Genomes and Genes
Isoform specific effects of the autoinhibitory element and the C-terminus of nitr
Principal Investigator: JOHN SALERNO
Abstract: Nitric oxide synthases play multiple roles in such diverse physiological processes as control of vascular tone, signal transduction in the central nervous system, and immune response. Temporally and/or spatially inappropriate production of nitric oxide (NO) leads to several different pathologies. The endothelial isoform of NOS (eNOS) controls vascular constriction and dilation and has effects on platelet aggregation, with pathologies including hypertension and other vascular diseases. Autoimmune mechanisms trigger NO production by iNOS, leading to apoptosis of pancreatic beta cells in both insulin-dependent and non-insulin-dependent diabetes mellitus. NOS is a large modular enzyme with a heme containing oxygenase domain and a three domain reductase component. Primary control of eNOS and nNOS is exerted through regulation of electron flux from NADPH to the oxygenase active site. The most important of several inputs is calcium/calmodulin (Ca+2/CaM), but NO synthesis is also influenced by phosphorylation and protein-protein interactions. Ca+2/CaM control requires the participation of control elements located in the reductase region. The most important of these is the autoinhibitory insertion in the FMN binding domain, but the C terminal extension is also influential. The proposed work is organized around five hypotheses: 1. The autoinhibitory element of constitutively expressed NOS (cNOS) has isoform-specific effects in the absence of the C-terminal tail 2. The C-terminal tail has isoform-specific effects in the absence of the AI. 3. The C-terminal tail has isoform-specific effects in the presence of the AI 4. The autoinhibitory element and the C-terminal tail modulate each other's effects 5. Autoinhibitory element components have isoform-specific effect(s). The project will examine these hypotheses by creating novel chimeral genes in which control elements will be exchanged with cognates in distantly related NOS enzymes. Related chimera have been produced in a number of laboratories, establishing the feasibility of the approach. By using a larger section of the naturally occurring variability of the control sequences, we hope to greatly extend our knowledge of the control mechanism. Experiments suitable for undergraduate participation will be used to evaluate control of NO synthesis and electron transfer in mutants. More sophisticated kinetic analysis will allow us to obtain deeper insights than previous experiments with control element chimera.
Funding Period: 2009-09-30 - 2010-04-30
more information: NIH RePORT
- Calmodulin activates neuronal nitric oxide synthase by enabling transitions between conformational statesJohn C Salerno
Department of Biology, Kennesaw State University, Kennesaw, GA 30144, United States
FEBS Lett 587:44-7. 2013..9 ns output states in which FMN interacts with ferriheme. Enablement of the conformational cycle is an important paradigm for control in nNOS and related enzymes, and may extend to other control modalities...
- Space, time and nitric oxide--neuronal nitric oxide synthase generates signal pulsesJohn C Salerno
Biology Department, Kennesaw State University, Kennesaw, GA 30144, USA
FEBS J 276:6677-88. 2009..The general mechanism may be of wide importance in biological signaling...
- Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulinJonathan L McMurry
Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, GA 30144, USA
FEBS J 278:4943-54. 2011..This study is being extended using mutants to probe the roles of individual structural elements in binding and release...
- FMN fluorescence in inducible NOS constructs reveals a series of conformational states involved in the reductase catalytic cycleDipak K Ghosh
Department of Medicine, Duke University, VA Medical Centers, Durham, NC, USA
FEBS J 279:1306-17. 2012..These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle...