Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox_/_ coronary microvascular cells. Compared with wild-type p47phoxcDNAtransfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2 . production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells

The insulin receptor family and protein kinase B (Akt) are activated in the heart by alkaline pH and α1-adrenergic receptors

Insulin and insulin-like growth factor stimulate protein synthesis and cardioprotection in the heart, acting through their receptors (INSRs, IGF1Rs) and signalling via protein kinase B (PKB, also known as Akt). Protein synthesis is increased in hearts perfused at alkaline pHo to the same extent as with insulin. Moreover, α1-adrenergic receptor (α1-AR) agonists (e.g. phenylephrine) increase protein synthesis in cardiomyocytes, activating PKB/Akt. In both cases, the mechanisms are not understood. Our aim was to determine if insulin receptor-related receptors (INSRRs, activated in kidney by alkaline pH) may account for the effects of alkaline pHo on cardiac protein synthesis, and establish if α1-ARs signal through the insulin receptor family. Alkaline pHo activated PKB/Akt signalling to the same degree as insulin in perfused adult male rat hearts. INSRRs were expressed in rat hearts and, by immunoblotting for phosphorylation (activation) of INSRRs/INSRs/IGF1Rs, we established that INSRRs, together with INSRs/IGF1Rs, are activated by alkaline pHo. The INSRR/INSR/IGF1R kinase inhibitor, linsitinib, prevented PKB/Akt activation by alkaline pHo, indicating that INSRRs/INSRs/IGF1Rs are required. Activation of PKB/Akt in cardiomyocytes by α1-AR agonists was also inhibited by linsitinib. Furthermore, linsitinib inhibited cardiomyocyte hypertrophy induced by α1-ARs in cultured cells, reduced the initial cardiac adaptation (24 h) to phenylephrine in vivo (assessed by echocardiography) and increased cardiac fibrosis over 4 days. We conclude that INSRRs are expressed in the heart and, together with INSRs/IGF1Rs, the insulin receptor family provide a potent system for promoting protein synthesis and cardioprotection. Moreover, this system is required for adaptive hypertrophy induced by α1-ARs

Vascular collagen type-IV in hypertension and cerebral small vessel disease

Cerebral small vessel disease (SVD) is common in older people and causes lacunar stroke and vascular cognitive impairment. Risk factors include old age, hypertension and variants in the genes encoding collagen alpha-1(IV) and alpha-2(IV), here termed collagen-IV, which are core components of the basement membrane. We tested the hypothesis that increased vascular collagen-IV associates with clinical hypertension and with SVD in older persons and with chronic hypertension in young and aged primates and genetically hypertensive rats. We quantified vascular collagen-IV immunolabeling in small arteries in a cohort of older persons with minimal Alzheimer's pathology (N=52; 21F/31M, age 82.8±6.95 years). We also studied archive tissue from young (age range 6.2-8.3 years) and older (17.0-22.7 years) primates ( ) and compared chronically hypertensive animals (18 months aortic stenosis) with normotensives. We also compared genetically hypertensive and normotensive rats (aged 10-12 months). Collagen-IV immunolabeling in cerebral small arteries of older persons was negatively associated with radiological SVD severity (ρ: -0.427, =0.005) but was not related to history of hypertension. General linear models confirmed the negative association of lower collagen-IV with radiological SVD ( <0.017), including age as a covariate and either clinical hypertension ( <0.030) or neuropathological SVD diagnosis ( <0.022) as fixed factors. Reduced vascular collagen-IV was accompanied by accumulation of fibrillar collagens (types I and III) as indicated by immunogold electron microscopy. In young and aged primates, brain collagen-IV was elevated in older normotensive relative to young normotensive animals ( =0.029) but was not associated with hypertension. Genetically hypertensive rats did not differ from normotensive rats in terms of arterial collagen-IV. Our cross-species data provide novel insight into sporadic SVD pathogenesis, supporting insufficient (rather than excessive) arterial collagen-IV in SVD, accompanied by matrix remodeling with elevated fibrillar collagen deposition. They also indicate that hypertension, a major risk factor for SVD, does not act by causing accumulation of brain vascular collagen-IV

MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension.

BACKGROUND: Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. METHODS AND RESULTS: In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. CONCLUSIONS: Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the “RAF paradox”) may have the same effect. BRAF was upregulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a “RAF paradox” effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the “RAF paradox”. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function

NADPH oxidases: key modulators in aging and age-related cardiovascular diseases?

