TET3 is a positive regulator of mitochondrial respiration in Neuro2A cells.

Ten-Eleven-Translocase (TET) enzymes contribute to the regulation of the methylome via successive oxidation of 5-methyl cytosine (5mC) to derivatives which can be actively removed by base-excision-repair (BER) mechanisms in the absence of cell division. This is particularly important in post-mitotic neurons where changes in DNA methylation are known to associate with changes in neural function. TET3, specifically, is a critical regulator of both neuronal differentiation in development and mediates dynamic changes in the methylome of adult neurons associated with cognitive function. While DNA methylation is understood to regulate transcription, little is known of the specific targets of TET3-dependent catalytic activity in neurons. We report the results of an unbiased transcriptome analysis of the neuroblastoma-derived cell line; Neuro2A, in which Tet3 was silenced. Oxidative phosphorylation (OxPhos) was identified as the most significantly down-regulated functional canonical pathway, and these findings were confirmed by measurements of oxygen consumption rate in the Seahorse bioenergetics analyser. The mRNA levels of both nuclear- and mitochondrial-encoded OxPhos genes were reduced by Tet3-silencing, but we found no evidence for differential (hydroxy)methylation deposition at these gene loci. However, the mRNA expression of genes known to be involved in mitochondrial quality control were also shown to be significantly downregulated in the absence of TET3. One of these genes; EndoG, was identified as a direct target of TET3-catalytic activity at non-CpG methylated sites within its gene body. Accordingly, we propose that aberrant mitochondrial homeostasis may contribute to the decrease in OxPhos, observed upon Tet3-downregulation in Neuro2A cells.

Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.

BACKGROUND: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH. METHODS: Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH. RESULTS: Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling. CONCLUSIONS: A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.

Dysregulation of 2-oxoglutarate-dependent dioxygenases by hyperglycaemia: does this link diabetes and vascular disease?

The clinical, social and economic burden of cardiovascular disease (CVD) associated with diabetes underscores an urgency for understanding the disease aetiology. Evidence suggests that the hyperglycaemia associated with diabetes is, of itself, causal in the development of endothelial dysfunction (ED) which is recognised to be the critical determinant in the development of CVD. It is further recognised that epigenetic modifications associated with changes in gene expression are causal in both the initiation of ED and the progression to CVD. Understanding whether and how hyperglycaemia induces epigenetic modifications therefore seems crucial in the development of preventative treatments. A mechanistic link between energy metabolism and epigenetic regulation is increasingly becoming explored as key energy metabolites typically serve as substrates or co-factors for epigenetic modifying enzymes. Intriguing examples are the ten-eleven translocation and Jumonji C proteins which facilitate the demethylation of DNA and histones respectively. These are members of the 2-oxoglutarate-dependent dioxygenase superfamily which require the tricarboxylic acid metabolite, α-ketoglutarate and molecular oxygen (O2) as substrates and Fe (II) as a co-factor. An understanding of precisely how the biochemical effects of high glucose exposure impact upon cellular metabolism, O2 availability and cellular redox in endothelial cells (ECs) may therefore elucidate (in part) the mechanistic link between hyperglycaemia and epigenetic modifications causal in ED and CVD. It would also provide significant proof of concept that dysregulation of the epigenetic landscape may be causal rather than consequential in the development of pathology.

SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics.

BACKGROUND: Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail. OBJECTIVES: Several formin proteins are expressed in platelets so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape. METHODS: Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effects on platelet spreading, platelet size, platelet cytoskeletal dynamics, and organization were analyzed using fluorescence and electron microscopy. RESULTS: Pretreatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organization and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post-translational modification. CONCLUSIONS: These data therefore demonstrated an important role for formin-mediated actin polymerization in platelet spreading and highlighted the importance of formins in cross-talk between the actin and tubulin cytoskeletons.

Formin proteins in megakaryocytes and platelets: regulation of actin and microtubule dynamics.

The platelet and megakaryocyte cytoskeletons are essential for formation and function of these cells. A dynamic, properly organised tubulin and actin cytoskeleton is critical for the development of the megakaryocyte and the extension of proplatelets. Tubulin in particular plays a pivotal role in the extension of these proplatelets and the release of platelets from them. Tubulin is further required for the maintenance of platelet size, and actin is the driving force for shape change, spreading and platelet contraction during platelet activation. Whilst several key proteins which regulate these cytoskeletons have been described in detail, the formin family of proteins has received less attention. Formins are intriguing as, although they were initially believed to simply be a nucleator of actin polymerisation, increasing evidence shows they are important regulators of the crosstalk between the actin and microtubule cytoskeletons. In this review, we will introduce the formin proteins and consider the recent evidence that they play an important role in platelets and megakaryocytes in mediating both the actin and tubulin cytoskeletons.