Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Linda J. Roman is active.

Publication


Featured researches published by Linda J. Roman.


Journal of Biological Chemistry | 1998

INDUCIBLE NITRIC-OXIDE SYNTHASE GENERATES SUPEROXIDE FROM THE REDUCTASE DOMAIN

Yong Xia; Linda J. Roman; Bettie Sue Siler Masters; Jay L. Zweier

In the absence of l-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O⨪2). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O⨪2 formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O⨪2production does not occur. Moreover, the mechanism and enzyme site responsible for O⨪2 generation is unknown. To determine whether iNOS produces O⨪2 and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrrolineN-oxide. In the presence of NADPH, prominent O⨪2adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of l-arginine decreased this O⨪2formation, whereas its enantiomer d-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O⨪2 signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O⨪2 generation. Furthermore, strong O⨪2 generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O⨪2, and this mainly occurs at the flavin-binding sites of the reductase domain.


Journal of Biological Chemistry | 1997

An Autoinhibitory Control Element Defines Calcium-regulated Isoforms of Nitric Oxide Synthase

John C. Salerno; Dawn E. Harris; Kris Irizarry; Binesh Patel; Arturo J. Morales; Susan M. E. Smith; Pavel Martásek; Linda J. Roman; Bettie Sue Siler Masters; Caroline L. Jones; Ben Avi Weissman; Paul Lane; Qing Liu; Steven S. Gross

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]N G-nitro-l-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin’s ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.


The FASEB Journal | 1996

Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structure studies of a cysteine thiolate-liganded heme protein that hydroxylates L-arginine to produce NO. as a cellular signal.

Bettie Sue Siler Masters; Kirk McMillan; Essam A. Sheta; Jonathan S. Nishimura; Linda J. Roman; Pavel Martásek

The nitric oxide synthases (NOS‐I, neuronal, NOS‐II, inducible, and NOS‐III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate‐liganded protoporphyrin EX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L‐arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca2+‐calmodulin and/or tetrahydrobiopterin (BH4).—Masters, B. S. S., McMillan, K., Sheta, E. A., Nishimura, J. S., Roman, L. J., Martasek, P. Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structural studies of a cysteine thiolate‐li‐ganded heme protein that hydroxylates L‐arginine to produce NO· as a cellular signal. FASEB J. 10, 552‐558 (1996)


Journal of Biological Chemistry | 2000

The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin

Linda J. Roman; Pavel Martásek; R. Timothy Miller; Dawn E. Harris; Melissa de la Garza; Thomas M. Shea; Jung Ja P Kim; Bettie Sue Siler Masters

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20–40 residues longer in the C terminus, forming a “tail” that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7–21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.


Journal of Biological Chemistry | 2002

The role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide.

Gerald M. Rosen; Pei Tsai; John Weaver; Supatra Porasuphatana; Linda J. Roman; Anatoly A. Starkov; Gary Fiskum; Sovitj Pou

Tetrahydrobiopterin (H4B) is a critical element in the nitric-oxide synthase (NOS) metabolism ofl-arginine to l-citrulline and NO⋅. It has been hypothesized that in the absence of or under nonsaturating levels of l-arginine where O2 reduction is the primary outcome of NOS activation, H4B promotes the generation of H2O2 at the expense of O 2 ⨪ . The experiments were designed to test this hypothesis. To test this theory, two different enzyme preparations, H4B-bound NOS I and H4B-free NOS I, were used. Initial rates of NADPH turnover and O2 utilization were found to be considerably greater in the H4B-bound NOS I preparation than in the H4B-free NOS I preparation. In contrast, the initial generation of O 2 ⨪ from the H4B-free NOS I preparation was found to be substantially greater than that measured using the H4B-bound NOS I preparation. Finally, by spin trapping nearly all of the NOS I produced O 2 ⨪ , we found that the initial rate of H2O2 production by H4B-bound NOS I was considerably greater than that for H4B-free NOS I.


Journal of Biological Chemistry | 2006

Electron transfer by neuronal nitric-oxide synthase is regulated by concerted interaction of calmodulin and two intrinsic regulatory elements

Linda J. Roman; Bettie Sue Siler Masters

The nitric-oxide synthases (NOSs) are modular, cofactor-containing enzymes, divided into a heme-containing oxygenase domain and an FMN- and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaM-CC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.


