Mechanistic insights revealed by a UBE2A mutation linked to intellectual disability.

Ubiquitin-conjugating enzymes (E2) enable protein ubiquitination by conjugating ubiquitin to their catalytic cysteine for subsequent transfer to a target lysine side chain. Deprotonation of the incoming lysine enables its nucleophilicity, but determinants of lysine activation remain poorly understood. We report a novel pathogenic mutation in the E2 UBE2A, identified in two brothers with mild intellectual disability. The pathogenic Q93E mutation yields UBE2A with impaired aminolysis activity but no loss of the ability to be conjugated with ubiquitin. Importantly, the low intrinsic reactivity of UBE2A Q93E was not overcome by a cognate ubiquitin E3 ligase, RAD18, with the UBE2A target PCNA. However, UBE2A Q93E was reactive at high pH or with a low-pKa amine as the nucleophile, thus providing the first evidence of reversion of a defective UBE2A mutation. We propose that Q93E substitution perturbs the UBE2A catalytic microenvironment essential for lysine deprotonation during ubiquitin transfer, thus generating an enzyme that is disabled but not dead.

Adenosine Kinase couples sensing of cellular potassium depletion to purine metabolism.

Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance. The transfer of γ-phosphate from ATP to ADO by ADK involves regulation by the substrates and products, as well as by Mg2+ and inorganic phosphate. Here we present new crystal structures of mouse ADK (mADK) binary (mADK:ADO; 1.2 Å) and ternary (mADK:ADO:ADP; 1.8 Å) complexes. In accordance with the structural demonstration of ADO occupancy of the ATP binding site, kinetic studies confirmed a competitive model of auto-inhibition of ADK by ADO. In the ternary complex, a K+ ion is hexacoordinated between loops adjacent to the ATP binding site, where Asp310 connects the K+ coordination sphere to the ATP binding site through an anion hole structure. Nuclear Magnetic Resonance 2D 15N-1H HSQC experiments revealed that the binding of K+ perturbs Asp310 and residues of adjacent helices 14 and 15, engaging a transition to a catalytically productive structure. Consistent with the structural data, the mutants D310A and D310P are catalytically deficient and loose responsiveness to K+. Saturation Transfer Difference spectra of ATPγS provided evidence for an unfavorable interaction of the mADK D310P mutant for ATP. Reductions in K+ concentration diminish, whereas increases enhance the in vitro activity of mADK (maximum of 2.5-fold; apparent Kd = 10.4 mM). Mechanistically, K+ increases the catalytic turnover (Kcat) but does not affect the affinity of mADK for ADO or ATP. Depletion of intracellular K+ inhibited, while its restoration was accompanied by a full recovery of cellular ADK activity. Together, this novel dataset reveals the molecular basis of the allosteric activation of ADK by K+ and highlights the role of ADK in connecting depletion of intracellular K+ to the regulation of purine metabolism.

FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes.

Focal adhesion kinase (FAK) has emerged as a mediator of mechanotransduction in cardiomyocytes, regulating gene expression during hypertrophic remodeling. However, how FAK signaling is relayed onward to the nucleus is unclear. Here, we show that FAK interacts with and regulates myocyte enhancer factor 2 (MEF2), a master cardiac transcriptional regulator. In cardiomyocytes exposed to biomechanical stimulation, FAK accumulates in the nucleus, binds to and upregulates the transcriptional activity of MEF2 through an interaction with the FAK focal adhesion targeting (FAT) domain. In the crystal structure (2.9 Å resolution), FAT binds to a stably folded groove in the MEF2 dimer, known to interact with regulatory cofactors. FAK cooperates with MEF2 to enhance the expression of Jun in cardiomyocytes, an important component of hypertrophic response to mechanical stress. These findings underscore a connection between the mechanotransduction involving FAK and transcriptional regulation by MEF2, with potential relevance to the pathogenesis of cardiac disease.

