bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural analyses reveal two distinct families of nucleoside phosphorylases
Authors: PUGMIRE Matthew J. , EALICK Steven E.
Published: August 2015

The reversible phosphorolysis of purine and pyrimidine nucleosides is an important biochemical reaction in the salvage pathway, which provides an alternative to the de novo purine and pyrimidine biosynthetic pathways. Structural studies in our laboratory and by others have revealed that only two folds exist that catalyse the phosphorolysis of all nucleosides, and provide the basis for defining two families of nucleoside phosphorylases. The first family (nucleoside phosphorylase-I) includes enzymes that share a common single-domain subunit, with either a trimeric or a hexameric quaternary structure, and accept a range of both purine and pyrimidine nucleoside substrates. Despite differences in substrate specificity, amino acid sequence and quaternary structure, all members of this family share a characteristic subunit topology. We have also carried out a sequence motif study that identified regions of the common subunit fold that are functionally significant in differentiating the various members of the nucleoside phosphorylase-I family. Although the substrate-binding sites are arranged similarly for all members of the nucleoside phosphorylase-I family, a comparison of the active sites from the known structures of this family indicates significant differences between the trimeric and hexameric family members. Sequence comparisons also suggest structural identity between the nucleoside phosphorylase-I family and both 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase and AMP nucleosidase. Members of the second family of nucleoside phosphorylases (nucleoside phosphorylase-II) share a common two-domain subunit fold and a dimeric quaternary structure, share a significant level of sequence identity (>30%) and are specific for pyrimidine nucleosides. Members of this second family accept both thymidine and uridine substrates in lower organisms, but are specific for thymidine in mammals and other higher organisms. A possible relationship between nucleoside phosphorylase-II and anthranilate phosphoribosyltransferase has been identified through sequence comparisons. Initial studies in our laboratory suggested that members of the nucleoside phosphorylase-II family require significant domain movements in order for catalysis to proceed. A series of recent structures has confirmed our hypothesis and provided details of these conformational changes. Structural studies of the nucleoside phosphorylases have resulted in a wealth of information that begins to address fundamental biological questions, such as how Nature makes use of the intricate relationships between structure and function, and how biological processes have evolved over time. In addition, the therapeutic potential of suppressing the nucleoside phosphorylase activity in either family of enzymes has motivated efforts to design potent inhibitors. Several research groups have synthesized a variety of nucleoside phosphorylase inhibitors that are at various stages of preclinical and clinical evaluation.
The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins
Authors: OBERER Monika , ZANGGER Klaus , PRYTULLA Stefan , KELLER Walter
Published: August 2015

NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2. ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter PparDE and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE. Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and 3JHα-NH scalar coupling constants showed that the N-terminal domain of ParD consists of a short β-ribbon followed by three α-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors. 15N longitudinal (T1) and transverse (T2) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain. An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein. This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins.
Adenophostin A and ribophostin, but not inositol 1,4,5-trisphosphate or manno-adenophostin, activate the Ca2+ release-activated Ca2+ current, ICRAC, in weak intracellular Ca2+ buffer
Authors: PAREKH Anant B. , RILEY Andrew M. , POTTER Barry V. L.
Published: August 2015

Under physiological conditions of weak intracellular Ca2+ buffering (0.1mM EGTA), the second messenger Ins(1,4,5)P3 often fails to activate any detectable store-operated Ca2+ current. However, it has been reported that the fungal metabolite adenophostin A [which has a severalfold higher affinity than Ins(1,4,5)P3 for Ins(1,4,5)P3 receptors] consistently activates the current under similar conditions. Here, whole-cell patch clamp experiments have been performed to examine how adenophostin A can activate the store-operated Ca2+ current (ICRAC) in RBL-1 (rat basophilic leukaemia) cells. In a strong intracellular Ca2+ buffer, saturating concentrations of adenophostin A activated ICRAC maximally and the current amplitude and kinetics were indistinguishable from those obtained with high concentrations of Ins(1,4,5)P3. In a weak Ca2+ buffer, adenophostin A consistently activated ICRAC, but the current was submaximal. High concentrations of Ins(1,4,5)P3 or the non-metabolizable analogue Ins(2,4,5)P3 were largely ineffective under these conditions. The size of ICRAC to adenophostin A in weak Ca2+ buffer could be significantly increased by either inhibiting sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (‘SERCA’) pumps with thapsi-gargin or enhancing mitochondrial Ca2+ uptake, although blocking the mitochondrial Ca2+ uniporter with Ruthenium Red did not suppress the activation of the current. Changing the levels of free ATP in the recording pipette did not enhance the size of ICRAC evoked by adenophostin A. We also examined two structurally distinct analogues of adenophostin A (manno-adenophostin and ribophostin), for which the affinities for the Ins(1,4,5)P3 receptor are similar to that of Ins(1,4,5)P3 in equilibrium binding experiments. Although these analogues were able to activate ICRAC to its maximal extent in strong buffer, ribophostin, but not manno-adenophostin, consistently activated the current in weak buffer. We conclude that adenophostin A and ribophostin are able to activate ICRAC in weak buffer through a mechanism that is quite distinct from that employed by Ins(1,4,5)P3 and manno-adenophostin and is not related to equilibrium affinities.
Cloning and expression of a novel UDP-GlcNAc:α-d-mannoside β1,2-N-acetylglucosaminyltransferase homologous to UDP-GlcNAc:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I
Authors: ZHANG Wenli , BETEL Doron , SCHACHTER Harry
Published: August 2015