Reactive oxygen species (ROS) and oxidative stress have long been linked to aging and diseases prominent in the elderly such as hypertension, atherosclerosis, diabetes and atrial fibrillation (AF). NADPH oxidases (Nox) are a major source of ROS in the vasculature and are key players in mediating redox signalling under physiological and pathophysiological conditions. In this review, we focus on the Nox-mediated ROS signalling pathways involved in the regulation of 'longevity genes' and recapitulate their role in age-associated vascular changes and in the development of age-related cardiovascular diseases (CVDs). This review is predicated on burgeoning knowledge that Nox-derived ROS propagate tightly regulated yet varied signalling pathways, which, at the cellular level, may lead to diminished repair, the aging process and predisposition to CVDs. In addition, we briefly describe emerging Nox therapies and their potential in improving the health of the elderly population

Protein Structure and Biochemical Characterisation of the p22phox and p47phox in NADPH Oxidase-2 Activation.

It has been well documented that a superoxide-generating NADPH oxidase 2 (Nox2) is involved in the pathogenesis of oxidative stress-related cardiovascular diseases such as hypertension and atherosclerosis. However, the structure of the Nox2 protein subunits and the regulatory mechanisms of Nox2 activation remain largely unknown. Therefore the purpose of this Ph.D. research project is to use a combination of in silico computational protein structure modelling and molecular cell biology to investigate the three-dimensional (3D) protein structural changes of the p22phox (the Nox2 partner of the cytochrome b558) and p47phox (a key regulatory subunit of the Nox2) in the phosphorylation regulation of Nox2 activation. A consensus in silico 3D protein structure model for the p22phox was generated by incorporating transmembrane-specific structural predictions with the computational modelling program molecular operating environment (MOE). The results showed that the p22phox consists of an N-terminal transmembrane domain (124 a.a.) with three membrane transecting helices, an extensive extracellular region between helices two and three, and a C-terminal cytoplasmic domain (71 a.a.). Using the p22phox structure model generated in this thesis, I examined the C242T polymorphism, which has been shown to be protective against the progression of atherosclerosis. My results showed that the C242T polymorphism, which corresponds to substitution of Histidine-72 to Tyrosine-72, is located in the extracellular loop of the p22phox, which causes significant morphological changes of the p22phox and affects its interaction with the catalytic Nox2 subunit. Transfection of p22phox plasmid baring C242T polymorphism into human endothelial cells significantly reduced TNFα (100U/ml, 30 min)-induced reactive oxygen species (ROS) production as compared to cells transfected with wild-type (WT) p22phox plasmid and vector controls. These results were further verified in p22phox-defficient HeLa cells. The process of p22phox binding to phosphorylated p47phox has been found to be a crucial step in Nox2 activation. However, it has only been suggested, but not proven that under resting conditions, the p47phox is in its auto-inhibited conformation such that the p22phox binding groove is masked by a polybasic auto-inhibitory region (AIR). Phosphorylation of the AIR exposes the p22phox binding groove and is known to initiate Nox2 activation. In order to investigate the phosphorylation-induced morphological changes in p47phox, I modelled the p47phox, based on the available crystal structures, in both resting and the activated forms by molecular dynamics. My results showed that phosphorylation of the serines-303/4, 310, 315 and 379 located at the C-terminus of p47phox moves the auto-inhibitory region away, which in turn exposes the p22phox binding groove and initiates a process essential for the activation of Nox2. In summary, my Ph.D. project combines the knowledge and techniques of bioinformatics with cellular and molecular biology. The results of the in silico models of both p22phox and p47phox can be used for further investigation of the mechanisms of Nox2 activation, and can be exploited for the design of small molecules to inhibit Nox2 activation. This thesis has also provided important insights into the mechanism of the C242T polymorphism, which explains its clinical relevance to inhibit endothelial dysfunction reported by previous clinical studies

Protein Structure and Biochemical Characterisation of the p22phox and p47phox in NADPH Oxidase-2 Activation.

