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PubMed1995

Dynamic regulation of integrins.

Dynamic regulation of receptor function is a distinguishing feature of the integrin family of adhesion molecules and makes a significant contribution to alterations in cellular adhesive properties. The best characterized example is that of the platelet receptor alpha IIb beta 3 (glycoprotein IIb-IIIa), which does not bind soluble fibrinogen on resting platelets. Following platelet activation, the alpha IIb beta 3 binds soluble fibrinogen with moderately high affinity and platelet aggregation ensues. Similar regulation of receptor function has also been directly demonstrated for alpha 5 beta 1 and alpha M beta 2, and it is likely that it is a general property of all members of the family. The altered ligand binding affinity is due to a change in the conformation of the external domain of the receptor, in response to intracellular signals that are transmitted the length of the molecule. The cytoplasmic tails of the integrins are active participants in this regulation, and they also mediate interactions with the cytoskeleton. A number of anti-integrin monoclonal antibodies have been described which induce high affinity ligand binding, and certain of these preferentially bind to the high affinity conformation of the receptor. The alteration in conformation allows better access for macromolecular ligands to the ligand binding pocket, which has been mapped (in alpha IIb beta 3) to the amino terminal globular head of the receptor. The precise mechanism by which the activating signal is transferred from within the cell to the distal external domain remains the subject of active research.

Animals↗
PubMed1995

Evolutionary reengineering of the phosphofructokinase active site: ARG-104 does not stabilize the transition state in 6-phosphofructo-2-kinase.

Arg-104 of the kinase domain of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was mutated to alanine, the mutant enzyme expressed in E. coli with a T7 RNA polymerase-based expression system, and purified to homogeneity by Blue-Sepharose and Q-Sepharose chromatography. The mutant enzyme exhibited a 200-fold increase in Km for fructose-6-phosphate, no change in Km for ATP, and a 2-3-fold increase in catalytic rate. The results indicate that Arg-104, along with Arg-195, are the principal binding site residues for the 6-phosphate group of fructose-6-phosphate. In contrast to the corresponding residue in the related E. coli 6-phosphofructo-1-kinase, Arg-104 did not stabilize the transition state at pH 7-9. The Arg-104 mutation also decreased Fru-2,6-P2ase activity without affecting substrate inhibition, which suggests that this mutation affects the bisphosphatase active site conformation and/or substrate access to it.

Arginine↗
PubMed1988

Reversal of insulin-induced negative cooperativity by monoclonal antibodies that stabilize the slowly dissociating ("Ksuper") state of the insulin receptor.

Two monoclonal antibodies to the insulin receptor, MA-5 and MA-20, unlike other monoclonal antibodies, do not mimick the accelerating effect of insulin on the dissociation of 125I-insulin from the receptors (negative cooperativity). On the contrary, MA-5 and MA-20 markedly slow down the dissociation rate. We show now that MA-5 and MA-20 are potent antagonists of the negative cooperativity induced by insulin, and reverse the insulin-induced acceleration whether added simultaneously with insulin or after insulin. The reversal of the insulin-induced acceleration is almost immediate. These data strengthen the concept therefore that the insulin-receptor complex has access to alternative conformational states that can be stabilized by ligand-induced site-site interactions.

Animals↗
PubMed1996

Catalytic activity of poly(ADP-ribose) polymerase is necessary for repair of N-methylpurines in nontranscribed, but not in transcribed, nuclear DNA sequences.

The role of poly(ADP-ribose) polymerase (PADPRP) in nuclear DNA repair and other nuclear processes has been intensely studied and debated for decades. Recent studies have begun to shed new light on these arguments with firm experimental data for its role, primarily, as a remodeler of chromatin structure. Those studies imply that PADPRP plays an indirect role in DNA repair, serving to expose DNA to repair enzymes through chromatin remodeling. Only DNA that is tightly packaged would require PADPRP activity for its repair; while DNA in an open conformation would be accessible to DNA repair enzymes and not require PADPRP activity. The purpose of the current studies was to address the above hypothesis directly. Using quantitative Southern blot analysis, we studied repair in transcribed and nontranscribed nuclear DNA sequences in ADPRT 351 cells 95% deficient in PADPRP activity. Cells were exposed to methylnitrosourea (MNU) for 1 h and allowed to repair for 8 or 24 h. Densitometric scans of autoradiographs revealed that, when compared to their parental V79 cell line, ADPRT 351 cells 95% deficient in PADPRP activity were equally as efficient in repair of N-methylpurines in the transcribed sequence containing the dihydrofolate reductase gene. However, the ADPRT 351 cells were deficient in the ability to repair these lesions in the nontranscribed sequence containing the IgE gene compared to repair of the same sequence in the parental V79 cells. Nucleoid sedimentation assays demonstrated that the ADPRT 351 cells are deficient in repair across the entire genome when compared to the parental V79 cells. These studies indicate that PADPRP activity is not required for repair of N-methylpurines in transcribed nuclear DNA sequences but is necessary for the repair of these lesions in nontranscribed nuclear DNA sequences as well as across the entire genome since the DNA in a given cell is predominantly nontranscribed.

