Heme-binding Histidine Research Articles

Targeting Nox4 and Interleukin-6 crosstalk can be a potential strategy for Gastric Cancer prevention

Over 950000 million people are being affected in Gastric Cancer (GC) in each year. And so many patients are dying for its lethality. That means prognosis and treatment strategies are not well enough for its treatment. So, in the present scenario research work on this disease is truly significant and necessary. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase or NOX) is a family of enzyme consists of seven NOX isoforms members, such as- NOX1, NOX2, NOX3, NOX4, NOX5 and dual oxidase (DUOX) 1 and 2, that possess similarities in terms of enzyme function and structure. All these seven isoforms are transmembrane proteins (having six transmembrane domains) with a NADPH binding site, a FAD-binding site and four heme-binding histidines. Among these enzymes (NOX family), NOX4 is highly expressed in Gastric Cancer (GC) cells. As well as it was observed that high expression of NOX4 is correlated with worse overall survival (OS) in all GC patients. Interestingly, high mRNA expression of NOX4 indicates a worse OS in stage III/IV GC patients, but not in stage I/II GC patients. That is suggesting, NOX4 may contribute to the GC progression and play a crucial role in its (GC) late-stage. And it is observed that NOX4 is related with the cell invasion by regulating the JAK2/STAT3 signaling pathway. Functionally this enzyme family is leading with production of Reactive Oxygen Species (ROS) by utilizing NADPH. Few common examples of ROS are hydrogen peroxide (H2O2), superoxide anion (O2-) and hydroxyl radicles (OH) etc. These NOX producing ROS are connected to carcinogenesis as it is involved with so many cellular processes like cell proliferation, DNA damage and angiogenesis etc. On the other hand, recent studies show that Interleukin-6 (IL-6) is significantly related with the cell invasion and JAK2/STAT3 activation. That means both compounds (NOX4 and IL-6) are related with JAK2/STAT3 signaling pathway. And this JAK2/STAT3 signaling actually activates those genes which are associated with cell proliferation and anti-apoptosis. So, it is suggested that, targeting both Nox4 and IL-6 may be a fruitful strategy in GC treatment

Probing the Heme-Binding Site of the Cytochrome c Maturation Protein CcmE

Maturation of c-type cytochromes in many bacterial species and plant mitochondria requires the participation of the heme chaperone CcmE that binds heme covalently via a His residue (H130 in Escherichia coli) before transferring it stereospecifically to the apo form of cytochromes c. Only the structure of the apo form of CcmE is known; the heme-binding site has been modeled on the surface of the protein in the vicinity of H130. We have determined the reduction potential of CcmE, which suggests that heme bound to CcmE is not as exposed to solvent as was initially thought. Alanine insertions in the vicinity of the heme-binding histidine (which we showed by NMR do not perturb the protein fold) strikingly abolish formation of both holo-CcmE and cytochrome c, whereas previously reported point mutations of residues adjacent to H130 gave only a partial attenuation. The heme iron coordinating residue Y134 proved to be strictly required for axial ligation of both ferrous and ferric heme. These results indicate the existence of a conformationally well-defined heme pocket that involves amino acids located in the proximity of H130. However, mutation of Y134 affected neither heme attachment to CcmE nor cytochrome c maturation, suggesting that heme binding and release from CcmE are hydrophobically driven and relatively indifferent to axial ligation.

Gene expression and tissue distribution of cytoglobin and myoglobin in the Amphibia and Reptilia: Possible compensation of myoglobin with cytoglobin in skeletal muscle cells of anurans that lack the myoglobin gene

Cytoglobin (Cygb), a recently discovered vertebrate cytoplasmic heme-binding globin, is considered to be in a clade with vertebrate myoglobin (Mb), which is exclusively distributed in the cytoplasm of cardiac and skeletal muscles as an oxygen storage protein. GenBank databases (NCBI and JGI) and gene synteny analyses showed the absence of the Mb gene ( mb) in two anuran amphibians, Xenopus laevis and X. tropicalis. Here we conducted comparative studies on the gene expression and tissue distribution of Cygb and Mb in anuran and reptilian tissues. Cygb and Mb genes were cloned from a reptile, iguana ( Iguana iguana). Two types of cygb ( cygb-1 and -2) were cloned, with lengths of 1066 and 1034 bp, and 196 and 193 amino acid residues, respectively. Their nucleotide and amino acid sequence identities were 90 and 87%, respectively. The Mb gene covered 1416 bp with an open reading frame of 465 bp, giving rise to a 154 amino acid protein. The distal ligand-binding histidine at E7, the proximal heme-binding histidine at F8, and the phenylalanine residue at CD1 were conserved in Mb and Cygb. The nucleotide and amino acid sequence identity of I. iguana cygb-1 and cygb-2 against X. laevis cygb were approximately 67% and 65%, respectively. RT-PCR demonstrated that X. laevis cygb was uniquely expressed in the heart and skeletal muscles, and faintly in the liver and spleen, which was quite contrasted with Iguana and the other vertebrates, where mb is exclusively expressed in the heart and skeletal muscles. Immunohistochemical analyses showed the distribution of Cygb in the cytoplasm of skeletal muscle cells. Interestingly, Cygb in the heart was localized in the nuclei. Considering the absence of mb in the Anura, we hypothesize that Cygb in muscle cells of anurans compensates for the lack of Mb for the storage and intracellular transportation of oxygen.

Dynamic Ligation Properties of the Escherichia coli Heme Chaperone CcmE to Non-covalently Bound Heme

The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.

Heme Histidine Ligands within gp91phox Modulate Proton Conduction by the Phagocyte NADPH Oxidase

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.

Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression.

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.

Arabidopsis thaliana sequence analysis confirms the presenceof cyt b-561 in plants: Evidence for a novel protein family

Recent advances in the Arabidopsis sequencing project has elucidated the presence of two genes Atb561-A and Atb561-B that show limited homology to the DNA sequence encoding for the mammalian chromaffin granule cytochrome b-561 (cyt b-561). Detailed analysis of the structural features and conserved residues reveals, however, that the structural homology between the presumptive Arabidopsis proteins and the animal proteins is very high. All proteins are hydrophobic and show highly conserved transmembrane helices. The presumably heme-binding histidine residues in the plant and animal sequences as well as the suggested binding site for the electron acceptor, monodehydroascorbate, are strictly conserved. In contrast, the suggested electron donor (ascorbate) binding site is not very well conserved between the plant and animal sequences questioning the function of this motif. Sequence analysis of the Atb561-B gene demonstrates a different splicing than that initially predicted in silico resulting in a protein with nine extra amino acids and a significantly higher homology to the other cyt b-561 sequences. The homology between the plant and animal sequences is further supported by the strong similarity between a number of biochemical properties of the chromaffin cyt b-561 and the cyt b-561 isolated from bean hook plasma membranes. Since the mammalian cyt b-561 is considered specific to neuroadrenergic tissues, the identification of a closely related homologue in an aneural organism demonstrates that these proteins constitute a new class of widely occurring membrane proteins. Both the plant and animal cyt b-561 are involved in transmembrane electron transport using ascorbate as an electron donor. The similarity between these proteins therefore suggests, for the first time, that this transport supports a number of different cell physiological processes. An evolutionary relationship between the plant and animal proteins is presented.

The protein sequence of an archaeal catalase-peroxidase

The gene encoding a catalase-peroxidase of archaeal origin, the halophilic catalase-peroxidase from Haloarcula marismortui, was sequenced. The primary structure proposed was confirmed by Edman degradation and mass spectrometry analyses of proteolytic fragments of the purified protein. The open reading frame in the gene corresponds to 731 amino acids and the calculated mass of the mature protein (deleted of the N-terminal methionine) is 81253.65 Da, in reasonable agreement with the value of 81292±9 Da previously measured by mass spectrometry. Southern and Northern blot analyses showed that the protein is encoded by a single gene as a monocistronic transcript. The protein sequence shows a high level of identity with bacterial catalase-peroxidases, with strongly conserved regions around the heme binding histidines. Similarly to other soluble halophilic proteins, it shows the excess of acidic residues that has been associated with solvation in halophilic adaptation.

Amino-acid sequence of a cooperative, dimeric myoglobin from the gastropod mollusc, Buccinum undatum L.

The complete amino-acid sequence of a dimeric myoglobin from the radular mussel of the gastropod mollusc, Buccinum undatum L. has been determined. The globin 1, which shows cooperative binding of oxygen, contains 146 amino acids, is N-terminal aminoacetylated, and has histidine residues at position 65 and 97, corresponding to the heme-binding histidines seen in mammalian myoglobins. It shows about 75% and 50% homology, respectively, with the dimeric molluscan myoglobins from Busycon canaliculatum and Cerithidea rhizophorarum, the former of which also shows weak cooperativity, but much less similarity to other species of myoglobin and hemoglobin.

Identification of the histidine residues of hemopexin that coordinate with heme-iron and of a receptor-binding region.

