A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodium falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) exchange into amide bonds, quantitated by on-line HPLC and mass spectrometry, has been used to compare the dynamic and conformational properties of human HGPRT alone, the HGPRT-GMP-Mg(2+) complex, the HGPRT-IMP-MgPPi <==> HGPRT-Hx-MgPRPP equilibrating mixture, and the transition-state analogue complex HGPRT-ImmGP-MgPPi. The rate and extent of H/D exchange of 26 peptic peptides, spanning 91% of the primary structure, have been monitored. Human HGPRT has 207 amide H/D exchange sites. After 1 h in D2O, HGPRT alone exchanges 160, HGPRT-GMP-Mg(2+) exchanges 154, the equilibrium complex exchanges 139, and the transition-state analogue complex exchanges 126 of these amide protons. H/D exchange rates are correlated with structure for peptides in (1) catalytic site loops, (2) a connected peptide of the subunit interface of the tetramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HGPRT-GMP-Mg(2+) and HGPRT-ImmGP-MgPPi complexes. Transition-state analogue binding strengthens the interaction between subunits and tightens the catalytic site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, and ligand exchange rates. Solvent exchange reveals loop dynamics in the free enzyme, Michaelis complexes, and the complex with the bound transition-state analogue. Proton transfer paths, rather than dynamic motion, are required to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.

A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila.

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.

Direct acetylation of the estrogen receptor alpha hinge region by p300 regulates transactivation and hormone sensitivity.

Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERalpha) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERalpha-regulated gene expression involves interactions with cointegrators (e.g. p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERalpha is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ERalpha at lysine residues within the ERalpha hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERalpha hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERalpha acetylation normally suppresses ligand sensitivity. These ERalpha lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERalpha acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.

Temperature dependence of the folding and unfolding kinetics of the GCN4 leucine zipper via 13C(alpha)-NMR.

Studies by one-dimensional NMR are reported on the interconversion of folded and unfolded forms of the GCN4 leucine zipper in neutral saline buffer. The peptide bears 99% 13C(alpha) labels at three sites: V9, L12, and G31. Time-domain 13C(alpha)-NMR spectra are interpreted by global Bayesian lineshape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 degrees C. The data are well fit by the assumption that the same rate constants apply at each labeled site, confirming that only two conformational states need be considered. Results show that 1) both processes require a free energy of activation; 2) unfolding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages approximately 40% helical. The activation parameters for unfolding, derived from NMR data at the elevated temperatures where both conformations are populated, lead to estimates of the rate constant at low temperatures (5-15 degrees C) that agree with extant values determined by stopped-flow CD via dilution from denaturing media. However, the corresponding estimated values for the folding rate constant are larger by two to three orders of magnitude than those obtained by stopped flow. We propose that this apparent disagreement is caused by the necessity, in the stopped-flow experiment, for initiation of new helices as the highly denaturant-unfolded molecule adjusts to the newly created benign solvent conditions. This must reduce the success rate of collisions in producing the folded molecule. In the NMR determinations, however, the unfolded chains always have a small, but essential, helix content that makes such initiation unnecessary. Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-chain helical peptides and from demonstration that the folding rate constants for coiled coils, as obtained by stopped flow, are influenced by the nature of the denaturant used.

Primary structure of Noetia ponderosa hemoglobins: functional correlates.

Homo- and heterodimeric hemoglobins have been isolated from the red cells of the arcid clam Noetia ponderosa (Np). These hemoglobins bind oxygen cooperatively. An extensively studied dimeric hemoglobin from another arcid clam, Scapaharca inaequivalvis, exhibits a molecular mechanism for cooperative ligand binding that is radically different from tetrameric vertebrate hemoglobins. In this study, the two chains found in both Noetia hemoglobins are sequenced and compared to the hemoglobins of the related clam S. inaequivalvis to determine whether Noetia hemoglobins have the structural basis for the same unusual mechanism for cooperative ligand binding and to inquire about the structural basis of absence of tetramers. Although the Noetia sequences are homologous to the Scapharca sequences, critical differences exist. The lack of tetramerization of Np subunits is most likely related to the absence of critical residues in the A and G helices that stabilize the interdimer contact seen in the Scapharca Hb tetramer. The lower affinity of the homodimer (Np-I), but particularly the heterodimer (Np-II) with respect to the homodimer and heterotetramer of Scapharca, can be due to (i) changes in the proximal heme environment and (ii) changes in the dimer interface. Interactions between Asn 100 and the heme of the other subunit are altered in Np-II due to the substitution of this residue by methionine, possibly causing the reduced O(2) affinity of the heterodimer of Noetia. (iii) Sequence changes in the E and F helices present in Np-I and Np-II could also contribute to the effect through interfacial changes. In particular, the substitution of Val for Thr in position 72 is expected to have a substantial influence on the interface. We conclude that Np dimers have the structural basis for a direct heme-heme interaction mechanism for cooperativity, as in Scapharca, but there are enough sequence changes to suggest that the pathway of interaction might be somewhat different.

Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange.

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.

Characterisation of N-terminal chromogranin A and chromogranin B in mammals by region-specific radioimmunoassays and chromatographic separation methods.

Chromogranin A (CgA) and chromogranin B (CgB) are acidic proteins stored in and released from hormone granules in endocrine and neuroendocrine tissue. The chromogranins are postulated to serve as pro-hormones to generate biologically active peptides, which may influence hormonal release and vascular functions or have antibacterial functions. Although N-terminal and C-terminal regions show some species amino acid homology, the chromogranins as a whole display considerable interspecies differences, which prevents their use in comparative studies of biological functions. We present four new radioimmunoassays for the measurement of defined N-terminal regions of CgA and CgB. A new radioimmunoassay for measurement of intact bovine CgA has also been developed. With these assays and two previously published ones, we have compared the cross-reactivity of chromogranins from man, cattle, sheep, goat, pig and horse and compared adrenomedullar content and serum levels of CgA from these species. We have also studied the influence of peptide concentrations and the ionic strength of the mobile phase on molecular weight estimations. Assays with antibodies directed against the N-terminal parts of CgA and CgB showed sufficient interspecies cross-reactivity to allow comparative quantification of the circulating levels in man, cattle, sheep, goat, pig and horse. Assays measuring the intact human or bovine CgA were not suitable for comparative purposes in samples from sheep, goat, pig and horse. Molecular interactions between vasostatin immunoreactive material and intact bovine CgA were demonstrated in gel permeation studies, suggesting that conclusions about the degree of N-terminal processing from elution profiles should be made with caution. Reliable interspecies comparison of chromogranins is difficult, but measurements with region-specific assays may be helpful to study concentrations of chromogranins and chromogranin-related peptides.

p300 and p300/cAMP-response element-binding protein-associated factor acetylate the androgen receptor at sites governing hormone-dependent transactivation.

The androgen receptor (AR) is a sequence-specific DNA-binding protein that plays a key role in prostate cancer cellular proliferation by dihydrotestosterone and the induction of secondary sexual characteristics. In this study we demonstrate that the AR can be modified by acetylation in vitro and in vivo. p300 and p300/cAMP-response element-binding protein acetylated the AR at a highly conserved lysine-rich motif carboxyl-terminal to the zinc finger DNA-binding domain. [(14)C]acetate-labeling experiments demonstrated that AR acetylation by p300 in cultured cells requires the same residues identified in vitro. Point mutation of the AR acetylation site (K632A/K633A) abrogated dihydrotestosterone-dependent transactivation of the AR in cultured cells. Mutation of the p300 CH3 region or the p300/cAMP-response element-binding protein histone acetylase domain reduced ligand-dependent AR function. The identification of the AR as a direct target of histone acetyltransferase co-activators has important implications for targeting inhibitors of AR function.

Ultracentrifuge and circular dichroism studies of folding equilibria in a retro GCN4-like leucine zipper.

Equilibrium ultracentrifuge and circular dichroism (CD) studies of a retropeptide of a GCN4-like leucine zipper in neutral saline buffer are reported as functions of temperature. Ultracentrifuge results indicate the presence of three oligomeric species: monomer, dimer, and tetramer, in quantifiable amounts, and the data provide values for the standard DeltaG, DeltaH, and DeltaS for interconversion. CD at 222 nm displays the strong concentration dependence characteristic of dissociative unfolding, but also shows a helicity far below that of the parent propeptide. Remarkably enough, the CD at 222 nm shows an extremum in the region between 0 and 20 degrees C. At higher T, the usual cooperative unfolding is observed. Comparable data are presented for a mutant retropeptide, in which a single asparagine residue is restored to the characteristic heptad position it occupies in the propeptide. The mutant shows marked differences from its unmutated relative in both thermodynamic properties and CD, although the oligomeric ensemble also comprises monomers, dimers, and tetramers. The mutant is closer in helicity to the parent propeptide but is less stable. These findings do not support either of the extant views on retropeptides. The behavior seen is consistent neither with the view that retropeptides should have the same structure as propeptides nor with the view that they should have the same structure but opposite chirality. The simultaneous availability of oligomeric population data and CD allows the latter to be dissected into individual contributions from monomers, dimers, and tetramers. This dissection yields explanations for the observed extrema in curves of CD (222 nm) versus T and reveals that the dimer population in both retropeptides undergoes "cold denaturation."