A TBLASTN search with human UDP-GlcNAc:α-3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) as a probe identified human and mouse Unigenes encoding a protein similar to human GnT I (34% identity over 340 amino acids). The recombinant protein converted Man(α1–6)[Man(α1–3)]Man(β1-)O-octyl to Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1-)O-octyl, the reaction catalysed by GnT I. The enzyme also added GlcNAc to Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1-)O-octyl (the substrate for β-1,2-N-acetylglucosaminyltransferase II), Man(α1-)O-benzyl [with Km values of ≈ 0.3 and > 30mM for UDP-GlcNAc and Man(α1-)O-benzyl respectively] and the glycopeptide CYA[Man(α1-)O-T]AV (Km ∼ 12mM). The product formed with Man(α1-)O-benzyl was identified as GlcNAc(β1–2)Man(α1-)O-benzyl by proton NMR spectroscopy. The enzyme was named UDP-GlcNAc:α-d-mannoside β-1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). The human gene mapped to chromosome 1. Northern-blot analysis showed a 3.3kb message with a wide tissue distribution. The cDNA has a 1980bp open reading frame encoding a 660 amino acid protein with a type-2 domain structure typical of glycosyltransferases. Man(β1-)O-octyl, Man(β1-)O-p-nitrophenyl and GlcNAc(β1–2)Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1–4)GlcNAc(β1–4)GlcNAc(β1-)O-Asn were not acceptors, indicating that GnT I.2 is specific for α-linked terminal Man and does not have N-acetylglucosaminyltransferase III, IV, V, VII or VIII activities. CYA[Man(α1-)O-T]AV was between three and seven times more effective as an acceptor than the other substrates, suggesting that GnT I.2 may be responsible for the synthesis of the GlcNAc(β1–2)Man(α1-)O-Ser/Thr moiety on α-dystroglycan and other O-mannosylated proteins.
Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes
Authors: FORGET Marie-Annick , DESROSIERS Richard R. , GINGRAS Denis , BÉLIVEAU Richard
Published: August 2015

The Rho GDP dissociation inhibitor (RhoGDI) regulates the activation—inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by extracting them from the membrane. To study the roles of Mg2+, phosphatidylinositol 4,5-bisphosphate (PIP2), ionic strength and phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed a new, efficient and reliable method to produce prenylated Rho proteins using the yeast Saccharomyces cerevisiae. It has been previously reported that protein kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from membranes by RhoGDI [Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and Bertoglio (1996) EMBO J. 16, 510–519]. In the present study, we used an in vitro affinity chromatography system to show that phosphorylation of RhoA and Cdc42 significantly increased their interaction with RhoGDI under physiological conditions of ionic strength. This increase was independent of the nucleotide (GDP or guanosine 5′-[γ-thio]triphosphate) loaded on to the Rho proteins, as well as of Mg2+ and PIP2. Moreover, dephosphorylation of rat brain membranes by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42 by RhoGDI. Subsequent re-phosphorylation by PKA restored the extraction levels, indicating the reversibility of this process. These results clearly demonstrate that the phosphorylation states of Cdc42 and RhoA regulate their interactions with RhoGDI and, consequently, their extraction from rat brain membranes. We therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and RhoA activity that is independent of GDP—GTP cycling.
Phosphorylation of the protein kinase C-theta activation loop and hydrophobic motif regulates its kinase activity, but only activation loop phosphorylation is critical to in vivo nuclear-factor-κB induction
Authors: LIU Yin , GRAHAM Caroline , LI Aiqun , FISHER Robert J. , SHAW Stephen
Published: August 2015

Protein kinase C (PKC)-theta, a member of the ‘novel’ subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for ‘classical’ PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr538), turn motif (Ser676) and hydrophobic motif (Ser695). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (>100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical invivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor κB (NF-κB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-κB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-κB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive phosphorylation of its activation loop.
Sarcolipin uncouples hydrolysis of ATP from accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum
Authors: SMITH Wendy S. , BROADBRIDGE Robert , EAST J. Malcolm , LEE Anthony G.
Published: August 2015