Alkylation↗
PubMed1997

Lung carcinomas composed of rhabdoid cells.

Rhabdoid tumours form a distinctive morphological entity that is associated with aggressive biological behaviour. They have been described in several sites and tumour types. This paper presents three new cases of rhabdoid lung cancers. Lung cancers were analysed for the presence of cells with the rhabdoid phenotype: eccentric vesicular nuclei and abundant eosinophilic cytoplasm. Cells displaying this morphology were then subjected to immunohistochemistry and electron microscopy. The relevant clinical data on these cases were then accessed. Three cases conforming to the morphological, immunophenotypic and ultrastructural characteristics of rhabdoid cells were identified. Two of the cases were associated with foci of adenocarcinoma and the remaining case was a large cell neuroendocrine carcinoma. Two of the cases showed rapid clinical courses with the patients dying of disease within 6 months. Lung tumours with a rhabdoid phenotype are uncommon but are noteworthy because of their aggressive behaviour and, hence, poor prognosis.

Adult↗
PubMed2003

Correlation between butyrate-induced histone hyperacetylation turn-over and c-myc expression.

Transcriptionally active chromatin has an high level of histone acetylation, a post-transcriptional modification known to alter nucleosomal conformation increasing the accessibility of transcription factors to DNA. Recent studies have led new interest in histone acetylase and deacetylase because of their role as transcription factors. Sodium butyrate, a known reversible inhibitor of histone deacetylase, modulates a large number of genes. This report is focused on the modulation of the c-myc oncogene expression by butyrate. In HeLa cells, treated with butyrate and then exposed to butyrate-free medium, we established a correlation between the reactivation kinetic of c-myc expression and the increase in level of histone H4 acetylation. Both parameters, in cells exposed to butyrate-free medium, after showing a rebound effect, return to the control level. This trend was confirmed by quantitative analysis of the level of histone acetylation and of c-myc expression in the three distinct class of nucleosomal fragments with different transcriptional activity. In this chase process, we also detected a concomitant enrichment in c-myc sequences in the "active" chromatin fractions and decreased presence in the inactive nucleosomal fragment. Therefore we here demonstrate an excellent correlation between histone hyperacetylation and reactivation of a specific gene (c-myc).

Acetylation↗
PubMedDate not supplied

Identification of formaldehyde-induced modifications in proteins: reactions with insulin.

Formaldehyde is frequently used to inactivate, stabilize, or immobilize proteins. The treatment results in a large variety of chemical modifications in proteins, such as the formation of methylol groups, Schiff bases, and methylene bridges. The purpose of the present study was to identify the stable formaldehyde-induced modifications in a small protein. Therefore, insulin was treated with excess formaldehyde (CH2O) or deuterated formaldehyde (CD2O). In a separate experiment, insulin was modified by formaldehyde (CH2O vs CD2O) and glycine. The mixture of CH2O-treated and CD2O-treated insulin was digested by the proteinase Glu-C. The peptide fragments obtained were analyzed by liquid chromatography-mass spectrometry (LC-MS). Seven intramolecular cross-links were identified in formaldehyde-treated insulin. Furthermore, eight out of the sixteen potentially reactive sites of the insulin molecule were modified by incubation with formaldehyde and glycine. Both the location and the chemical nature of the modifications could be assigned based on the mass increase of potential adducts as elucidated in our previous study (B. Metz et al. (2004) J. Biol. Chem. 279, 6235-6243). To confirm the assigned structures, LC-MS measurements with collision-induced dissociation (LC-MS/MS) were performed on insulin fragments. The results of the LC-MS/MS analyses agreed excellently with the assignments. The study showed that arginine, tyrosine, and lysine residues were very reactive. However, eight theoretically reactive residues did not show detectable modifications, probably because of their low intrinsic reactivity, inaccessibility, or both. The asparagine, glutamine, and histidine residues were not converted in insulin. The N-termini of insulin were partly converted to the expected imidazolidinone adducts, indicating that the protein conformation affects the accessibility and reactivity of these residues. In conclusion, this study shows that, based on our current insights in the chemistry of the reactions between proteins and formaldehyde, we are able to elucidate the location and nature of formaldehyde-induced modifications in a small protein. The approach followed in this study may be generally applicable to larger formaldehyde-treated proteins, such as toxoids used in vaccines.