It has been well documented that a superoxide-generating NADPH oxidase 2 (Nox2) is involved in the pathogenesis of oxidative stress-related cardiovascular diseases such as hypertension and atherosclerosis. However, the structure of the Nox2 protein subunits and the regulatory mechanisms of Nox2 activation remain largely unknown. Therefore the purpose of this Ph.D. research project is to use a combination of in silico computational protein structure modelling and molecular cell biology to investigate the three-dimensional (3D) protein structural changes of the p22phox (the Nox2 partner of the cytochrome b558) and p47phox (a key regulatory subunit of the Nox2) in the phosphorylation regulation of Nox2 activation. A consensus in silico 3D protein structure model for the p22phox was generated by incorporating transmembrane-specific structural predictions with the computational modelling program molecular operating environment (MOE). The results showed that the p22phox consists of an N-terminal transmembrane domain (124 a.a.) with three membrane transecting helices, an extensive extracellular region between helices two and three, and a C-terminal cytoplasmic domain (71 a.a.). Using the p22phox structure model generated in this thesis, I examined the C242T polymorphism, which has been shown to be protective against the progression of atherosclerosis. My results showed that the C242T polymorphism, which corresponds to substitution of Histidine-72 to Tyrosine-72, is located in the extracellular loop of the p22phox, which causes significant morphological changes of the p22phox and affects its interaction with the catalytic Nox2 subunit. Transfection of p22phox plasmid baring C242T polymorphism into human endothelial cells significantly reduced TNFα (100U/ml, 30 min)-induced reactive oxygen species (ROS) production as compared to cells transfected with wild-type (WT) p22phox plasmid and vector controls. These results were further verified in p22phox-defficient HeLa cells. The process of p22phox binding to phosphorylated p47phox has been found to be a crucial step in Nox2 activation. However, it has only been suggested, but not proven that under resting conditions, the p47phox is in its auto-inhibited conformation such that the p22phox binding groove is masked by a polybasic auto-inhibitory region (AIR). Phosphorylation of the AIR exposes the p22phox binding groove and is known to initiate Nox2 activation. In order to investigate the phosphorylation-induced morphological changes in p47phox, I modelled the p47phox, based on the available crystal structures, in both resting and the activated forms by molecular dynamics. My results showed that phosphorylation of the serines-303/4, 310, 315 and 379 located at the C-terminus of p47phox moves the auto-inhibitory region away, which in turn exposes the p22phox binding groove and initiates a process essential for the activation of Nox2. In summary, my Ph.D. project combines the knowledge and techniques of bioinformatics with cellular and molecular biology. The results of the in silico models of both p22phox and p47phox can be used for further investigation of the mechanisms of Nox2 activation, and can be exploited for the design of small molecules to inhibit Nox2 activation. This thesis has also provided important insights into the mechanism of the C242T polymorphism, which explains its clinical relevance to inhibit endothelial dysfunction reported by previous clinical studies

Endothelial Nox1 oxidase assembly in human pulmonary arterial hypertension; driver of Gremlin1-mediated proliferation.

Pulmonary arterial hypertension (PAH) is a rapidly degenerating and devastating disease of increased pulmonary vessel resistance leading to right heart failure. Palliative modalities remain limited despite recent endeavors to investigate the mechanisms underlying increased pulmonary vascular resistance (PVR), i.e. aberrant vascular remodeling and occlusion. However, little is known of the molecular mechanisms responsible for endothelial proliferation, a root cause of PAH-associated vascular remodeling. Lung tissue specimens from PAH and non-PAH patients and hypoxia-exposed human pulmonary artery endothelial cells (ECs) (HPAEC) were assessed for mRNA and protein expression. Reactive oxygen species (ROS) were measured using cytochrome c and Amplex Red assays. Findings demonstrate for the first time an up-regulation of NADPH oxidase 1 (Nox1) at the transcript and protein level in resistance vessels from PAH compared with non-PAH patients. This coincided with an increase in ROS production and expression of bone morphogenetic protein (BMP) antagonist Gremlin1 (Grem1). In HPAEC, hypoxia induced Nox1 subunit expression, assembly, and oxidase activity leading to elevation in sonic hedgehog (SHH) and Grem1 expression. Nox1 gene silencing abrogated this cascade. Moreover, loss of either Nox1, SHH or Grem1 attenuated hypoxia-induced EC proliferation. Together, these data support a Nox1-SHH-Grem1 signaling axis in pulmonary vascular endothelium that is likely to contribute to pathophysiological endothelial proliferation and the progression of PAH. These findings also support targeting of Nox1 as a viable therapeutic option to combat PAH