that was identified as folded dimer. Metal analyses showed that the purified enzyme only contains two equivalent of zinc per monomer, corresponding to the metal ions responsible for catalytic activity. When these metals were removed by EDTA treatment, the complex dissociated into the same dimeric species as those observed at high pH. Dodecameric TET particles were obtained from the metal free dimers when 2mM of divalent ions were added to the protein samples. Most of the dimers remained assembled at high temperature. Thus, we have shown that dimers are the building units in the TET oligomerization pathway and that the active site metals are essential in this process.► Dimers are the building blocks of the dodecameric TET protease. ► The co-catalytic metal ions are essential for the oligomerization of the dimers. ► TET dimers assemble in an incremental manner to form the tetrahedral complex.
Keywords: Aminopeptidase; Intracellular proteolysis; Large molecular complexes; Quaternary structure assembling; Metal binding; Hyperthermophiles
Molecular dissection of Streptomyces trypsin on substrate recognition by Yoshiko Uesugi; Hirokazu Usuki; Jiro Arima; Masaki Iwabuchi; Tadashi Hatanaka (pp. 1295-1304).
We recently identified residue 71 of two homologous serine proteases from Streptomyces omiyaensis (SOT) and Streptomyces griseus (SGT) as a crucial residue for differences in their topological specificities, i.e. recognition of a distinct three-dimensional structure. To study the role of this key residue in substrate recognition, we used surface plasmon resonance analysis to evaluate the affinities of inactive mutants, in which residues 71 of SOT and SGT were substituted respectively with Leu and Tyr, toward different types of collagens. We identified another amino acid residue involved in the interaction with collagens from analyses of inactive chimeras between SOT and SGT using an in vivo DNA shuffling system. Results showed that residue 72 contributes to collagen binding. By substituting Leu71 and Gln72 with Tyr and Arg, respectively, SGT mutant showed a change in topological specificity and high hydrolytic activity toward type IV collagen comparable to SOT. We demonstrated that the neighboring residues 71 and 72 in the N-terminal β-barrel domain of the enzyme synergistically play an important role in substrate recognition.Display Omitted► Residue 71 of Streptomyces trypsin is crucial for topological specificities. ► Residue 72 contributes to collagen binding. ► These residues synergistically play an important role in substrate recognition.
Keywords: Abbreviations; SOT; Streptomyces omiyaensis; serine protease; SGT; Streptomyces griseus; trypsin; SPR; surface plasmon resonance; RU; resonance unit; CD; circular dichroism; RIBS; repeat-length independent and broad spectrumSerine protease; Substrate recognition; Surface plasmon resonance; Collagen binding; RIBS; in vivo; DNA shuffling
Dynamics of polymerization shed light on the mechanisms that lead to multiple amyloid structures of the prion protein by Maria-Teresa Alvarez-Martinez; Pascaline Fontes; Viviana Zomosa-Signoret; Jacques-Damien Arnaud; Erwan Hingant; Laurent Pujo-Menjouet; Jean-Pierre Liautard (pp. 1305-1317).
It is generally accepted that spongiform encephalopathies result from the aggregation into amyloid of a ubiquitous protein, the so-called prion protein. As a consequence, the dynamics of amyloid formation should explain the characteristics of the prion diseases: infectivity as well as sporadic and genetic occurrence, long incubation time, species barriers and strain specificities. The success of this amyloid hypothesis is due to the good qualitative agreement of this hypothesis with the observations. However, a number of difficulties appeared when comparing quantitatively the in vitro experimental results with the theoretical models, suggesting that some differences should hide important discrepancies. We used well defined quantitative models to analyze the experimental results obtained by in vitro polymerization of the recombinant hamster prion protein. Although the dynamics of polymerization resembles a simple nucleus-dependent fibrillogenesis, neither the initial concentration dependence nor off-pathway hypothesis fit with experimental results. Furthermore, seeded polymerization starts after a long time delay suggesting the existence of a specific mechanism that takes place before nucleus formation. On the other hand, polymerization dynamics reveals a highly stochastic mechanism, the origin of which appears to be caused by nucleation heterogeneity. Moreover, the specific structures generated during nucleation are maintained during successive seeding although a clear improvement of the dynamics parameters (polymerization rate and lag time) is observed. We propose that an additional on-pathway reaction takes place before nucleation and it is responsible for the heterogeneity of structures produced during prion protein polymerization in vitro. These amyloid structures behave like prion strains. A model is proposed to explain the genesis of heterogeneity among prion amyloid.► Dynamics of polymerization do not fit simple nucleus-dependent fibrillogenesis. ► Neither initial concentration dependency nor off-pathway hypothesis fit results. ► Seeded polymerization starts after a long delay time. ► Conformational heterogeneity takes place before nucleus formation. ► Heterogeneous polymer structures emerged from this molecular event.
Keywords: Abbreviations; ATF; Atomic Force; CD; Circular Dichroism; FACS; Fluorescence Activated Cells Sorting; GdnCl; Guanidium Chloride; PrP; Prion protein; rPrP; recombinant prion protein; FTIR; Fourier-transform infrared spectroscopy; ThT; Thioflavin-TPrion; Amyloid; Heterogeneity; Strain; Dynamics
Transient model of thermal deactivation of enzymes by Nelson G. Chen; Kalvin Gregory; Ye Sun; Val Golovlev (pp. 1318-1324).
The kinetics of enzyme deactivation provide useful insights on processes that determine the level of biological function of any enzyme. Photinus pyralis (firefly) luciferase is a convenient enzyme system for studying mechanisms and kinetics of enzyme deactivation, refolding, and denaturation caused by various external factors, physical or chemical by nature. In this report we present a study of luciferase deactivation caused by increased temperature (i.e., thermal deactivation). We found that deactivation occurs through a reversible intermediate state and can be described by a Transient model that includes active and reversibly inactive states. The model can be used as a general framework for analysis of complex, multiexponential transient kinetics that can be observed for some enzymes by reaction progression assays. In this study the Transient model has been used to develop an analytical model for studying a time course of luciferase deactivation. The model might be applicable toward enzymes in general and can be used to determine if the enzyme exposed to external factors, physical or chemical by nature, undergoes structural transformation consistent with thermal mechanisms of deactivation.►Enzyme deactivation upon heating proceeds in multiple steps ►Deactivation is rapid, when compared to denaturation ►Transient model to describe sequential changes — different from Equilibrium model
Keywords: Enzyme; Deactivation; Denaturation; Heating; Protein
RNA-dependent RNA polymerases from different hepatitis C virus genotypes reveal distinct biochemical properties and drug susceptibilities by Marina M. May; Heike Lorengel; Joerg Kreuter; Holger Zimmermann; Helga Ruebsamen-Schaeff; Andreas Urban (pp. 1325-1332).
The RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is the key enzyme for viral replication, recognized as one of the promising targets for antiviral intervention. Several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GTs). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. This work gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1–6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg2+ were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a>2a, 3a, 4a, 5a>1a. Furthermore, the optimized assay conditions for genotypic profiling were confirmed by testing the inhibitory activity of 4 known prototype NNIs addressing the NNI binding sites 1 to 4.► Biochemical properties for optimal NS5B activity were investigated for all HCV GT. ► Significant differences in optimal cofactor Mg2+ concentration were observed. ► Substrate affinities of NS5B were found to be very similar across different GT.► Differences in spec. act. of purified NS5Bs with highest activities for GT 1b and 6a.► GT profiles of 4 NNIs were investigated. Only benzofuran stood out with a broad coverage.
Keywords: Abbreviations; HCV; hepatitis C virus; GT; genotype; NS5B; non-structure protein 5B (RNA-dependent RNA polymerase); NNI; non-nucleoside polymerase inhibitorHCV; NS5B; Genotype; RNA polymerase; Non nucleoside inhibitor
Enzymatic characterization of a serralysin-like metalloprotease from the entomopathogen bacterium, Xenorhabdus by Mustafa K. Massaoud; Marokhazi Judit Marokházi; István Venekei (pp. 1333-1339).
We investigated the enzymatic properties of a serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest kcat/ KM value. The pH profile of activity, similarly to other serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu2+ and Co2+ ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn2+ was inhibitory (both are unusual among serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn2+, Cu2+ and Co2+ up to 90–200% of its original value, while Zn2+ was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the serralysins contain Asn.► Some properties of Xenorhabdus PrtA protease are rare among serralysins. ► It cleaves next to Lys, Arg in P4-P1 peptides but next to Ser in a P4-P4’ peptide.► It is inhibited by Zn2+ and is less sensitive to inhibition by EDTA.► Its activity is enhanced by Cu2+ and Co2+ and rescued by Cu, Co, Mn but not by Zn ion.► The unusual effect of Zn2+ and EDTA can be explained by an Asn191Asp substitution.
Keywords: Abbreviations; pNA; paranitroanilide; AMC; aminomethylcoumarine; Dabcyl; 4-[4′(dimethylamino)phenylazo]benzoyl; Edans; 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; Succ; succinyl; SBzl; thiobenzyl; BOC; butyloxycarbonyl; CBZ; carbobenzoxy; DTT; dithiotreitol; Fua; furylacryloyl; pNA; para-nitroanilide; SBTI; soy bean trypsin inhibitor; PMSF; phenylmethanesulfonyl fluoride; CPS; count per secondSerralysin; PrtA peptidase; Metalloprotease; Zn-inhibition; Xenorhabdus; Photorhabdus
Sulforaphane induces apoptosis in human hepatic cancer cells through inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase4, mediated by hypoxia inducible factor-1-dependent pathway by Young Keul Jeon; Dong Ryeol Yoo; Yun Ho Jang; Se Young Jang; Myeong Jin Nam (pp. 1340-1348).
The anti-cancer activity of sulforaphane (SFN) has recently been investigated in several cancer cell lines, including human hepatic cancers. However, the mechanism of SFN-induced cell death in human hepatic cancer cells is still not well understood. The aim of the present work is to explore the possible mechanisms of SFN-induced apoptosis in hepatocellular carcinoma cells using proteomic analysis. A two-dimensional electrophoresis (2-DE)-based-proteomic analysis was employed for identification of possible target-related proteins of SFN-induced apoptosis. Among eleven proteins identified as regulated, we focused on the down-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase4 (PFKFB4) protein, which has been known as a key modulator of glycolysis. We also showed that SFN down-regulated the expression of the transcriptional factor, hypoxia inducible factor-1α (HIF-1α), which strongly regulates PFKFB4 expression. In order to obtain a broad understanding of the correlation of HIF-1α and SFN, we observed the inhibition of the activity of mitogen-activated protein kinases, regulators of HIF-1α activity. Our findings suggest that SFN is a potent inducer of apoptosis in hepatocellular carcinoma cells via PFKFB4-inhibition pathways. HIF-1 pathway inhibition may be mediated by the inhibition of mitogen-activated protein kinases.► We investigated an anti-cancer activity of sulforaphane (SFN) and its mechanisms. ► SFN induced apoptosis on huh-7 cells in dose- and time-dependent manners. ► We identified proteins related to SFN-induced apoptosis using proteomic analysis. ► The apoptotic mechanism might involve modulation of PFKFB-associated glycolysis. ► PFKFB-associated glycolysis was regulated through a HIF-1α-dependent pathway.
Keywords: Sulforaphane; Proteomic analysis; 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatases; Hypoxia-inducible factor-1; Mitogen-activated protein kinase
Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase by Sandeep Kumar Srivastava; Kalagiri Rajasree; B. Gopal (pp. 1349-1357).
Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug—an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme.► The structures of S. aureus NDK and substrate complexes were determined. ► The phosphotransfer mechanism and oligomerization were rationalized by MD simulations. ► The structure and MD simulations reveal the role of the active site serine residue. ► These structures provide a basis for the design of S. aureus specific NDK inhibitors.
Keywords: Nucleoside kinase; Substrate specificity; Phosphorylation; Phosphotransfer
Unraveling the structural and functional differences between purine nucleoside phosphorylase and 5′-deoxy-5′-methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus by Giovanna Cacciapuoti; Anna Marabotti; Francesca Fuccio; Marina Porcelli (pp. 1358-1366).