Cross-Linking Reagents↗
PubMed1991

Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase. Effects of ATP on the tryptophan-57 loop.

Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics. Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158. The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components. The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes. The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum. In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component. The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand. Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes. Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component. These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57. ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components.

Adenosine Triphosphate↗
PubMed1987

High-resolution proton and laser photochemically induced dynamic nuclear polarization NMR studies of cation binding to bovine alpha-lactalbumin.

alpha-Lactalbumin (alpha-LA) is a calcium binding protein that also binds Mn(II), lanthanide ions, A1(III), Zn(II), Co(II). The structural implications of cation binding were studied by high-resolution proton (200 MHz) NMR and photochemically induced dynamic nuclear polarization (CIDNP) spectroscopy. Marked changes were observed in the NMR spectra of the apoprotein upon addition of a stoichiometric amount of calcium to yield Ca(II)-alpha-LA, manifested particularly in ring current shifted aliphatic peaks and in several shifts in the aromatic region, all of which were under slow exchange conditions. The CIDNP results showed that two surface-accessible tyrosine residues, assigned as Tyr-18 and -36, became inaccessible to the solvent upon addition of 1:1 Ca(II) to apo-alpha-lactalbumin, while Tyr-103 and Trp-104 remained completely accessible in both conformers. The proton NMR spectra of apo-alpha-LA and A1(III)-alpha-LA were extremely similar, which was also consistent with intrinsic fluorescence results [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. The paramagnetic cation Mn(II) bound to the strong calcium binding site on apo-alpha-LA but also to the weak secondary Ca(II) binding site(s) on Ca(II)-alpha-LA. It was also found that Co(II) bound to some secondary sites on Ca(II)-alpha-LA that overlapped the weak calcium site. All of the lanthanide shift reagents [Pr(III), Eu(III), Tb(III), Dy(III), Tm(III), Yb(III)] bound under slow exchange conditions; their relative affinities for apo-alpha-lactalbumin from competitive binding experiments were Dy(III), Tb(III), and Pr(III) greater than Ca(II) greater than Yb(III).

Animals↗
PubMed1988

Murine epidermal growth factor: structure and function.

Murine epidermal growth factor (EGF), a 53 amino acid protein, has been modified by enzymic digestion, site-specific chemical reactions, and recombinant DNA technology. After trypsin digestion the EGF derivatives EGF1-48 (called EGF-T) and EGF1-45 (called EGF-T2) were separated from the residual EGF and the C-terminal pentapeptide by reversed-phase high-performance liquid chromatography. EGF-T competes for binding to EGF receptors with the same efficiency as EGF. The EGF-T2 derivative had no detectable receptor binding activity even at 100 nM. The in vitro mitogenic potencies of EGF and EGF-T for Balb/c 3T3 cells were indistinguishable. Treatment of EGF-T with carboxypeptidase Y yielded two derivatives, EGF-T-(des-Arg48) and EGF-T-des(Leu47-Arg48). There was only a 3-7-fold diminution in the binding efficiency and mitogenic potency for EGF-T-(des-Arg48). However, there was more than a 100-fold decrease in the binding efficiency and mitogenic activity of EGF-T-des (Leu47-Arg48). These results indicated that Leu47 is intimately involved in the formation of the ligand-receptor complex. Studies with a number of proteases indicated that the C-terminus of EGF was susceptible to enzymic digestion; however, the N-terminus appears to be folded into a conformation which prevents access to proteolytic digestion. Consequently, the N-terminus was modified by preparing an analogue with recombinant DNA technology. Oligonucleotides corresponding to EGF(3-48). Met3 Lys21 residues were ligated in frame to a beta-galactosidase expression vector. The beta-Gal-EGF fusion protein was cleaved with cyanogen bromide and EGF(4-48).Lys21 purified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1982

Phosphorylation and hydrolysis of 7-deazaadenine nucleotides by rat liver and beef heart mitochondria.

