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REVIEW
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York
mahin_maines{at}urmc.rochester.edu
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Abstract |
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Introduction |
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 Top Introduction BVR: Structure, Regulation, and...Biliverdin Reductase: An...Regulation of Oxidative Response...Role in Cell Signaling:...ConclusionReferences |
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BVR: Structure, Regulation, and Reductase Activity |
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TopIntroductionBVR: Structure, Regulation, and...Biliverdin Reductase: An...Regulation of Oxidative Response...Role in Cell Signaling:...ConclusionReferences |
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). The average sequence identity between mammalian species is >80%, with conservation of certain key features (13, 24, 36) denoted in FIGURE 1. Among those are the leucine zipper (bzip) motif (LX6LX6L/KX6LX6L), adenine dinucleotide-binding motif (GXGXXG), serine/threonine kinase domain [G(T/S)XX(F/Y)XAPE], Src homology (SH2)-binding domains (YMXM and YSLF), and Zn/metal-binding motif (H/CX10C·C/H). These features, as discussed below, are likely to have key function(s) in cell-signaling activities of BVR.
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). The first two residues of BVR are deleted during the maturation process (13). The addition of an extra phosphoacceptor residue, threonine, could potentially affect structure, function, or stability of the protein.
BVR reduces C10 (
-bridge) of biliverdin IX
, a product of heme (Fe-protoporphyrin IX) degradation by heme oxygenase (HO) isozymes HO-1 and HO-2, which catalyze the isomer-specific cleavage of heme at the -methene carbon bridge. Plants use biliverdin IX, produced by ferredoxin-dependent HO (3), to synthesize phytochromes, the sensory photoreceptors, to modulate their growth. The reaction, as characterized for Arabidopsis, depends on a ferredoxin-dependent BVR (23).
Although the structural basis for the unique dual pH/cofactor-dependence activity profile of BVR remains unsolved, substituting serine residues with alanine and solving the secondary structure of the rat BVR-NADH complex have offered some clues and have identified key residues in reductase activity. The primary structure of the rat BVR (13) and crystal structure of the rat BVR-enzyme-cofactor complex (22, 60) have implicated the NH2-terminal domain (Rossman fold) in dinucleotide binding. There is an extensive interaction between the two domains of BVR, the NH2-terminal (the cofactor-binding domain) and the COOH-terminal ß-sheet of BVR (22, 60) (FIGURE 2). Point mutation of residues that in BVR interact with the adenine nucleotide, including G17 (in the Walker homology domain), S149 [in the serine/threonine kinase domain (21)], and K92·H93 dipeptide [in "oxidoreductase" domain AGKHVLVEY (30)], all inactivate the reductase. Among these, S149 has proven to be essential for activity (30, 41, 51). Additionally, changing C73 in the rat BVR (C74 in hBVR) to alanine inactivates the enzyme, as it is involved in substrate/cofactor binding (40). These mutations have nearly the same negative impact on activity with both NADPH and NADH. This contrasts with loss of S44, which results in a nearly fourfold increase in only the NADH-dependant activity, mainly reflecting an increase in the Vmax of the mutant BVR, due to reduced hindrance to NADH binding and NAD release. A noteworthy finding is that in human renal carcinoma BVR activity is increased, but only with NADH (35). The significance and cause of this increase in activity are not clear, but it would not be advantageous for the host; rather, it would advance growth of the malignant cells.
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BVR is the product of a single-copy gene that, in the rat, is 17,025 bp in length and consists of seven exons and six introns (38) (GenBank). The human BVR gene has eight exons, with the initiation codon in exon 2. Exon 5 includes the most strongly conserved motif, which is found in every oxidoreductase (30).
In the rat, the ~1.6-kb transcript is expressed sparingly in organs such as the testis and the thymus but abundantly in others such as the kidney, spleen, liver, and brain. The promoter region of the human and rat genes contain consensus sequence elements (38) associated with regulation of transcriptional activity, embryonic gene expression, and response to hyperthermia (12, 39). In addition to hyperthermia, cytokines and LPS induce BVR transcription (33).
hBVR expression is downregulated by the zinc-finger hematopoietic transcription factor GATA1 (7, 15) and upregulated by heme (Gibbs PEM and Maines MD, unpublished observation). GATA1 is a key factor in lineage-specific development of stem cells and may be indicative of a function for BVR in the establishment of gene-expression profiles in developing stem cells. GATA2, a marker of skeletal muscle hypertrophy (44), does not affect BVR transcription.
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Biliverdin Reductase: An Intracellular Transporter? |
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TopIntroductionBVR: Structure, Regulation, and...Biliverdin Reductase: An...Regulation of Oxidative Response...Role in Cell Signaling:...ConclusionReferences |
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The proposed function of BVR in protein:protein interaction and intracellular transport of signaling factors is based on its kinase activity, its primary structural features, and the secondary structure of the COOH-terminal domain as a large ß-sheet made of six strands; such a structure characterizes a monomer-monomer interface site, similar to those found in intracellular-trafficking "scaffold" proteins. Notably, two copies of the CX10C motif are present in the C1 domain of PKCs (42, 46). Oxidation of cysteine residues to the disulfide form (or covalently bonding with -SH-reactive compounds) prevents BVR dimerization and reductase activity (27). Homodimeric BVR is incapable of docking with other proteins, and this could arise from exposure to factors that affect the oxidation state of the cell and/or have affinity for sulfhydryls. Protein sulfhydryls are a target for the NO· radical (4), for example.
Over two decades ago, when little was known about the primary and secondary structure of BVR, observations were made that were unexplainable at the time but when revisited in light of the current information offer support for the function of cysteine residues in BVR protein:protein interactions. In one study rat BVR was characterized as "extremely sensitive" to -SH-reactive reagents, although biliverdin was found to fully protect the enzyme (27). Another observation (16) was the "interconversion" of two molecular forms of rat liver BVR; a ~34-kDa form was converted to a larger molecular form (~68 kDa) when rats were treated with -SH-reactive agents. Reduced thioredoxin, an agent that can reduce disulfide bonds to sulfhydryl groups, could reverse the conversion. The larger form was found to lack sensitivity to sulfhydryl reagents. These observations would suggest occurrence of dynamic interchange between mono- and dimeric forms of BVR as the consequence of sulfhydryl disulfide interconversion in vivo.
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Regulation of Oxidative Response and HO-1 Expression |
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TopIntroductionBVR: Structure, Regulation, and...Biliverdin Reductase: An...Regulation of Oxidative Response...Role in Cell Signaling:...ConclusionReferences |
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) (33). Gene-array analysis suggests that BVR activates a large number of genes in cell-signaling pathways, immune, and stress-response, and further analysis showed that, indeed, ho-1 and activating transcription factor (ATF-2)/cAMP response element (CRE) (25) are controlled by BVR. The ability of BVR to activate expression of these genes was established in 293A cells from human embryonic kidney transfected with an adenoviral construct containing BVR. BVR is a bzip-type transcription factor that binds in dimeric form to AP-1 sites. Activation of AP-1 is the key event in oxidative stress response of ho-1 and other stress proteins (54).
BVR regulates cellular levels of biliverdin, a potent gene regulator as illustrated by its being the determinant factor for dorsal axis development in Xenopus larva, by the suppression of PKC isozymes, by inhibition of BVR binding to AP-1 regulatory elements in the promoter of ho-1, and by activation of Ah receptor (1, 14, 41, 50). On an equimolar basis (50 µM), biliverdin is as potent an inhibitor of PKC (, ß, , mixture) as a commercially available PKC inhibitory peptide (RKRCLRRL). At this concentration, biliverdin inhibits PKC activity by 95%; bilirubin, a known inhibitor of PKC (19), causes 50% inhibition of PKC activity.
The occurrence of a regulatory loop in the cell to control the heme degradation activity of HO-1, which would involve a combination of negative product-feedback processes, i.e., inhibition of heme oxidation by biliverdin and reduction of the latter by bilirubin (26, 27)—in other words, coupled catalytic activity of BVR and HO-1—can be considered. HO-1 is an early-response oxidative-stress gene. BVR is also activated by oxidants (33, 41, 51). Accelerated rate of conversion of biliverdin to bilirubin, i.e., inactivation of biliverdin, allows for induction of ho-1 expression and increased heme-degradation activity (26); the subsequent negative feedback inhibition of BVR activity by its product (bilirubin) would allow for buildup of biliverdin levels, causing product (biliverdin) inhibition of the oxygenase, hence permitting a return to normal conditions of the heme degradation (27). A direct link between BVR and the ho-1 oxidative-stress response became evident by attenuated responses of ho-1 to superoxide anion and arsenite in cells treated with antisense BVR or small interference (si) BVR (1, 41).
In real-life settings, BVR activity may become crucial to survival. For instance, under hemolytic conditions or induction of ho-1 expression, a large amount of heme is degraded, requiring high levels of BVR to prevent biliverdin levels from rising beyond normal physiological levels. If not available, then the fatal "green jaundice" would occur (18). Biliverdin, unlike bilirubin, is not lipophilic and does not cross the cell membrane lipid bilayer. In fact, in earlier days, BVR was considered to be specific to placental animals, allowing elimination of the heme degradation product from the fetus. The fatality could reflect both an attenuated immune and oxidative stress response for induction of cytoprotective genes and the deficiency in production of bilirubin, a potent antioxidant in the cell (57). It is relevant that exceedingly high concentrations of biliverdin can accumulate as a consequence of exposure to compounds that disrupt cell-signaling pathways (31).