Sarcolipin (SLN) is a small peptide found in the sarcoplasmic reticulum of skeletal muscle. It is predicted to contain a single hydrophobic transmembrane α-helix. Fluorescence emission spectra for the single Trp residue of SLN suggest that SLN incorporates fully into bilayers of dioleoylphosphatidylcholine, but only partially into bilayers of phosphatidylcholines with long (C22 or C24) fatty acyl chains. The fluorescence of SLN is quenched in bilayers of dibromostearoylphosphatidylcholine, also consistent with incorporation into the lipid bilayer. SLN was reconstituted with the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum. Even at a 50:1 molar ratio of SLN/ATPase, SLN had no significant effect on the rate of ATP hydrolysis by the ATPase or on the Ca2+-dependence of ATP hydrolysis. However, at a molar ratio of SLN/ATPase of 2:1 or higher the presence of SLN resulted in a marked decrease in the level of accumulation of Ca2+ by reconstituted vesicles. The effect of SLN was structurally specific and did not result from a breakdown in the vesicular structure or from the formation of non-specific ion channels. Vesicles were impermeable to Ca2+ in the absence of ATP in the external medium. The effects of SLN on accumulation of Ca2+ can be simulated assuming that SLN increases the rate of slippage on the ATPase and the rate of passive leak of Ca2+ mediated by the ATPase. It is suggested that the presence of SLN could be important in non-shivering thermogenesis, a process in which heat is generated by hydrolysis of ATP by skeletal-muscle sarcoplasmic reticulum.
Signal- and importin-dependent nuclear targeting of the kidney anion exchanger 1-binding protein kanadaptin
Authors: HÜBNER Stefan , JANS David A. , XIAO Chong-Yun , JOHN Anna P. , DRENCKHAHN Detlev
Published: August 2015

Kanadaptin (kidney anion exchanger adaptoror protein) has recently been identified as a protein with binding activity to the cytoplasmic domain of the kidney Na+-independent Cl−/HCO−3 anion exchanger 1 (kAE1). Since it is widely expressed in tissues devoid of kAE1, however, kanadaptin is likely to have additional cellular roles. This is supported by its multidomain structure, and possession of three clusters of basic amino acids exhibiting similarity to known nuclear localization sequences (NLSs). In the present study, we use immunofluorescence and subcellular fractionation approaches to demonstrate that kanadaptin is localized within the nuclei of various epithelial and non-epithelial cultured cell types. The role of the different NLSs is examined in transfection studies using plasmids encoding full-length kanadaptin with or without green fluorescent protein (GFP) as a fusion tag, as well as truncation derivatives thereof. Strong nuclear localization of fusion proteins containing amino acids 140–230 of kanadaptin, which include the sequence AVSRKRKA193 (NLS1) was observed. Substitution of Arg191 with a threonine residue resulted in a cytoplasmic location of the expressed protein, while NLS1 proved sufficient to target an otherwise cytoplasmically localized β-galactosidase—GFP fusion protein to the nucleus. Using a direct binding assay we show that a fusion protein containing kanadaptin amino acids 1–231 (but not the Thr191 substituted derivative) is recognized with nM affinity by the conventional NLS-binding importin α/β heterodimer. Nuclear import studies on microinjected and permeabilized rat hepatoma cells demonstrated functionality of the NLS in nuclear targeting, with inhibition by antibodies demonstrating the requirement of both importin α and β for nuclear import of kanadaptin. That kanadaptin possesses a functional importin-α/β-recognized NLS explains the nuclear localization of kanadaptin in various cultured cell types, and opens up the possibility that kanadaptin may have a signalling role in the nucleus.
Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites
Authors: GASSET María , ALFONSO Carlos , NEIRA José L. , RIVAS Germán , PAJARES María A.
Published: August 2015

The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24±0.15. The variations of free energy in the absence of denaturant [ΔG(H2O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15±2.3kJ·mol−1 and 19.87±0.71kJ·mol−1·M−1 for the dissociation of the native dimer and 14.77±1.63kJ·mol−1 and 5.23±0.21kJ·mol−1·M−1 for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69kJ·mol−1 and 30.33kJ·mol−1·M−1 respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established.
Characterization of monoclonal antibodies against Escherichia coli core RNA polymerase
Authors: ROUBY Jöelle , PUGNIERE Martine , MANI Jean-Claude , GRANIER Claude , MONMOUTON Pierrette , GERMAIN Stephane THEULIER SAINT , LEONETTI Jean-Paul
Published: August 2015