Ca2+-induced increases in steady-state concentrations of intracellular calcium are not required for inhibition of parathyroid hormone secretion.

It has been well established that increases in extracellular calcium concentration ([Ca2+]) inhibit parathyroid hormone (PTH) secretion. The effects of [Ca2+] are mediated through a G-protein-coupled receptor that has been cloned and characterized. Additionally, it has been demonstrated in parathyroid cells that an increase in [Ca2+] results in an increase in steady-state levels of intracellular calcium ([Ca2+]i). At present, it has not been fully resolved whether changes in [Ca2+]i are related to changes in PTH secretion. In the current study, the effect of increased [Ca2+] on PTH secretion and the connection regarding changes in concentrations of intracellular calcium [Ca2+]i have been examined in primary cultures of bovine parathyroid cells. PTH secretion was measured by radioimmunoassay and intracellular calcium was determined by single cell calcium imaging. Bovine parathyroid cells pre-incubated with either 0.5 or 1 mM calcium responded to rapid increases in [Ca2+] (> or = 0.5 mM) with an immediate and sustained increase in steady-state levels of [Ca2+]i that persisted for time intervals greater than 15 minutes. Although the magnitude of the sustained increase in [Ca2+]i varied among individual cells (approximately 40% to > 300%), the overall pattern and course of time were similar in all cells examined (n = 142). In all trials, [Ca2+]i immediately returned to baseline levels following the addition of the calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Additional control studies, however, suggest that sustained increases in [Ca2+]i do not correlate with regulation of parathyroid hormone secretion. Sustained elevations of [Ca2+]i were not observed when [Ca2+] was gradually increased by the addition of 0.1 mM increments at 1 minute intervals. Furthermore, the effect on inhibition of PTH secretion was the same regardless of whether [Ca2+] was increased by gradual or rapid addition.

Design of useful peptide antigens.

Well-designed anti-peptide antibodies provide the specificity and control for everyday and cutting-edge applications. Pragmatic peptide antigen design principles can be used to help ensure production of successful antibodies. Issues of peptide chemistry, sequence selection, antigen preparation, antibody handling, and experimental needs must be taken into consideration in the planning stages.

Dichotomous development of the organic anion transport protein in liver and choroid plexus.

Both adult liver and choroid plexus express the organic anion transport protein (oatp1) and transport [35S]bromosulfophthalein (BSP). Studies of the developing rat liver reveal that oatp1 mRNA and protein do not begin to be expressed until 15 days postnatal and are at adult levels by 30 days. Uptake of [35S]BSP follows the same time course. In contrast, neonatal rat choroid plexus expresses oatp1 mRNA and protein. When quantified on a weight basis, the uptake of [35S]BSP in choroid plexus is lower in the adult than at earlier stages of development. Although fluorescence confocal microscopy of adult rat choroid plexus shows that oatp is localized to the apical surface, facing the cerebrospinal fluid, this method reveals an intracellular localization of oatp1 in the neonate. Approximately 12 wk are required for the appearance of the adult pattern of distribution. Changes in the localization and activity of oatp1 during development could play an important role in the pathobiology of maturation of the liver and the central nervous system.

The chemokine interleukin-8 regulates parathyroid secretion.

Interleukin-8 (IL-8) is a chemokine important in inflammatory processes. Homology cloning experiments performed using bovine parathyroid cDNA and degenerate primers encoding transmembrane regions III and VI of peptide and protein hormone G-protein coupled receptors identified a set of known receptors not previously identified in the parathyroid. Among these was the IL-8 type B receptor. Incubation of freshly isolated bovine parathyroid cells with recombinant IL-8 for 6-48 h produced an increase in the levels of mRNA for parathyroid hormone (PTH). The levels of PTH secreted in response to nanomolar amounts of IL-8 were also elevated in cells incubated for 1 h with IL-8. Differential display analysis of mRNA from parathyroid cells, incubated in the presence and absence of IL-8, permitted the identification of cDNA clones for RNA species whose expression was either elevated or suppressed. These experiments suggest that IL-8 and inflammatory events play a role in bone homeostasis through actions on the parathyroid gland.

Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry.

