scholarly journals Crystallographic evidence for oxidative Met→Asp conversion in human hemoglobin

2014 ◽  
Vol 70 (a1) ◽  
pp. C310-C310
Author(s):  
Jayashree Soman ◽  
Michael Strader ◽  
Wayne Hicks ◽  
Tigist Kassa ◽  
Eileen Singleton ◽  
...  
Keyword(s):  
Electron Density ◽  
Dose Dependence ◽  
Side Chain ◽  
Β Subunit ◽  
Α Subunit ◽  
In The Wild ◽  
Ferryl Species ◽  
Coordinated Water ◽  
Α Subunits

The mutants HbA Bristol-Alesha (βV(E11)67M) and HbF Toms River (γV(E11)67M) [1,2] are examples of a `silent' posttranslational modification in which the side chain of the substituted amino acid is chemically modified (Met→Asp) resulting in a disparity between the DNA and protein sequences. In both cases the patients' hemolysate contained both V67M and V67D isoforms. But in the analogous α subunit mutant, Hb Evans αV(E11)62M, the conversion to Asp was not identified and DNA sequencing confirmed the Met replacement [3]. Our crystal structures of the three (ferrous) CO-bound recombinant V(E11)M mutants show the MetE11 side chain in similar conformations. But the air-oxidized β mutant crystals clearly showed a `bifurcated' and smaller electron density pattern for the E11 side chain, indicating the appearance of Asp. Also, the ligand electron-density at the iron atom in the oxidized β subunit appears to be an oxoferryl Fe4+=O rather than a Fe3+OH2 ferric complex. In contrast, there was little change in the electron density for αMetE11 in oxidized αV62M crystals. The ligand in the ferric α subunit is clearly a coordinated water molecule. But again, a ferryl Fe4+=O complex appears to occur in the wild-type β subunit. This strongly suggest that β subunits have a greater propensity to form highly reactive ferryl species, and that the ferryl species play a role in the Met→Asp conversion. Our autoxidation and proteomics studies showed that although all three recombinant VE11M mutants had similar, high rates of autooxidation and a strong H2O2 dose dependence on sulfoxide and sulfone formation, no Asp formation was detected in α subunits whereas MetE11 is converted to Asp to levels as high as 15% in vitro in β and γ subunits. We propose that the Met→Asp conversion specifically involves H2O2 mediated oxidation of the ferrous heme to an oxoferryl state, and because the transient ferryl intermediates are much less stable in the α subunits, there is no oxidative conversion.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

scholarly journals Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

2017 ◽  
Vol 474 (5) ◽  
pp. 751-769 ◽  
Author(s):  
M. Kristian Koski ◽  
Jothi Anantharajan ◽  
Petri Kursula ◽  
Prathusha Dhavala ◽  
Abhinandan V. Murthy ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Coiled Coil ◽  
Β Subunit ◽  
Dimerization Domain ◽  
Α Subunit ◽  
Protein Dimer ◽  
Symmetric Molecule ◽  
Asymmetric Shape ◽  
Proline Rich Peptide ◽  
Α Subunits

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

scholarly journals Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their β subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 721-729 ◽  
Author(s):  
Johanna VEIJOLA ◽  
Pia ANNUNEN ◽  
Peppi KOIVUNEN ◽  
Antony P. PAGE ◽  
Taina PIHLAJANIEMI ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cloning And Expression ◽  
Β Subunit ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
C Elegans ◽  
Baculovirus Expression ◽  
Expression Studies ◽  
Α Subunits

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the β subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/β polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/β polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/β polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/β and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/β polypeptide forms an active prolyl 4-hydroxylase α2β2 tetramer with the human α subunit and an αβ dimer with the C. elegans α subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either α subunit. Removal of the 32-residue C-terminal extension from the C. elegans α subunit totally eliminated αβ dimer formation. The C. elegans PDI/β polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans α subunits than did the human PDI/β polypeptide, being particularly ineffective with the C. elegans α subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/β polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/β polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase αβ dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the Km for the hydroxylation of long polypeptide substrates.

Expression of Distinct α Subunits of GABAA Receptor Regulates Inhibitory Synaptic Strength

2004 ◽  
Vol 92 (3) ◽  
pp. 1718-1727 ◽  
Author(s):  
Pavel I. Ortinski ◽  
Congyi Lu ◽  
Kentaroh Takagaki ◽  
Zhanyan Fu ◽  
Stefano Vicini
Keyword(s):  
Primary Cultures ◽  
Molecular Assembly ◽  
Inhibitory Synapses ◽  
Critical Determinant ◽  
Pharmacological Agents ◽  
Α Subunit ◽  
Subunit Expression ◽  
Α Subunits ◽  
In Cells