Rabbit hemopexin cDNA was cloned from a rabbit liver lambda gt11 cDNA expression library using a mixture of five monoclonal antibodies raised against rabbit hemopexin, and the entire rabbit hemopexin sequence was determined. The heme-binding domain I of rabbit hemopexin (Smith, A., and Morgan, W. T. (1984) J. Biol. Chem. 259, 12049-12053) contains only 4 histidine residues which are conserved in rabbit, human, rat, and mouse hemopexin. The 2 axial heme-iron coordinating histidine residues, identified by Edman microsequencing and amino acid analyses of chemically modified domain I and isolated fragments of domain I, are the conserved histidine residues at positions 56 and 127 of the mature rabbit protein. The epitope recognized by JEN-14 (a monoclonal antibody which specifically reacts with domain I and blocks the hemopexin-receptor interaction (Morgan, W. T., Muster, P., Tatum, F. M., McConnell, J., Conway, T. P., Hensley, P., and Smith, A. (1988) J. Biol. Chem. 263, 8220-8225) was shown to lie between residues 122 and 142 by Western blotting of protease-digested domain I and transposon-insertion mutants of domain I expressed in a plasmid vector system. The location of this epitope near the heme-binding histidine residue 127 is compatible with a transport mechanism in which the release of heme from hemopexin is accompanied by a concomitant transfer of heme to the hemopexin receptor or the membrane heme-binding protein (Smith, A., and Morgan, W. T. (1985) J. Biol. Chem. 260, 8325-8329).

Does helix dipole have any role in binding metal ions in protein structures?

Positions of metal-binding residues with respect to helical terminii in protein structures have been analyzed in order to determine if the location of these ligands is influenced by the helix dipole. Most ligands do not show any preference for the amino- or carboxy-terminus of a helix. For steric reasons, peptide ligands can be located only at the C-terminus. The availability of a second ligand residue closely placed along the sequence may be of more importance, rather than the electrostatic interaction involving helix dipole, in cases where ligands are found near the C-terminus. The location of heme-binding histidine residues at the C-terminus may be due to the steric requirements of the heme group and also the intrahelical hydrogen bond that the residues can form at this position. Considerations based on such geometrical features and not just the helix dipole may help us to understand the observed distribution of charged residues along α-helices and the favorable role these amino acids have on folding of isolated helices.

Mutational and structural analysis of the nitrate reductase heme domain of Nicotiana plumbaginifolia.

We have analyzed four Nicotiana plumbaginifolia null mutants presumably affected in the heme domain of nitrate reductase. The DNA sequence of this domain has been determined for each mutant and for the wild type. Two mutations were identified as single base changes leading to, respectively, the substitution of a histidine residue by an asparagine (mutant E56) and to the appearance of an ochre stop codon (mutant E64). Based on the amino acid sequence homology between the nitrate reductase heme domain and mammalian cytochrome b5, we have predicted the three-dimensional structure of this domain. This showed that the nitrate reductase heme domain is structurally very similar to cytochrome b5 and it also confirmed that the residue involved in E56 mutation is one of the two heme-binding histidines. The two other mutations (mutants A1 and K21) were found to be, respectively, -1 and +1 frameshift mutations resulting in the appearance of an opal stop codon. These sequence data confirmed previous genetic and biochemical hypotheses on nitrate reductase-deficient mutants. Northern blot analysis of these mutants indicated that mutant E56 overexpressed the nitrate reductase mRNA, whereas the nonsense mutations present in the other mutants led to reduced levels of nitrate reductase mRNA.

Identification by nucleotide sequence analysis of a goat pseudoglobin gene.

We have recently prepared a recombinant library of goat genomic DNA and have isolated clones containing th known goat globin genes. These include the alpha, gamma, beta C and beta A genes. In addition to these, another beta-like sequence has been observed. In this communication we report the complete nucleotide sequence of this gene, excluding a portion of the large intervening sequence. Several features suggest that this is a non-functional or pseudoglobin gene. The alterations include a frameshift mutation, substitution of the heme-binding histidines, a mutated termination codon, a change in the GT/AG excision sequence of the 5' end of the first intervening sequence, an AT rich sequence in the 3' untranslated region, and a mutated Hogness-Goldberg box. We conclude that this gene cannot function in the synthesis of globin.

The primary structure of soybean leghemoglobin.

Publisher Summary This chapter focuses on primary structure of soybean leghemoglobin. Leghemoglobin is the only hemoglobin-like protein found in the plant kingdom. Leghemoglobin can be resolved by electrophoresis and ion exchange chromatography on DEAE cellulose into two main components of molecular weight about 17,000, each containing one heme group per molecule. The hydrolysis of the apoprotein by trypsin, chymotrypsin, and thermolysin, purification of the peptide fragments on Dowex and Dowex or by high-voltage electrophoresis and determination of the amino-acid sequences of these peptides have been described. The most striking feature of Lb a is the complete absence of sulfur-containing amino acids. Another characteristic is that it contains only one pair of histidine residues, which is the minimum number expected if they are assumed to be the heme-binding residues identical to those found in vertebrate hemoglobins. Leghemoglobin differs from vertebrate hemoglobins by having no basic center. The polypeptide chains of hemoglobin and myoglobin usually have one heme-binding histidine residue in the middle, in contrast to cytochrome c .