Tubercidin nucleotides [tubercidin 5'-mono-phosphate (TuMP), 5'-diphosphate (TuDP), and 5'-triphosphate (TuTP)] were tested as potential substrates for the mitochondrial phosphotransferases from rat liver and beef heart. TuDP is recognized by the mitochondrial translocase and phosphorylated by the respiratory chain enzymes in both mitochondria and submitochondrial particles from rat liver and beef heart; the low transport rate of the analogue into the matrix space of the intact organelles seems to be not a limiting step in the formation of TuTP. The phosphorylation of TuDP is significantly lower in beef heart mitochondria because of a higher specificity for ADP of the heart oxidative phosphorylation system. On the basis of the kinetic parameters of the partially purified liver mitochondrial adenylate kinase, one can conclude that the liver mitochondria are able to phosphorylate in vivo TuMP at a rate practically equal to the rate of AMP phosphorylation. The liver mitochondrial NDP kinase ensures a further fast phosphorylation of TuDP without the direct involvement of respiratory chain enzymes. In the case of heart mitochondria, two factors limit the rate of TuMP phosphorylation to TuTP: the lower acceptor activity of adenylate kinase with TuMP as compared with AMP and the different localization of heart NDP kinase situated on the inner face of the inner mitochondrial membrane. TuDP and TuTP preserve the ability of the natural nucleotides to interact with the "tight" nucleotide binding sites of isolated or membrane-bound F1. The low hydrolytic rate of TuTP with F1 may be related to the unusual flexibility of the glycosyl bond of tubercidin nucleotides in aqueous solution, with a high accessibility to syn conformation.

Adenine Nucleotides↗
PubMed2003

Phosphorylation of the NADPH oxidase component p67(PHOX) by ERK2 and P38MAPK: selectivity of phosphorylated sites and existence of an intramolecular regulatory domain in the tetratricopeptide-rich region.

p67(PHOX), a cytosolic component of the NADPH oxidase complex, is phosphorylated during neutrophil activation by several agonists. The intracellular signaling pathways leading to its phosphorylation in neutrophils may involve a PKC-dependent pathway and a PKC-independent pathway. Here, we analyzed p67(PHOX) phosphorylation by ERK2 and p38MAPK. Both ERK2 and p38MAPK phosphorylated p67(PHOX) in vitro, with similar K(m) values (10 and 9 microM, respectively). Phosphopeptide mapping indicated that ERK2 and p38MAPK phosphorylate different subgroups of peptides. Using truncated forms of p67(PHOX), we found that the major phosphorylation target site of ERK2 was located in the N-terminal fragment (1-243), while the major phosphorylation target sites of p38MAPK were located in the C-terminal fragment (244-526). Furthermore, an additional peptide, which was not phosphorylated in the intact protein, appeared to be phosphorylated in the isolated C-terminal fragment (aa 244-526). This site may not thus be accessible in the intact protein. Indeed, incubation of the C-terminal fragment (244-526) with different N-terminal fragments (1-243, 1-210, or 1-199) containing the tetratricopeptide-rich region prevented phosphorylation of this C-terminal fragment. ERK1/2 and p38MAPK are also involved in p67(PHOX) phosphorylation in intact neutrophils. Indeed, PD98059 and SB203580, two selective inhibitors of MEK1/2 and p38MAPK, respectively, inhibited p67(PHOX) phosphorylation in fMLP- and PMA-stimulated neutrophils, with additive effects, thus suggesting that they also target different sites in vivo. Furthermore, the major peptides phosphorylated by ERK2 and p38MAPK in vitro were also phosphorylated in fMLP-stimulated neutrophils. Taken together, these results suggest not only that p67(PHOX) is phosphorylated by ERK2 and p38MAPK in vitro and in intact neutrophils on several selective sites but also that a C-terminal phosphorylation site may become accessible after a conformational change of the protein.

Humans↗
PubMed2003

Site-directed sulfhydryl labeling of helix IX in the lactose permease of Escherichia coli.

Site-directed sulfhydryl modification of transmembrane helix IX in the lactose permease of Escherichia coli was studied in right-side-out membrane vesicles with the thiol-specific reagents N-[(14)C]ethylmaleimide (NEM) and methanethiosulfonate ethylsulfonate (MTSES) which are permeant and impermeant, respectively. Out of approximately 20 mutants with a single Cys residue at each position in the helix, only five mutants label with NEM. (i) Cys residues at positions 291, 308, and 310 label at 25 degrees C, and binding of substrate has no effect. (ii) Cys residues at positions 295 and 298 label only in the presence of substrate. NEM labeling at 0 degrees C indicates that alkylation of Cys residues at positions 295 and 308 is dependent on the thermal motion of the protein. In contrast, temperature has little effect on labeling of Cys residues at positions 291, 298, and 310. Interestingly, pretreatment with MTSES blocks NEM labeling of all the mutants. The findings demonstrate that the face of helix IX on which Arg302 is located is involved in ligand-induced conformational changes and accessible to water from the periplasmic surface of the membrane. Since Arg302 facilitates deprotonation of Glu325 (helix X) during turnover [Sahin-Tóth, M., and Kaback, H. R. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6068-6073], the findings are consistent with the idea that this face of helix IX may comprise part of the H(+) translocation pathway.