Immediate response of myocardium to pressure overload includes transient regulation of genes associated with mitochondrial bioenergetics and calcium availability

Ventricular hypertrophy is one of the major myocardial responses to pressure overload (PO). Most studies on early myocardial response focus on the days or even weeks after induction of hypertrophic stimuli. Since mechanotransduction pathways are immediately activated in hearts undergoing increased work load, it is reasonable to infer that the myocardial gene program may be regulated in the first few hours. In the present study, we monitored the expression of some genes previously described in the context of myocardial hypertrophic growth by using the Northern blot technique, to estimate the mRNA content of selected genes in rat myocardium for the periods 1, 3, 6, 12 and 48 h after PO stimuli. Results revealed an immediate switch in the expression of genes encoding alpha and beta isoforms of myosin heavy chain, and up-regulation of the cardiac isoform of alpha actin. We also detected transitory gene regulation as the increase in mitochondrial cytochrome c oxidase 1 gene expression, parallel to down-regulation of genes encoding sarco(endo)plasmic reticulum Ca+2 ATPase and sodium-calcium exchanger. Taken together, these results indicate that initial myocardial responses to increased work load include alterations in the contractile properties of sarcomeres and transitory adjustment of mitochondrial bioenergetics and calcium availability.

Analysis of adenosine by RP-HPLC method and its application to the study of adenosine kinase kinetics.

An RP-HPLC method for the analysis of adenosine (ADO) has been developed and validated. In the present study, we report an RP-HPLC-based method with modifications of mobile phase and shorter retention time that substantially improved the efficiency of ADO analysis. The HPLC separation of the ADO was achieved on a C18 column, using a mobile phase consisting of water, containing 7% v/v ACN, at a flow rate of 0.8 mL/min. The column effluent was monitored by UV detection at 260 nm. A linear response was achieved over the concentration range of 0.25-100.00 micromol/L. The analytical method inter- and intra-run accuracy and precision were better than +/- 15%. The LOQ was 0.25 micromol/L, with ADO detection in the range of 6.25 pmol per sample. The method has been applied to the study of adenosine kinase (AK) kinetics.

High performance liquid chromatography analysis of a 4-anilinoquinazoline derivative (PD153035), a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in rat plasma.

The quinazoline derivative, 4-N-(3'-bromo-phenyl)amino-6,7-dimethoxyquinazoline (PD153035), has recently been identified as a potential drug for the treatment of proliferative disease. Here, we report a sensitive high performance liquid chromatography (HPLC)-based quantitative detection method for measurement of PD153035 levels in rat plasma. Sample pretreatment involved a two-step extraction with chloroform. The analytes were separated on a column packed with OmniSpher C18 material and eluted with acetonitrile-0.1 M ammonium acetate, pH 7.2 (70:30, v/v). The column effluent was monitored by UV detection at 330 nm. A linear response was achieved over the concentration range 0.50-100.00 microM using multilevel calibration with an internal standard. The analytical method inter- and intra-run accuracy and precision were better than +/-15%. The lower limit of quantification was 0.50 microM. The method has been applied to study the preclinical pharmacokinetics of this compound in rats.

Implicações cardiovasculares da síndrome metabólica: importância da hiperinsulinemia na hipertrofia cardíaca e do vaso / Cardiovascular implications of metabolic syndrome: role of hyperinsulinemia on cardiac and vascular hypertrophy

Evidências epidemiológicas indicam que a resistência à insulina e a conseqüente hiperinsulinemia são fatores de risco independentes para doenças cardiovasculares. Diversos estudos ecocardiográficos têm demonstrado que a hiperinsulinemia está associada de maneira independente à hipertrofia ventricular esquerda e ao aumento da relação entre a massa ventricular esquerda e o volume diastólico final.Por outro lado, estudos prospectivos e observações epidemiológicas demonstraram associação independente entre hiperinsulinemia e doença isquêmica coronária e vascular cerebral, indicando participação importante da insulina no desenvolvimento da aterosclerose. Embora ainda não estejam estabelecidos os mecanismos pelos quais a hiperinsulinemia induz à hipertrofia tanto cardíaca como vascular em condições de resistência à insulina, observações experimentais recentes têm sugerido que anormalidades na sinalização intracelular ativada pela insulina possam exercer papel fundamental nesse processo. A redução da ativação da via da fosfatidilinositol-3-quinase/Akt, responsável pelos efeitos metabólicos da insulina, em paralelo à ativação preservada da via de crescimento celular mediada pelas quinases ativadas por mitógenos (MAPKs) estão sendo consideradas como eventos fundamentais para o desenvolvimento da hipertrofia tanto miocárdica como do vaso. Além disso, desequilíbrios entre insulina e fatores de crescimento, como a angiotensina 11, parecemtambém contribuir para a hipertrofia em ambos os tecidos. Portanto, a hiperinsulinemia parece ser um importante regulador da hipertrofia cardíaca e vascular, consistindo em um fator de risco independente para a morbidade e a mortalidade cardiovasculares. Ademais, mecanismos fisiopatogênicos similares podem estar envolvidos no crescimento cardíaco e do vaso em condições de resistência à insulina e hiperinsulinemia.