Journal of Biological Chemistry | 2013

Nox4 NADPH Oxidase Mediates Peroxynitrite-Dependent Uncoupling of Endothelial Nitric Oxide Synthase and Fibronectin Expression in Response to Angiotensin II. Role of Mitochondrial Reactive Oxygen Species

Doug Yoon Lee; Fabien Wauquier; Assaad A. Eid; Linda J. Roman; Goutam Ghosh-Choudhury; Khaled Khazim; Karen Block; Yves Gorin

Background: Oxidative stress is critical for the fibrotic response of mesangial cells (MCs) to angiotensin II. Results: Nox4- and mitochondrial reactive oxygen species (ROS)-dependent endothelial nitric-oxide synthase (eNOS) uncoupling led to fibronectin accumulation in MCs stimulated by angiotensin II. Conclusion: The Nox4/mitochondrial ROS/eNOS pathway mediates angiotensin II-induced MC injury. Significance: Targeting Nox4 and mitochondrial ROS is a promising therapeutic approach. Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis.


Journal of Biological Chemistry | 2000

The C terminus of mouse macrophage inducible nitric-oxide synthase attenuates electron flow through the flavin domain.

Linda J. Roman; R. Timothy Miller; Melissa de la Garza; Jung Ja P Kim; Bettie Sue Siler Masters

The sequences of nitric-oxide synthase (NOS) flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR), with the exception of a few regions. One such region is the C terminus; all NOS isoforms are 20–40 amino acids longer than CPR, forming a “tail” that is absent in CPR. To investigate its function, we removed the 21-amino acid C-terminal tail from murine macrophage inducible NOS (iNOS) holoenzyme and from a flavin domain construct. Both the truncated holoenzyme and reductase domain exhibited cytochrome c reductase activities that were 7–10-fold higher than the nontruncated forms. The truncated holoenzyme catalyzed NO formation approximately 20% faster than the intact form. Using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins and from the flavin to the heme domain is 2–5-fold faster in the absence of the C-terminal tail. The heme-nitrosyl complex, formed in all NOS isoforms during NO catalysis, is 5-fold less stable in truncated iNOS. Although both CPR and intact NOS can exist in a stable, one electron-reduced semiquinone form, neither the truncated holoenzyme nor the truncated flavin domain demonstrate such a form. We propose that this C-terminal tail curls back to interact with the flavin domain in such a way as to modulate the interaction between the two flavin moieties.


Journal of Medicinal Chemistry | 2009

Discovery of highly potent and selective inhibitors of neuronal nitric oxide synthase by fragment hopping

Haitao Ji; Huiying Li; Pavel Martásek; Linda J. Roman; Thomas L. Poulos; Richard B. Silverman

Selective inhibition of neuronal nitric oxide synthase (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. This study shows that not only greater inhibitory potency and isozyme selectivity but more druglike properties can be achieved by fragment hopping. On the basis of the structure of lead molecule 6, fragment hopping effectively extracted the minimal pharmacophoric elements in the active site of nNOS for ligand hydrophobic and steric interactions and generated appropriate lipophilic fragments for lead optimization. More potent and selective inhibitors with better druglike properties were obtained within the design of 20 derivatives (compounds 7-26). Our structure-based inhibitor design for nNOS and SAR analysis reveal the robustness and efficiency of fragment hopping in lead discovery and structural optimization, which implicates a broad application of this approach to many other therapeutic targets for which known druglike small-molecule modulators are still limited.


Annals of Neurology | 2009

Selective neuronal nitric oxide synthase inhibitors and the prevention of cerebral palsy

Haitao Ji; Sidhartha Tan; Jotaro Igarashi; Huiying Li; Matthew Derrick; Pavel Martásek; Linda J. Roman; Jeannette Vasquez-Vivar; Thomas L. Poulos; Richard B. Silverman

To design a new class of selective neuronal nitric oxide synthase (NOS) inhibitors, and demonstrate that administration in a rabbit model for cerebral palsy (CP) prevents hypoxia‐ischemia–induced deaths and reduces the number of newborn kits exhibiting signs of CP.

Collaboration


Dive into the Linda J. Roman's collaboration.

Top Co-Authors

Avatar

Bettie Sue Siler Masters

University of Texas Health Science Center at San Antonio

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Huiying Li

University of California

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Haitao Ji

Northwestern University

View shared research outputs
Top Co-Authors

Avatar

Satya Prakash Panda

University of Texas Health Science Center at San Antonio

View shared research outputs
Top Co-Authors

Avatar

John C. Salerno

Kennesaw State University

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Fengtian Xue

Northwestern University

View shared research outputs
Top Co-Authors

Avatar

Jonathan S. Nishimura

University of Texas Health Science Center at San Antonio

View shared research outputs
Researchain Logo
Decentralizing Knowledge