Purine nucleoside metabolism in the archaeon Pyrococcus furiosus is catalyzed by purine nucleoside phosphorylase (PfPNP) and 5′-deoxy-5′-methylthioadenosine phosphorylase (PfMTAP). These enzymes, characterized by 50% amino acid sequence identity, show non-common features of thermophilicity and thermostability and are stabilized by intramolecular disulfide bonds. PfPNP is highly specific for 6-oxopurine nucleosides while PfMTAP is characterized by a broad substrate specificity with 6-aminopurine nucleosides as preferred substrates. Amino acid sequence comparison clearly shows that the hypothetical active sites of PfPNP and PfMTAP are almost identical and that, in analogy with human 5′-deoxy-5′-methylthioadenosine phosphorylase and human purine nucleoside phosphorylase, residue changes at level of the same crucial positions could be responsible for the switch of substrate specificity. To validate this hypothesis we changed the putative active site of PfPNP by site-directed mutagenesis. Substrate specificity and catalytic efficiency of PfPNP mutants were then analyzed by kinetic studies and compared with the wild-type enzyme. We carried out the molecular modeling of PfPNP and PfMTAP to obtain a picture of the overall enzyme structure and to identify structural features as well as interactions playing critical roles in thermostability. Finally, we utilized the structural models of mutant enzyme–substrate complex to rationalize the functional effects of the mutations.► We elucidated PfPNP and PfMTAP structure by molecular modelling. ► We identified structural features playing critical roles in thermostability. ► We performed kinetic analysis of PfPNP mutants. ► We performed structural analysis of mutant enzyme–substrate complex. ► We proposed that in PfPNP a protonated Glu-169 acts as the main catalytic residue.
Keywords: Abbreviations; PNP; purine nucleoside phosphorylase; hPNP; human purine nucleoside phosphorylase; PfPNP; purine nucleoside phosphorylase from; Pyrococcus furiosus; PfMTAP; 5′-deoxy-5′-methylthioadenosine phosphorylase from; Pyrococcus furiosus; hMTAP; human 5′-deoxy-5′-methylthioadenosine phosphorylase; SsMTAPII; 5′-deoxy-5′-methylthioadenosine phosphorylase II from; Sulfolobus solfataricus; MTA; 5′-deoxy-5′-methylthioadenosinePurine nucleoside phosphorylase; 5′-deoxy-5′-methylthioadenosine phosphorylase; Pyrococcus furiosus; Site-directed mutagenesis; Substrate specificity
Gel-based proteomics of liver cancer progression in rat by Jakob Albrethsen; Leah M. Miller; Phyllis M. Novikoff; Ruth H. Angeletti (pp. 1367-1376).
A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control “sham-operated” liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.► We analyze a rat model of liver cancer progression by 2D DIGE. ► We pinpoint proteomic aberrations in liver cancer versus precursor liver tissues. ► By 2DE we study protein post-translational modification and microtubule-association.
Keywords: Liver cancer; Proteomics; Biomarker; HSP60; Post-translational modification; Inflammation
The ribonucleolytic activity of the ribotoxin α-sarcin is not essential for in vitro protein biosynthesis inhibition by Alvarez-Garcia Elisa Álvarez-García; Elizabeth Diago-Navarro; Herrero-Galan Elías Herrero-Galán; Garcia-Ortega Lucía García-Ortega; Lopez-Villarejo Juan López-Villarejo; Nieves Olmo; Diaz-Orejas Ramón Díaz-Orejas; José G. Gavilanes; Martinez-del-Pozo Álvaro Martínez-del-Pozo (pp. 1377-1382).
Fungal ribotoxins are toxic secreted ribonucleases that cleave a conserved single phosphodiester bond located at the sarcin/ricin loop of the larger rRNA. This cleavage inactivates ribosomes leading to protein biosynthesis inhibition and cell death. It has been proposed that interactions other than those found at the active site of ribotoxins are needed to explain their exquisite specific activity. The study presented shows the ability of a catalytically inactive α-sarcin mutant (H137Q) to bind eukaryotic ribosomes and interfere with in vitro protein biosynthesis. The results obtained are compatible with previous observations that α-sarcin can promote cell death by a mechanism that is independent of rRNA cleavage, expanding the potential set of activities performed by this family of toxins.► Ribotoxins inactivate ribosomes by cleaving a conserved single phosphodiester bond. ► Ribosome inactivation leads to protein biosynthesis inhibition and cell death. ► Catalytically inactive α-sarcin mutant H137Q inhibits in vitro protein synthesis. ► Wild-type and H137Q α-sarcin bind to and cosediment with ribosomes. ► Ribotoxins’ binding to ribosomes seems to suffice for impairing protein synthesis.
Keywords: Abbreviations; DEPC; Diethyl pyrocarbonate; H137Q; a mutant version of α-sarcin where His-137 has been substituted by Gln; PMSF; phenylmethylsulfonyl fluoride; polyPhe; polyphenylalanine; RNase; ribonuclease; SRL; sarcin/ricin loop; YPD; 1.0% yeast-extract–2.0% peptone–2.0% dextrose mediumRibotoxin; Restrictocin; Ribosome; Protein-synthesis
Biophysical characterization of α-amylase inhibitor Parvulustat (Z-2685) and comparison with Tendamistat (HOE-467) by Sokocevic Alma Sokočević; Sigeng Han; Joachim W. Engels (pp. 1383-1393).