Multiple interactions with DNA, RNA and transcription factors occur in a transcription cycle. To survey the proximity of some of these factors to the Escherichiacoli RNA polymerase surface, we produced a set of nine monoclonal antibodies (mAbs) against the enzyme. These mAbs, located at different places on the surface of the enzyme, were used in a co-immunopurification assay to investigate interference with the binding of NusA, σ70, GreB and HepA to core RNA polymerase. One of these mAbs turned out to be the first antibody inhibitor of the binding of NusA and σ70; it did not affect GreB and HepA interactions. Its epitope was located on the β′ subunit at the C-terminus of region G. The properties of this mAb reinforce the idea that the mutually exclusive binding of NusA and σ70 to core RNA polymerase is due to, at least partially, overlapping binding sites, rather than allosteric interaction between two distant binding sites. This mAb is also useful to understand the occupancy of σ70, NusA and Gre proteins on core RNA polymerase.
Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel)
Authors: HRUSKA-HAGEMAN Alesia M. , WEMMIE John A. , PRICE Margaret P. , WELSH Michael J.
Published: August 2015

Neuronal members of the degenerin/epithelial Na+ channel (DEG/ENaC) family of cation channels include the mammalian brain Na+ channel 1 (BNC1), acid-sensing ion channel (ASIC) and dorsal-root acid-sensing ion channel (DRASIC). Their response to acidic pH, their sequence similarity to nematode proteins involved in mechanotransduction and their modulation by neuropeptides suggest that they may function as receptors for a number of different stimuli. Using the yeast two-hybrid assay, we found that the PDZ domain-containing protein PICK1 (protein interacting with C kinase) interacts specifically with the C-termini of BNC1 and ASIC, but not DRASIC or the related αENaC or βENaC. In both the yeast two-hybrid system and mammalian cells, deletion of the BNC1 and ASIC C-termini abolished the interaction with PICK1. Likewise, mutating the PDZ domain in PICK1 abolished its interaction with BNC1 and ASIC. In addition, in a heterologous expression system PICK1 altered the distribution of BNC1 channels; this effect was dependent on the PDZ domain of PICK1 and the C-terminus of BNC1. We found crude synaptosomal fractions of brain to be enriched in ASIC, suggesting a possible synaptic localization. Moreover, in transfected hippocampal neurons ASIC co-localized with PICK1 in a punctate pattern at synapses. These data suggest that PICK1 binds ASIC and BNC1 via its PDZ domain. This interaction may be important for the localization and/or function of these channels in both the central and peripheral nervous systems.
A pH-dependent conformational transition of Aβ peptide and physicochemical properties of the conformers in the glial cell
Authors: MATSUNAGA Yoichi , SAITO Nobuhiro , FUJII Akihiro , YOKOTANI Junichi , TAKAKURA Tadakazu , NISHIMURA Tomoaki , ESAKI Hiroyuki , YAMADA Tatsuo
Published: August 2015

In the present study we identified the epitopes of antibodies against amyloid β-(1–42)-peptide (Aβ1–42): 4G8 reacted with peptides corresponding to residues 17–21, 6F/3D reacted with peptides corresponding to residues 9–14, and anti 5-10 reacted with peptides corresponding to residues 5–10. The study also yielded some insight into the Aβ1–42 structures resulting from differences in pH. An ELISA study using monoclonal antibodies showed that pH-dependent conformational changes occur in the 6F/3D and 4G8 epitopes modified at pH 4.6, but not in the sequences recognized by anti 1-7 and anti 5-10. This was unique to Aβ1–40 and Aβ1–42 and did not occur with Aβ1–16 or Aβ17–42. The reactivity profile of 4G8 was not affected by blockage of histidine residues of pH-modified Aβ1–40 and Aβ1–42 with diethyl pyrocarbonate; however, the mutant [Gln11]Aβ1–40 abrogated the unique pH-dependence towards 4G8 observed with Aβ1–40. These findings suggest that these epitopes are cryptic at pH4.6, and that Glu11 is responsible for the changes. We suggest that the abnormal folding of 6F/3D epitope affected by pH masked the 4G8 epitope. A study of the binding of metal ions to Aβ1–42 suggested that Cu2+ and Zn2+ induced a conformational transition around the 6F/3D region at pH7.4, but did not affect the region when it was modified at pH4.6. However, Fe2+ had no effect, irrespective of pH. Aβ modified at pH 4.6 appeared to be relatively resistant to proteinase K compared with Aβs modified at pH7.4, and the former might be preferentially internalized and accumulated in a human glial cell. Our findings suggest the importance of microenvironmental changes, such as pH, in the early stage of formation of Aβ aggregates in the glial cell.
Modulation of the reactivity of the essential cysteine residue of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa
Authors: GONZÁLEZ-SEGURA Lilian , VELASCO-GARCÍA Roberto , MUÑOZ-CLARES Rosario A.
Published: August 2015