A second mechanism by which BVR is likely to affect ho-1 gene expression would involve its heterodimerization with other members of the bzip family of transcription factors, such as c-Jun, c-Fos, ATF-2/CREB, Myc, and Bach-1; all are able to form heterodimeric complexes. The bzip motif of BVR is involved in DNA binding (1). BVR is a regulator of c-jun and atf-2/creb gene expression (25). In the case of ATF-2, when its levels are increased, it effectively competes with c-Fos, the usual dimer partner of c-Jun. The ATF-2/c-Jun heterodimer preferentially binds to the seven-base AP-1 sites (TGACTCA) rather than the usual site of ATF-2, CRE (TGACNT-CA). The ATF-2/c-Jun dimer DNA complex is more stable than the c-Fos/c-Jun DNA complex. Moreover, heterodimerization not only alters ATF-2 binding with remarkable variation in affinity for different AP-1/CRE sites but also alters gene-regulation activity of the dimeric partner. The identity of the association partner likely will result in a wide spectrum of changes in the cell.
That BVR regulates ho-1 expression by controlling gene-repressor activity of the hypoxia-inducible factor Bach-1 must be considered. This heme-regulated transcription-repressor factor is a bzip factor with demonstrated ability to form a heterodimer with a small Maf protein, an activator of gene expression from AP-1/CREB recognition sites (6). The heterodimer prevents Maf by recognizing the MARE sequence motif in DNA (47). Because BVR is also a bzip protein that binds to AP-1/CRE elements (1, 41), and Maf proteins heterodimerize with the AP-1 family of transcription factors (6), formation of a complex between BVR and Maf would block the repressor Bach-1 activity and allow induction of ho-1 expression, consistent with that seen in ischemia/reperfusion injury (37).
BVR may also regulate ho-1 gene expression using a site on BVR for binding metalloporphyrins distinct from its biliverdin binding site (5). Depending on the chelated metal, metalloporphyrins can activate or inhibit BVR; iron hematoporphyrin was characterized as an inhibitor of the reductase (5), whereas cobalt protoporphyrin is an activator of the reductase. Metalloporphyrin complexes regulate expression of ho-1 as well as a vast number of genes including ALA synthase, the rate-limiting enzyme in its biosynthesis (17).
A wide range of functions in the cell are subject to changes in HO-1 activity, including those controlled by nitric oxide radicals (8, 10, 11, 32, 34, 43, 48, 52). Therefore, interplay between BVR, its substrate, and its product in the regulation of the stress response of ho-1 would have a major impact on the cell.
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Role in Cell Signaling: Kinase Activity |
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TopIntroductionBVR: Structure, Regulation, and...Biliverdin Reductase: An...Regulation of Oxidative Response...Role in Cell Signaling:...ConclusionReferences |
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FIGURE 3 shows the insulin- and MAPK-signaling pathways and the junctures already demonstrated to be influenced by BVR. Insulin/insulin-like growth factor (IGF) action is mediated through activation of the insulin receptor (IR/IGFR), which is a disulfide-linked 2ß2 heterotetrameric complex. Insulin binds to the ß-subunits, and tyrosine phosphorylation of the ß-subunit activates the receptor, which enables biological responses, including metabolic processes such as glucose uptake and changes in carbohydrate and lipid or protein metabolism, as well as mitogenic processes such as alteration in growth, differentiation, DNA synthesis, and regulation of gene expression. Additionally, signaling in B cells through Toll-like receptors and NF-
B activation. NF-B is activated by PKC (conventional forms) as well as the MAPK/p38 pathway. Activated IR first phosphorylates tyrosine residues of cytoplasmic substrates. At least 11 such substrates of IR have been identified, 6 belonging to the insulin receptor substrate family (IRS-1 through -6). These docking proteins, through their pleckstrin homology domains, interact with SH2 domain-containing proteins, including phosphatidylinositol 3-kinase (PI3K), Grb-2, and Ship-2 (2, 62).