Distinct α subunit subtypes in the molecular assembly of GABAA receptors are a critical determinant of the functional properties of inhibitory synapses and their modulation by a range of pharmacological agents. We investigated the contribution of these subunits to the developmental changes of inhibitory synapses in cerebellar granule neurons in primary cultures from wild-type and α1 subunit −/− mice. The decay time of miniature inhibitory postsynaptic currents (mIPSCs) halved between 6 days in vitro (DIV6) and DIV12. This was paralleled by the decrease of α2 and α3 subunits, the increase of α1 and α6 subunits expression at synapses, and changes in the action of selective α subunit modulators. A small but significant shortening of mIPSCs was observed with development in cells from −/− mice together with a decrease in the expression of α3 subunit. In contrast, the expression of α2 subunit at inhibitory synapses in −/− cells was significantly higher than in +/+ cells at DIV11-12. α5 subunit was not detected, and increased sensitivity to a selective α4/α6 subunit agonist suggests increased expression of extrasynaptic receptors in −/− mice. β2/β3 subunit expression and loreclezole sensitivity increased with development in +/+ but not in −/− cells, supporting the preferential association of the α1 with the β2 subunit. Synaptic charge transfer strongly decreased with development but was not different between cells in the +/+ and −/− groups until DIV11-12. Our results uncover a pattern of sequential expression of α subunits underlying the changes in functional efficacy of GABAergic networks with development.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

scholarly journals Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of metE and metH: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase

2000 ◽  
Vol 182 (19) ◽  
pp. 5539-5550 ◽  
Author(s):  
Paula S. Fritsch ◽  
Mark L. Urbanowski ◽  
George V. Stauffer
Keyword(s):  
Rna Polymerase ◽  
Random Mutagenesis ◽  
Complex Surface ◽  
Α Subunit ◽  
C Terminus ◽  
Distinct Cluster ◽  
In Vitro Reconstitution ◽  
The Difference ◽  
Α Subunits

ABSTRACT Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α2, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the αCTD on the β-associated α subunit at metH. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.

scholarly journals Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F 1 -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

2013 ◽  
Vol 12 (11) ◽  
pp. 1451-1461 ◽  
Author(s):  
Thuy La ◽  
George Desmond Clark-Walker ◽  
Xiaowen Wang ◽  
Stephan Wilkens ◽  
Xin Jie Chen
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Kluyveromyces Lactis ◽  
Β Subunit ◽  
Α Subunit ◽  
Content Type ◽  
Subunit Stoichiometry ◽  
Catalytic Sites ◽  
A Subunit

ABSTRACT F 1 -ATPase is a rotary molecular machine with a subunit stoichiometry of α 3 β 3 γ 1 δ 1 ε 1 . It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α 3 β 3 core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F 1 -ATPase have suggested that the α 3 β 3 core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F 1 -ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ o ) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F 1 complexes retained 21.7 to 44.6% of the native F 1 -ATPase activity. The γ-less F 1 subcomplex was assembled but was structurally and functionally labile in vitro . Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α 3 β 3 subcomplex.

scholarly journals Conversion of TSH Heterodimer to a Single Polypeptide Chain Increases Bioactivity and Longevity

Endocrinology ◽  
2012 ◽  
Vol 153 (2) ◽  
pp. 954-960 ◽  
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Fuad Fares
Keyword(s):  
Binding Affinity ◽  
Half Life ◽  
Chinese Hamster ◽  
Β Subunit ◽  
Wild Type ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Α Subunit

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and β genes with [TSHβ- carboxyl-terminal peptide (CTP)-α] or without (TSHβ-α) the CTP of human chorionic gonadotropin β-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHβ-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T4 in circulation after 2 and 4 h of treatment. However, the level of T4 after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHβ-α and TSHβ-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHβ-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

scholarly journals A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

1999 ◽  
Vol 341 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Jürgen KIRCHBERGER ◽  
Anke EDELMANN ◽  
Gerhard KOPPERSCHLÄGER ◽  
Jürgen J. HEINISCH
Keyword(s):  
Single Point ◽  
Size Exclusion ◽  
Single Point Mutation ◽  
Β Subunit ◽  
Western Blots ◽  
Α Subunit ◽  
Phosphofructokinase Activity ◽  
Atp Inhibition ◽  
Size Exclusion Hplc

Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data demonstrating that an alanine residue at positions 874 (for the PFK1-encoded α-subunit) or 868 (for the PFK2-encoded β-subunit) is crucial to achieve this structure. Thus subunits carrying substitutions by either aspartate or lysine of this residue cause a lack of phosphofructokinase activity in vitro and signals of the subunits are poorly detectable in Western blots. Size-exclusion HPLC in conjunction with ELISA detection of the enzyme protein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits, not being assembled, tend to aggregate and subsequently become degraded. Substitution of the alanine by valine in either subunit leads to a reduction in specific activities, as expected from a conservative exchange. The kinetic data of the latter mutant revealed a higher affinity to the substrate fructose 6-phosphate, a lower extent of ATP inhibition and a lower degree of activation by fructose 2,6-bisphosphate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the α- or the β-subunit to fructose 2,6-bisphosphate was increased. As no X-ray data on eukaryotic phosphofructokinases are available yet, our data provide the first evidence that a non-charge amino acid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structure of the well-known prokaryotic enzyme.

scholarly journals Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the α-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the β-subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 659-665 ◽  
Author(s):  
David C. A. JOHN ◽  
Neil J. BULLEID
Keyword(s):  
Heavy Chain ◽  
Binding Protein ◽  
Immunoglobulin Heavy Chain ◽  
Β Subunit ◽  
Intact Cells ◽  
Α Subunit ◽  
Free System ◽  
A Cell ◽  
Α Subunits

Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important α-subunit and the β-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an α2β2 tetramer from newly synthesized α-subunits associating with an endogenous pool of β-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled α-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018–14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized α-subunits were shown to assemble with the β-subunit, to form insoluble aggregates, or to remain soluble but not associate with the β-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and α-subunits were co-immunoprecipitated with antibodies against the α-subunit and BiP, respectively. These results indicate that unassembled α-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP.

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