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to glycine betaine. In the human opportunistic pathogen Pseudomonas aeruginosa this reaction is an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. As with every aldehyde dehydrogenase studied so far, BADH possesses an essential cysteine residue involved in the formation of the intermediate thiohemiacetal with the aldehyde substrate. We report here that the chemical modification of this residue is conveniently measured by the loss in enzyme activity, which allowed us to explore its reactivity in a pH range around neutrality. The pH dependence of the observed second-order rate constant of BADH inactivation by methyl methanethiosulphonate (MMTS) suggests that at low pH values the essential cysteine residue exists as thiolate by the formation of an ion pair with a positively charged residue. The estimated macroscopic pK values are 8.6 and 4.0 for the free and ion-pair-forming thiolate respectively. The reactivity towards MMTS of both thiolate forms is notably lower than that of model compounds of similar pK, suggesting a considerable steric inhibition by the structure of the protein. Binding of the dinucleotides rapidly induced a significant and transitory increment of thiolate reactivity, followed by a relatively slow change to an almost unreactive form. Thus it seems that to gain protection against oxidation without compromising catalytic efficiency, BADH from P. aeruginosa has evolved a complex and previously undescribed mechanism, involving several conformational rearrangements of the active site, to suit the reactivity of the essential thiol to the availability of coenzyme and substrate.
S′2 substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B
Authors: KRUPA Joanne C. , HASNAIN Sadiq , NÄGLER Dorit K. , MÉNARD Robert , MORT John S.
Published: August 2015

The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His110 and His111) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His111 is unpaired, His110 is present as an ion pair with Asp22 on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P′3 position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His111 makes a positive 10-fold contribution to the exopeptidase activity, whereas His110 is critical for this action with the Asp22—His110 ion pair stabilizing the electrostatic interaction by a maximum of 13.9kJ/mol (3.3kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P′3 and its other subsites.
Cross-talk unfolded: MARCKS proteins
Authors: ARBUZOVA Anna , SCHMITZ Arndt A. P. , VERGÈRES Guy
Published: August 2015

The proteins of the MARCKS (myristoylated alanine-rich C kinase substrate) family were first identified as prominent substrates of protein kinase C (PKC). Since then, these proteins have been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as endo-, exo- and phago-cytosis, and neurosecretion. The effector domain of MARCKS proteins is phosphorylated by PKC, binds to calmodulin and contributes to membrane binding. This multitude of mutually exclusive interactions allows cross-talk between the signal transduction pathways involving PKC and calmodulin. This review focuses on recent, mostly biophysical and biochemical results renewing interest in this protein family. MARCKS membrane binding is now understood at the molecular level. From a structural point of view, there is a consensus emerging that MARCKS proteins are ‘natively unfolded'. Interestingly, domains similar to the effector domain have been discovered in other proteins. Furthermore, since the effector domain enhances the polymerization of actin in vitro, MARCKS proteins have been proposed to mediate regulation of the actin cytoskeleton. However, the recent observations that MARCKS might serve to sequester phosphatidylinositol 4,5-bisphosphate in the plasma membrane of unstimulated cells suggest an alternative model for the control of the actin cytoskeleton. While myristoylation is classically considered to be a co-translational, irreversible event, new reports on MARCKS proteins suggest a more dynamic picture of this protein modification. Finally, studies with mice lacking MARCKS proteins have investigated the functions of these proteins during embryonic development in the intact organism.
Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis
Authors: WARD Richard J. , CHIOATO Lucimara , de OLIVEIRA Arthur H. C. , RULLER Roberto , SÁ Juliana M.
Published: August 2015

Bothropstoxin-I (BthTx-I) is a myotoxic phospholipase A2 variant present in the venom of Bothrops jararacussu, in which the Asp49 residue is replaced with a lysine, which damages artificial membranes by a Ca2+-independent mechanism. Wild-type BthTx-I and the mutants Lys49 → Asp, His48 → Gln and Lys122 → Ala were expressed in Escherichia coli BL21(DE3) cells, and the hydrolytic, myotoxic and membrane-damaging activities of the recombinant proteins were compared with native BthTx-I purified from whole venom. The Ca2+-independent membrane-damaging and myotoxic activities of the native and wild-type recombinant BthTx-I, His48Gln and Lys49Asp mutants were similar; however, the Lys122Ala mutant demonstrated reduced levels of both activities. Although a low hydrolytic activity against a mixed phospholipid substrate was observed with native BthTx-I, no substrate hydrolysis was detected with the wild-type recombinant enzyme or any of the mutants. In the case of the Lys49Asp mutant, this demonstrates that the absence of catalytic activity in Lys49-PLA2 is not a consequence of the single Asp49 → Lys replacement. Furthermore, these results provide unambiguous evidence that the Ca2+-independent membrane-damaging and myotoxic activities are maintained in the absence of hydrolysis. The evidence favours a model for a hydrolysis-independent, membrane-damaging mechanism involving an interaction of the C-terminal region of BthTx-I with the target membrane.
Polyamines are required for the initiation of rat liver regeneration
Authors: ALHONEN Leena , RÄSÄNEN Tiina-Liisa , SINERVIRTA Riitta , PARKKINEN Jyrki J. , KORHONEN Veli-Pekka , PIETILÄ Marko , JÄNNE Juhani
Published: August 2015