Parvulustat is a small, highly active proteinaceous α-amylase inhibitor whose high-resolution NMR structure was recently solved in Frankfurt. Here, we present its biochemical and biophysical characterization. Several spectroscopic methods such as UV, fluorescence and CD were utilized to extract conformational changes upon modification of pH, temperature and chemical denaturant. Parvulustat revealed native like behavior over a wide range of denaturizing agents as reflected in terms of activity and thermodynamic data. In addition, spectroscopic and thermodynamic properties of Parvulustat were compared to the well-characterized Tendamistat. Despite the overall structural similarity, the thermodynamic stability of the two proteins is different. Our analysis led to the conclusion that Parvulustat is even more stable than Tendamistat. Furthermore, investigations on three C-terminally truncated Parvulustat derivatives indicate that the higher stability is caused by the long flexible C-terminus.► Parvulustat is a very potent-amylase inhibitor (Ki 2.8x10-11 M) over a wide range of pH. ► Native like structure is found for denaturizing agents aslow pH and high temperature. ► Its very high stability is mostly caused by the c-terminus and hydrophobic core. ► Parvulustat is an excellent model protein for studies on oxidative folding of-sheet proteins.
Keywords: α-amylase inhibitor; Tendamistat; Parvulustat; Protein stability; C-Terminus; CD-spectroscopy
pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor by Nicole Howe; Mariangela Ceruso; Edward Spink; J. Paul G. Malthouse (pp. 1394-1403).
The stromelysin-1 catalytic domain83–247 (SCD) is stable for at least 16h at pHs 6.0–8.4. At pHs 5.0 and 9.0 there is exponential irreversible denaturation with half lives of 38 and 68min respectively. At pHs 4.5 and 10.0 irreversible denaturation is biphasic. At 25°C, C-terminal truncation of stromelysin-1 decreases the stability of the stromelysin-1 catalytic domain at pH values >8.4 and <6.0. We describe the conversion of the carboxylate group of (β R)-β-[[[(1 S)-1-[[[(1 S)-2-Methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1′-biphenyl]-4-hexanoic acid (UK-370106-COOH) a potent inhibitor of the metalloprotease stromelysin-1 to a glyoxal group (UK-370106-CO13CHO). At pH 5.5–6.5 the glyoxal inhibitor is a potent inhibitor of stromelysin-1 (Ki=~1μM). The aldehyde carbon of the glyoxal inhibitor was enriched with carbon-13 and using carbon-13 NMR we show that the glyoxal aldehyde carbon is fully hydrated when it is in aqueous solutions (90.4ppm) and also when it is bound to SCD (~92.0ppm). We conclude that the hemiacetal hydroxyl groups of the glyoxal inhibitor are not ionised when the glyoxal inhibitor is bound to SCD. The free enzyme pKa values associated with inhibitor binding were 5.9 and 6.2. The formation and breakdown of the signal at ~92ppm due to the bound UK-370106-CO13CHO inhibitor depends on pKa values of 5.8 and 7.8 respectively. No strong hydrogen bonds are present in free SCD or in SCD–inhibitor complexes. We conclude that the inhibitor glyoxal group is not directly coordinated to the catalytic zinc atom of SCD.► Stromelysin-1 catalytic domain83–247 is stable for at least 16 hours at pHs 6.0–8.4. ► C-terminal truncation of stromelysin-1 decreases its pH stability. ► A potent13C-enriched glyoxal inhibitor of stromelysin-1 has been synthesised. ► The inhibitor glyoxal aldehyde group is fully hydrated when bound to SCD. ► The glyoxal group is not directly coordinated to the catalytic zinc of SCD.
Keywords: Abbreviations; UK-370106-COOH; (β; R; )-β-[[[(1; S; )-1-[[[(1; S; )-2-Methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1′-biphenyl]-4-hexanoic acid; ZBG; zinc-binding group; SCD; stromelysin-1 catalytic domain; 83–247; MCA; (7-methoxycoumarin-4-yl)acetyl; Dnp; 2,4 dinitrophenylpKa; Metalloprotease; Tetrahedral intermediate; Glyoxal inhibitor; pH stability