Amino Acid Sequence↗
PubMed1997

Three-dimensional solution structure and stability of phage 434 Cro protein.

1H NMR resonances of the phage 434 Cro protein were assigned using standard 2D NMR methods, and its solution structure determined using 867 distance constraints in distance geometry (DIANA) calculations ultimately refined by restrained molecular dynamics (GROMOS). In the 20 best NMR structures, the average pairwise backbone and heavy atom RMSDs are 0.63 +/- 0.14 and 1.53 +/- 0.15 A, respectively, for the structurally well-defined residues 4-65. Residues 1-3 and 66-71 at the N- and C-termini are structurally disordered. The region 4-65 includes five alpha-helices and tight turns which define the hydrophobic core of the protein. The backbone and heavy atom RMSDs for residues 4-65 are 0.92 +/- 0.12 and 1.99 +/- 0.12 A, respectively, for the NMR versus the crystal structures, but there are significant differences in the side-chain conformations and solvent accessibilities for some core residues. Analytical ultracentrifugation experiments confirm that 434 Cro is monomeric even at the high NMR concentrations. 434 Cro folding under NMR solution conditions is two-state as indicated by coincident urea denaturation curves from circular dichroism and intrinsic fluorescence measurements. They yield values for 434 Cro stability which show good correspondence to the free energy for global unfolding determined by NMR hydrogen exchange measurements for the slowest exchanging amide protons.

Amino Acid Sequence↗
PubMed2004

Conformational flexibility of nickel(II) benziporphyrins.

(1)H NMR spectra of three paramagnetic Ni(II) complexes of benziporphyrins have been investigated in a broad temperature range. For the m- and p-benziporphyrin complexes, the line widths of certain signals exhibit an unusual temperature dependence characteristic of a dynamic process. This behavior is interpreted in terms of an equilibrium in which one of the forms is present at a very small concentration and cannot be observed directly. For the benziporphodimethene complex, the two exchanging forms are present at comparable concentrations and can yield separate signals in the slow exchange limit. To account for the observed exchange process, a mechanism is proposed involving the motion of the phenylene moiety. This hypothesis is further explored with DFT modeling, which indicates that the postulated conformers are thermally accessible.

Journal Article↗
PubMed2003

Role of entropy in increased rates of intramolecular reactions.

The rates of intramolecular condensation of a series of monoesters of dicarboxylic acids have been shown to be highly dependent on the nature of the intervening groups. To understand the origin of this effect, we estimated DeltaS(NAC,S), the entropy difference between the ensemble of accessible ground state conformers and a single ground state conformer having transition-state-like geometry. DeltaS(NAC,S) differs from the activation entropy for the reaction by DeltaS(TS,NAC), the difference in vibrational entropy between the selected ground state conformer and the transition state. The estimated values of DeltaS(NAC,S) correlate well (R(2) = 0.96 and 0.73 using dielectric constant values of 80 and 1, respectively) with experimentally determined reaction rate constants. Normal-mode analysis performed on minimized ground state conformations of each molecule suggests that the change in vibrational entropy makes only a small contribution to the total activation entropy. These results indicate that the conformational entropy difference between the transition and the ground states contributes significantly to the free energy of activation.

Journal Article↗
PubMed1992

Isobenzofurans as conformationally constrained miconazole analogues with improved antifungal potency.

A series of halogen-substituted isobenzofuran analogues was synthesized, which represented conformationally constrained analogues of miconazole (1). In vitro and in vivo topical antifungal activity against both dermatophytes and Candida species varied widely, but 13c proved to be significantly superior to both 1 and clotrimazole against a vaginal Candida infection in hamsters, while 13b was significantly more active than 1 against a a topical Trichophyton infection in guinea pigs. None of the compounds were orally active. When the most direct analogue of 1 proved to be among the least active, a molecular modeling study was done using 1, the two active analogues 13b and 13c, and the inactive analogue 13a. All four compounds possessed skeletally similar conformations either at or energetically readily accessible from the global minimum energy conformations. This common conformation of the inactive analogue 13a, however, occupies unique molecular volume space associated with two chlorine atoms, which must also present unique electrostatic properties at the receptor. The conformation-activity relationships discussed may contribute toward deduction of additional structural requirements for pharmacophore optimization and more efficacious antifungal drugs.

Animals↗