A large number of studies applying inhibitors of polyamine biosynthesis have indicated that these compounds are required for animal cell proliferation. Here we show, using a transgenic rat model with activated polyamine catabolism, that a certain critical concentration of the higher polyamines spermidine and spermine is required for liver regeneration. Partial hepatectomy of transgenic rats expressing spermidine/spermine N1-acetyltransferase (SSAT) under the control of mouse metallothionein promoter strikingly induced the enzyme at 24h and reduced hepatic spermidine by 80%. At that time, the weight of the liver remnant was significantly increased in syngenic rats and proliferating cell nuclear antigen (PCNA) labelling index was 20%, whereas the transgenic rats showed no liver weight gain and their PCNA-positive cells accounted for 0.5% of hepatocytes. Similarly, hepatic thymidine incorporation was markedly enhanced at this time point in syngenic, but not in transgenic, animals, whereas the rate of leucine incorporation was only marginally affected in the transgenic animals. At 3 days after operation, the spermidine pool in transgenic livers had increased to the pre-operative level, the remnant weight was significantly elevated and hepatic PCNA labelling index increased to 5%. N1,N11-Diethylnorspermine, a powerful inducer of SSAT, inhibited liver weight gain and proliferative activity in both syngenic and transgenic rats. We found an extremely close correlation between hepatic spermidine, and less close between spermine, concentrations and PCNA labelling index during early liver regeneration. These results indicate that spermidine and/or spermine, but apparently not putrescine, are required for liver regeneration, yet at concentrations smaller than those normally found after partial hepatectomy.
Re-evaluation of primary structure, topology, and localization of Scamper, a putative intracellular Ca2+ channel activated by sphingosylphosphocholine
Authors: SCHNURBUS Raphaela , De PIETRI TONELLI Davide , GROHOVAZ Fabio , ZACCHETTI Daniele
Published: August 2015

Naturally occurring sphingoid molecules control vital functions of the cell through their interaction with specific receptors. Proliferation, differentiation and programmed death result in fact from a fine balance of signals, among which sphingosine and structurally related molecules play fundamental roles, acting as either first or second messengers. The corresponding receptors need to be identified in order that the role of sphingoid molecules can be established. Among them, several G-protein-coupled receptors specific for sphingosine 1-phosphate, sphingosylphosphocholine, or both, have already been investigated. In contrast, the identification of the postulated intracellular receptors has been problematical. In the present study we re-evaluated the molecular characterization of Scamper, the first proposed intracellular receptor for sphingosylphosphocholine [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1993–1996] and commonly believed to be a Ca2+ channel of the endoplasmic reticulum (the name ‘SCaMPER’ used by Mao et al. being derived from ‘sphingolipid Ca2+-release-mediating protein of the endoplasmic reticulum'). In contrast with what has been believed hitherto, our primary-structure and overexpression experiments indicate that Scamper is a 110-amino-acid protein spanning the membrane once with a Nexo/Ccyt topology [von Heijne and Gavel (1988) Eur. J. Biochem. 174, 671–678]. Overexpression of either wild-type or tagged Scamper induces a specific phenotype characterized by the rapid extension of actin-containing protrusions, followed by cell death.
A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing
Authors: KUMAR Priyadarsini , WALSH Donal A.
Published: August 2015

We have previously shown that the protein kinase inhibitor β (PKIβ) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011–20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.
Macrophage metalloproteinases degrade high-density-lipoprotein-associated apolipoprotein A-I at both the N- and C-termini
Authors: EBERINI Ivano , CALABRESI Laura , WAIT Robin , TEDESCHI Gabriella , PIRILLO Angela , PUGLISI Lina , SIRTORI Cesare R. , GIANAZZA Elisabetta
Published: August 2015