Influência de fatores ambientais e genéticos na hipertrofia e remodelamento cardíacos na hipertensäo arterial / Influence of environmental and genetic factors on cardiac hypertrophy and remodeling in hypertension

A hipertrofia e o remodelamento ventricular representam mudanças estruturais adaptativas do ventrículo esquerdo em resposta à hipertensäo arterial. Apesar de consideradas compensatórias, essas alteraçöes estäo associadas a maior risco de morbimortalidade cardiovascular. Embora a pressäo arterial seja a principal determinante da massa e geometria ventricular esquerda, diversos fatores ambientais e genéticos podem exercer influência no fenótipo ventricular. Fatores ambientais como a ingestäo de sal, o consumo de álcool, o diabetes melito, o estresse psicológico e o exercício físico prolongado podem exercer influências significativas sobre a massa e a geometria do ventrículo esquerdo. Por outro lado, evidências epidemiológicas e análises de ligaçäo de polimorfismos gênicos têm apontado para uma importância significativa do componente genético na determinaçäo da massa ventricular esquerda. Portanto, a massa e a arquitetura do ventrículo esquerdo constituem fenótipos complexos que säo determinados pela interaçäo da carga hemodinâmica imposta dentre outras pela pressäo arterial com fatores genéticos e ambientais.

Cor triatriatum dexter: diagnóstico ecocardiográfico; relato de caso / Cor triatriatum dexter: echocardiographic diagnosis; a case report

Os autores apresentam um caso de cor triatriatum dexter, diagnosticado por ecodoplercardiografia bidimensional, associado a comuniçäo interatrial e estenose pulmonar valvar. A anomalia näo foi detectada no estudo hemodinâmico. O ecodopplercardiograma mostrou uma ampla membrana dividindo o átrio em duas cavidades, estendendo-se de sua porçäo inferior, imediatamente acima do anel tricúspide, até os orifícios da veias cavas. Esses achados foram confirmados pela necrópsia

Atividade colinesterásica no plasma e em eritrócitos de pacientes com as formas cardíaca e indeterminada da doença de Chagas / Cholinesterase activity in plasma and in erythrocytes of patients with cardiac and indeterminate form of Chagas' disease

A atividade colinesterásica foi determinada no plasma e em eritrócitos de 10 indivíduos normais e em 26 pacientes chagásicos crônicos, sendo 6 com insuficiência cardíaca congestiva compensada, 10 com cardiopatia sem insuficiência cardíaca congestiva e 10 com a forma indeterminada. Os valores enzimáticos encontrados foram (média + ou - sd): 2196 + ou - 442 UI/L no plasma e 19,4 + ou - 3,3 UI/g Hb em eritrócitos do grupo controle; 2291 + ou - 317 UI/L no plasma e 19,2 + ou - 3,7 UI/g Hb em eritrócitos dos chagásicos com insuficiência cardíaca congestiva compensada; 2445 + ou - 357 UI/L no plasma e 18,3 + ou - 3,0 UI/g Hb em eritrócitos dos cardiopatas chagásicos sem insuficiência cardíaca congestiva; 2006 + ou - 327 UI/L no plasma e 17,8 + ou - 3,1 UI/g Hb em eritrócitos de chagásicos com a forma indeterminada. Nossos dados mostram que o comprometimento neuronal que ocorre nos cardiopatas chagásicos crônicos näo é suficiente para levar a alteraçöes significativvas (p>0,05) das atividades colinesterásicas no plasma e em eritrócitos