C. glutamicum meso-diaminopimelate dehydrogenase is an enzyme of the L-lysine biosynthetic pathway in bacteria. The binding of NADPH and diaminopimelate to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry. NADPH binding reduces the extent of deuterium exchange, as does the binding of diaminopimelate. Pepsin digestion of the deuterated enzyme and enzyme-substrate complexes coupled with liquid chromatography/mass spectrometry have allowed the identification of eight peptides whose deuterium exchange slows considerably upon the binding of the substrates. These peptides represent regions known or thought to bind NADPH and diaminopimelate. One of these peptides is located at the interdomain hinge region and is proposed to be exchangeable in the "open," catalytically inactive, conformation but nonexchangeable in the "closed," catalytically active conformation formed after NADPH and diaminopimelate binding and domain closure. Furthermore, the dimerization region has been localized by this method, and this study provides an example of detecting protein-protein interface regions using hydrogen/deuterium exchange and electrospray ionization.

Lymphoproliferative responses to human papillomavirus (HPV) type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia.

BACKGROUND: Infection with human papillomavirus (HPV) type 16 (HPV16) is a major cause of high-grade cervical intraepithelial neoplasia (CIN). Experiments were planned to evaluate the role of cell-mediated immunity (e.g., lymphocyte proliferation) against HPV in the natural history of HPV-associated neoplasia and to identify antigenic sequences of the HPV16 proteins E6 and E7 against which an immune response may confer protection. METHODS: Forty-nine women with abnormal cervical cytology and biopsy-confirmed CIN were followed through one or more clinic visits. Lymphoproliferative responses of peripheral blood mononuclear cells to HPV16 E6 and E7 peptides were assessed in long-term (3-week) cultures. HPV DNA was detected in cervicovaginal lavage by means of polymerase chain reaction and Southern blotting. Disease status was determined by cervical cytologic examination and colposcopy. Reported P values are two-sided. RESULTS: Subjects with positive lymphoproliferative responses to E6 and/or E7 peptides were more likely to be HPV negative at the same clinic visit than were nonresponders (P = .039). Subjects who were negative for HPV and those with a low viral load were more likely to be responders than were those with a high viral load (P for trend = .037). Responses to N-terminal E6 peptide 369 were associated with absence of HPV infection at the same clinic visit (P = .015). Subjects with positive responses to E6 or E7 peptides at one clinic visit were 4.4 times more likely to be HPV negative at the next visit than were nonresponders (P = .142). Responses to E6 peptide 369 and/or E7 C-terminal peptide 109 were associated with an absence of HPV infection (P = .02 for both) and an absence of CIN (P = .04 and .02, respectively) at the next visit. CONCLUSIONS: Lymphoproliferative responses to specific HPV16 E6 and E7 peptides appear to be associated with the clearance of HPV infection and the regression of CIN.

Localization of endogenous furin in cultured cell lines.

Furin is a dibasic endopeptidase responsible for the proteolytic maturation of many precursor proteins in the secretory and endocytic pathways of mammalian cells. The levels of furin expression in most cells are very low, and this has hampered attempts to identify the intracellular compartments in which endogenous furin is localized. We have used a specific antibody reagent to a sequence in the carboxy terminus of furin to perform immunofluorescent staining of mammalian cell lines. This antibody was sensitive enough to detect staining for furin in various cell lines. For the most part, furin staining was confined to a juxtanuclear structure characteristic of the Golgi complex. Analyses by video microscopy and confocal microscopy showed that the distribution of furin was distinct from that of mannosidase II, a marker of the Golgi stack, and most closely resembled that of TGN38, a marker of the trans-Golgi network. Therefore, our results suggest that endogenous furin is predominantly localized to the area of the Golgi complex, most likely within the trans-Golgi network.

The choroid plexus epithelium is the site of the organic anion transport protein in the brain.

The mRNA for organic anion transport protein (oatp) was previously shown to be present in abundance in liver and kidney, and in small amounts in brain. Data obtained from experiments with reverse transcriptase-PCR techniques and in situ hybridization analysis showed that the oatp mRNA is present within the brain, localized to the choroid plexus. A sequence-specific antibody to the oatp polypeptide demonstrated the presence of the expected polypeptide with a molecular weight of 80,000 plus an immunoreactive species with a higher molecular weight in preparations of choroid plexus membranes. Examination of the choroid plexus by fluorescence confocal microscopy revealed that immunoreactive oatp polypeptide is localized to the apical surface of the choroid plexus epithelial cells, which contacts the cerebrospinal fluid. This localization of oatp is consistent with previous experiments showing vectorial transport of organic anions between the choroid plexus and the cerebrospinal fluid.