Atheromatous plaques contain various cell types, including macrophages, endothelial cells and smooth-muscle cells. To investigate the possible interactions between secreted matrix metalloproteinases and high-density lipoprotein (HDL) components, we tested the above cell types by culturing them for 24h. HDL3 (HDL subfractions with average sizes of between 8.44nm for HDL3A and 7.62nm for HDL3C) were then incubated in their cell-free conditioned media. Proteolytic degradation of apolipoprotein A-I was observed with macrophages, but not with endothelial-cell- or muscle-cell-conditioned supernatant. Absence of calcium or addition of EDTA to incubation media prevented all proteolytic processes. The identified apolipoprotein A-I fragments had sizes of 26, 22, 14 and 9kDa. Two-dimensional electrophoresis and MS resolved the 26 and the 22kDa components and identified peptides resulting from both N- and C-terminal cleavage of apolipoprotein A-I. The higher abundance of C- than N-terminally cleaved peptides agrees with data in the literature for a fully structured α-helix around Tyr18 compared with an unstructured region around Gly185 and Gly186. The flexibility in the latter region of apolipoprotein A-I may explain its susceptibility to proteolysis. In our experimental set-up, HDL3C was more extensively degraded than the other HDL3 subclasses (HDL3A and HDL3B). Proteolytic fragments produced by metalloproteinase action were shown by gel filtration and electrophoresis to be neither associated with lipids nor self-associated.
A novel function for the 90 kDa heat-shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits
Authors: SCHLATTER Harald , LANGER Thomas , ROSMUS Susann , ONNEKEN Marie-Luise , FASOLD Hugo
Published: August 2015

Ribosomal subunits are assembled in the nucleus, and mature 40S and 60S subunits are exported stoichiometrically into the cytoplasm. The nuclear export of ribosomal subunits is a unidirectional, saturable and energy-dependent process. An in vitro assay for the nuclear export of 60S ribosomal subunits involves the use of resealed nuclear envelopes. The export of ribosomal subunits from resealed nuclear envelopes is enhanced by cytoplasmic proteins. Here we present evidence that the export-promoting activity was due to the cytoplasmic 90kDa heat-shock protein (Hsp90). Isolated, purified Hsp90 vastly enhanced the export of 60S ribosomal subunits from resealed nuclear envelopes, while inhibition of Hsp90 function, either with the Hsp90-binding drug geldanamycin or with anti-Hsp90 antibodies, resulted in reduced release of 60S ribosomal subunits. To confirm these findings under in vivo conditions, corresponding experiments were performed with Xenopus oocytes using microinjection techniques; the results obtained confirmed the findings obtained with resealed nuclear envelopes. These findings suggest that Hsp90 facilitates the nuclear export of 60S ribosomal subunits, probably by chaperoning protein interactions during the export process.
Further characterization of the interaction between the cytoskeletal proteins talin and vinculin
Authors: BASS Mark D. , PATEL Bipin , BARSUKOV Igor G. , FILLINGHAM Ian J. , MASON Robert , SMITH Beverley J. , BAGSHAW Clive R. , CRITCHLEY David R.
Published: August 2015

The cytoskeletal protein talin, which is thought to couple integrins to F-actin, contains three binding sites (VBS1—VBS3) for vinculin, a protein implicated in the negative regulation of cell motility and whose activity is modulated by an intramolecular interaction between the vinculin head (Vh) and vinculin tail (Vt) domains. In the present study we show that recombinant talin polypeptides containing the three VBSs (VBS1, residues 498–636; VBS2, residues 727–965; and VBS3, residues 1943–2157) each bind tightly to the same or overlapping sites within vinculin1–258. A short synthetic talin VBS3 peptide (residues 1944–1969) was sufficient to inhibit binding of a 125I-labelled talin VBS3 polypeptide to vinculin1–258, and NMR spectroscopy confirmed that this peptide forms a 1:1 complex in slow exchange with vinculin1–258. Binding of the 125I-labelled VBS3 polypeptide was markedly temperature dependent, but was not inhibited by 1M salt or 10% (v/v) 2-methyl-2-propanol. Attempts to further define the talin-binding site within vinculin1–258 using a gel-blot assay were unsuccessful, but near maximal talin-binding activity was retained by a construct spanning vinculin residues 1–131 in a yeast two-hybrid assay. Interestingly, the talin VBS3 polypeptide was a potent inhibitor of the Vh—Vt interaction, and the VBS3 synthetic peptide was able to expose the actin-binding site in intact vinculin, which is otherwise masked by the Vh—Vt interaction. The results suggest that under certain conditions, talin may be an effective activator of vinculin.
Ceramides increase the activity of the secretory phospholipase A2 and alter its fatty acid specificity
Authors: KOUMANOV Kamen S. , MOMCHILOVA Albena B. , QUINN Peter J. , WOLF Claude
Published: August 2015