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): the MAPK pathway is associated mostly with transcriptional and mitogenic effects of insulin/IGF, and the PI3K pathway is involved in metabolic signaling. The balance between the strength of these two major pathways determines which downstream targets are favored (20). Recent findings suggest a key role for BVR in both pathways as well as regulating PKC isoforms that link the two pathways (1, 29, 41). Many insulin responses associated with cell growth and glucose metabolism are mediated through activation of IR substrates, IRS-1 and IRS-2 complexes (61), and tyrosine phosphorylation of their YMXM motifs. The phosphotyrosine-binding motif of IRS proteins interacts with the NPXY motif of IR (49). There are two potential SH2 protein-docking sites in BVR for PI3K (45), and Y198MKM is predicted to be an ideal binding site for the regulatory subunits of PI3K. The second, the Y228LSF motif, meets criteria for optimum tyrosine phosphorylation in proteins that assemble into a multi-protein complex that recruits and/or facilitates relocation by SH2 domain-containing polypeptides (56). PI3K most prominently factors in cell signaling; the SH2 domains of the p85 regulatory subunit of the kinase interacts with tyrosine-phosphorylated motif in receptors, such as Toll-like membrane glycoproteins and cytoplasmic 3-phosphoinositide-dependent kinases.
BVR is found mainly in the cytoplasm, although activation/hyperphosphorylation of the reductase leads to nuclear translocation (33). Translocalization within the cell is relevant to BVR’s function in transducing signals generated at the level of cell membrane receptors to the chromatin and to its function as an anchoring/docking protein. In the COOH-terminal domain of the protein, between Y198MKM and Y228LSF, there is a sequence that contains a number of positively charged residues, K219GPGLKRNR (FIGURE 1), that are essential for nuclear localization and form a potential myristoylation and membrane phospholipid-binding site (9). The GPG tripeptide sequence preceding the charged residues permits maximum flexibility of the BVR polypeptide. A change in conformation of a kinase can function both in directing proteins to subcellular targets and in modulating their activity. Replacement of positively charged residues in this sequence abrogates nuclear localization of BVR (33). In the primary structure of BVR, a leucine-rich sequence (LX2LX2LX3LXL) is present that is likely sufficient for nuclear export of BVR and associated proteins. This sequence is similar to the nuclear export signal that triggers rapid, active extrusion of the catalytically active subunit of cAMP-dependent kinase and rev, an RNA-binding protein of HIV-1 (59).
Serine phosphorylation of the IRS proteins enables interaction with IR and intermolecular docking. Insulin resistance has been linked to serine phosphorylation of IRS-1. Three observations suggest a role for BVR in the mechanism of insulin resistance (29). The presence of IRS increases phosphorylation of BVR by IRK; BVR directly phosphorylates IRS on serine residues; and insulin-mediated glucose uptake is increased when BVR expression is knocked down using siBVR mRNA. Consistent with this concept, BVR phosphorylates IRS-1 peptides at sites known to negatively affect glucose uptake, and, under conditions unfavorable to its autophosphorylation, phosphorylation of BVR is increased when both BVR and IRS-1 are available to IRK, reflecting what is most likely direct interaction of BVR and IRS proteins.
As the second major arm for insulin signaling, activation of the MAPK pathway primarily activates substrates that function in gene expression/mutagenesis; therefore, activation of BVR by IRK could affect a wide spectrum of functions in the cell, consistent with previous reports (25). Of the MAPK family (ERK, p38, JNK/SAPK), ERK is primarily activated by growth factors and phorbol esters and is associated with proliferation and differentiation of cells, whereas JNK/SAPK and p38 are activated by extracellular stress and cytokines. BVR clearly has a regulatory role in the stress-response pathway of the MAPK cascade (1, 25, 41). Ablation of BVR by siRNA causes a four- to fivefold increase in the number of cells that undergo apoptosis concomitant with an increase in the levels of factors associated with apoptosis after treatment with sodium arsenite (41).
Further evidence for involvement of BVR in both arms of IR/growth factor signaling follows from our initial findings that BVR has PKB/Akt-like activity and activates PKC enzymes as well (Miralem T, Lerner-Marmarosh N, and Maines MD, unpublished observations); PKB is a key mediator of signal-transduction processes, stimulates cell proliferation, and inhibits apoptosis (28). PKB-like activity of BVR is reflected by the finding that BVR transfection of the normally undifferentiated MCF7 breast cancer cells causes them to display morphological characteristics of differentiated cells (FIGURE 4). This figure also shows that it does not appear to be cell line specific as denoted by the profound change in morphology of HeLa cells transfected with BVR.
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Conclusion |
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TopIntroductionBVR: Structure, Regulation, and...Biliverdin Reductase: An...Regulation of Oxidative Response...Role in Cell Signaling:...ConclusionReferences |
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Acknowledgments |
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This work was supported by National Institutes of Health grants RO1-ES-04066, RO1-NS-41043, and RO1-ES-12187.
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References |
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TopIntroductionBVR: Structure, Regulation, and...Biliverdin Reductase: An...Regulation of Oxidative Response...Role in Cell Signaling:...ConclusionReferences |
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