Principal Investigator

Mahin Maines, Ph.D. University of Rochester work Box 712 601 Elmwood Ave Rochester NY 14642 office: MC 5-8834 p (585) 275-5383 f (585) 275-6007

History

The mechanism of Biliverdin Reductase (BVR).

Mahin Maines' entire scientific career has been focused on the heme metabolic pathways, particularly the enzymes of the heme catabolism pathway; the heme oxygenases (HO) that she named HO-1 and HO-2, and biliverdin reductase (BVR). She has carried on with work on the HO system and BVR in parallel. Maines and coworkers’ pioneering contribution and continued leadership in heme metabolism field have been instrumental to recognition of potential application of heme degradation system and their products in therapeutic settings to treat human diseases. She joined the University of Rochester in 1985 as one of the first group of 5 Dean’s Professors, and the first female professor in the basic science departments in the medical school.

Maines' earlier work identified HO-1 as a distinct enzyme and one of the first enzymes to be characterized as stress-responsive gene products. Her initial discovery of the stress-inducible HO-1 resulted in her receiving an Irma T. Hirschl Career Scientist Award. This was followed by discovering the second steroid- inducible form, HO-2, containing O2, NO and CO binding, heme regulatory motif (HRM). The subsequent discovery of HO-2 and its function in generation of the neurotransmitter, CO, in the brain led to the NIH Story of Discovery Award, the Burroughs's Wellcome Scholar Award and an NIH Merit Award. HO-2 is now recognized as the cellular oxygen sensor and integral component of the Ca/K gated channels. The motif that was the laboratory identified as the catalytic pocket of the HO-1 and HO-2, was subsequently found in HO enzymes in all forms of life and is now referred to as the Heme Oxygenase Signature by GenBank. Recently in the laboratory, regulation of HO-2 expression was shown to occur by post-transcriptional mechanisms, and that BVR mediates the stability of both HO-2 mRNA and protein.

Secondary structure of the human biliverdin reductase monomer.

Maines and coworkers discoveries have paved the pathway for the novel application of the heme degrading system to molecular medicine. The identification of synthetic metalloporphyrins that selectively inhibit HO activity was essential for elucidating signaling and antioxidant functions of the HO activity products and presented a novel method for treatment of the neonatal jaundice. Similarly, finding that the induction of HO-1 delays rejection of the transplanted organs lead to the application of HO induction in transplant technology. Mapping the neuronal-specific expression of HO-2 and its apparent function in cGMP production in the brain and heart reinforced the biological functions of CO. In 1993 an editorial in Science highlighted work by the Maines laboratory on expression and function of HO-2 in the brain, which has been followed by hundreds of reports that have expounded on targeting the HO system as a therapeutic approach to disorders involving the cardiovascular and renal systems, Alzheimer's, inflammation ,allergy and regulation of ion channels. Collectively the findings paved the path for inclusion of the HO-1 as a recognized component of the signal transduction pathways.

The laboratory's avant-garde discoveries were instrumental in metamorphosis of the system from a molecular wrecking ball for disposal of heme to a mesmerizing one and her depiction as the guiding light of heme oxygenase (Lane, The Sciences, 1998). In 2012, the 7th International Heme Oxygenase Conference will take place in Edinburgh, UK.

Schematic presentation of consensus sequences of hBVR for which functions have been ascribed. The numbers indicated for each consensus sequences are those of the hBVR amino acid sequence. The N-terminal segment of 99 residues is the catalytic domain of hBVR; it houses a sequence of four valines followed by the consensus for the ATP/adenine ring-binding site. The kinase activity of hBVR is responsible for its autophosphorylation. hBVR is also a kinase for serine phosphorylation of IRS 1, the phosphorylation of which halts glucose uptake. hBVR is also a kinase for T500 in the activating loop of PKC-βII 2 - the PKC is a key component of cell growth and differentiation. The reductase domain catalyzes reduction of biliverdin to bilirubin, a component of cellular defense mechanisms protecting against ROS and apoptosis. The sequences designated by one or two asterisks closely resemble sites in the primary sequence of repeats V (QAMLWDLNE) and VI (SIKIWDLE) of the receptor for activated C-kinase-1 (RACK1), respectively. RACK1 is a 36 kDa protein that is similar in size to hBVR. Activation of PKCs is associated with conformational change that exposes its RACK-binding site and we predict that this allows association of the kinases with the homologous sites in hBVR. The binding would not require kinase activity of hBVR. The b-Zip motif binds to 7 and 8 bp AP-1 and AP-2sites. Stress response genes are activated by AP-1, and cAMP responsive genes are regulated by AP2 regulatory elements. hBVR regulates expression of stress responsive HO-1, c-Fos, c-Jun and ATF2/CREB. Within this sequence is a motif that strongly resembles a conserved protein kinase motif. The high affinity ERK binding site, also known as CBox or DEF, is the site of interaction of ERK1/2 and hBVR, positioning ERK in proximity to its kinase, MEK. Nuclear localization of hBVR is also critical for transport of the transcriptional regulators ERK1/2 and heme into the nucleus. Reentry of ERK into the cytoplasm requires the intact hBVR NES. hBVR is directly phosporylated by IRK upon activation by insulin or IGF-1. The tyrosine in the SH2 recognition motif of hBVR, as with other SH2 recognition motif containing proteins, is predicted to form a platform for formation of signaling complexes. hBVR is phosphorylated by ERK and Motif Scan predicts serine in the SP sequence as the phosphorylation target site of ERK1/2. A second SH2 recognition motif follows the nuclear localization signal and is involved in activation of PKC-ζ by TNF-α. The low affinity D-Box-like sequence is the binding site for kinases and substrates in the MAPK signaling cascade. The C-terminal 6 residues are the Zn-binding domain of hBVR. Based on a report that Zn is essential for plasma membrane translocation of PKCs and nuclear translocation transport of NF-κB we predict that the function of hBVR in translocation of PKCs β and ζ to the cell membrane involves the membrane targeting activity of hBVR. Notably, hBVR under resting conditions is found in the cytoplasm and membrane caveolae. The C-terminal lysine 296 is critical for hBVR's catalytic activity (unpublished); although it lies in a disordered region of the BVR molecule, this does not preclude a catalytic function. The most recent findings indicate that hBVR binds and activates PKC-δ, and is necessary for activation of ERK2 by PKC-δ. It is apparent that hBVR is likely to affect a wide range of cellular processes.

The Maines lab's effort to characterize BVR is now on its 26th year of continuous support by NIH. The enzyme that intrigued the laboratory because of it having a dual cofactor –dual pH activity profile (a profile that is not display by any other enzyme characterized to date), has proven to be a pleiotropic enzyme with a range of functions in the cell that is unmatched by any. This includes being a dual-specificity (S/T/Y) kinase, a molecular scaffold, intracellular transporter of kinases and gene regulators, DNA-binding and transcriptional activator; and, of course, being a reductase for conversion of biliverdin to the antioxidant, bilirubin. The primary and secondary features and activities of BVR that so far have been uncovered have been depicted in the above figures.