Modulation of human recombinant secretory type II phospholipase A2 activity by ceramide and cholesterol was investigated using model glycerophospholipid substrates composed of phosphatidylethanolamine and phosphatidylserine dispersed in aqueous medium. Enzyme activity was monitored by measurement of released fatty acids using capillary GC-MS. Fatty acids from the sn-2 position of the phospholipids were hydrolysed by the enzyme in proportion to the relative abundance of the phospholipid in the substrate. Addition of increasing amounts of ceramide to the substrate progressively enhanced phospholipase activity. The increased activity was accomplished largely by preferential hydrolysis of polyunsaturated fatty acids, particularly arachidonic acid, derived from phosphatidylethanolamine. The addition of sphingomyelin to the substrate glycerophospholipids inhibited phospholipase activity but its progressive substitution by ceramide, so as to mimic sphingomyelinase activity, counteracted the inhibition. The presence of cholesterol in dispersions of glycerophospholipid-substrate-containing ceramides suppressed activation of the enzyme resulting from the presence of ceramide. The molecular basis of enzyme modulation was investigated by analysis of the phase structure of the dispersed lipid substrate during temperature scans from 46 to 20°C using small-angle synchrotron X-ray diffraction. These studies indicated that intermediate structures created after ceramide-dependent phase separation of hexagonal and lamellar phases represent the most susceptible form of the substrate for enzyme hydrolysis.
An alternative model of H ferritin promoter transactivation by c-Jun
Authors: FANIELLO Maria C. , CHIRICO Giuseppa , QUARESIMA Barbara , CUDA Giovanni , ALLEVATO Giovanna , BEVILACQUA Maria A. , BAUDI Francesco , COLANTUONI Vittorio , CIMINO Filiberto , VENUTA Salvatore , AVVEDIMENTO Vittorio E. , COSTANZO Francesco
Published: August 2015

c-Jun is a member of the activator protein 1 family, and its interaction with different nuclear factors generates a wide spectrum of complexes that regulate transcription of different promoters. H ferritin promoter transcription is tightly dependent on nuclear factor Y (NFY). Ferritin transcription is activated by c-Jun, although the promoter does not contain a canonical binding site. NFY, on the other hand, does not bind c-Jun in vitro, whereas in vivo c-Jun is found in the complex containing NFY. Moreover, a c-Jun—GCN4 chimaeric construct containing only the transactivation domain of Jun and the basic-region leucine-zipper domain of GCN4 stimulates the H ferritin promoter. A synthetic GAL4 promoter and the cognate activator, the fusion protein NFY—GAL4, are potently activated by c-Jun. Titration of p300 by co-expressing E1A abolishes the stimulatory effect. Moreover, another p300-dependent promoter, the cAMP-response element, can be superactivated by c-Jun using the same mechanism. These data indicate that c-Jun, when activated or overexpressed, is recruited to the H ferritin promoter by p300, which links NFY, bound to DNA, to the complex. These results add a new level of complexity to transcriptional regulation by c-Jun, which can activate p300-dependent promoters without binding directly to the target DNA.
Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50
Authors: VEITH Paul D. , TALBO Gert H. , SLAKESKI Nada , DASHPER Stuart G. , MOORE Caroline , PAOLINI Rita A. , REYNOLDS Eric C.
Published: August 2015

Porphyromonas gingivalis is an anaerobic, asaccharolytic Gram-negative rod associated with chronic periodontitis. We have undertaken a proteomic study of the outer membrane of P. gingivalis strain W50 using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteins were identified by reference to the pre-release genomic sequence of P. gingivalis available from The Institute for Genomic Research. Out of 39 proteins identified, five were TonB-linked outer membrane receptors, ten others were putative integral outer membrane proteins and four were putative lipoproteins. Pyroglutamate was found to be the N-terminal residue of seven of the proteins, and was predicted to be the N-terminal residue of 13 additional proteins. The RgpA, Kgp and HagA polyproteins were identified as fully processed domains in outer membranes prepared in the presence of proteinase inhibitors. Several domains were found to be C-terminally truncated 16–57 residues upstream from the N-terminus of the following domain, at a residue penultimate to a lysine. This pattern of C-terminal processing was not detected in a W50 strain isogenic mutant lacking the lysine-specific proteinase Kgp. Construction of another W50 isogenic mutant lacking the arginine-specific proteinases indicated that RgpB and/or RgpA were also involved in domain processing. The C-terminal adhesin of RgpA, designated RgpA27, together with RgpB and two newly identified proteins designated P27 and P59 were found to migrate on two-dimensional gels as vertical streaks at a molecular mass 13–42kDa higher than that calculated from their gene sequences. The electrophoretic behaviour of these proteins, together with their immunoreactivity with a monoclonal antibody that recognizes lipopolysaccharide, is consistent with a modification that could anchor the proteins to the outer membrane.