RESUMO
The type I cGMP-dependent protein kinases (PKG I) serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMP-dependent protein kinase (PKA). Here, we examined PKG Iα activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 (Δ53). We found that the construct exists as a monomer as assessed by whole-protein MS, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that Δ53 has a similar shape to the heterodimeric RIα-C complex of PKA. Moreover, we found that the Δ53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length PKG Iα as assessed by surface plasmon resonance (SPR) spectroscopy. However, we found that the Δ53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Iα activation is distinct from that of PKA, and its cooperativity is driven by in trans interactions between protomers.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/antagonistas & inibidores , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Multimerização Proteica , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Ativação Enzimática , Humanos , Camundongos , Modelos Moleculares , Fosforilação , Ligação Proteica , Ratos , Espalhamento a Baixo Ângulo , Homologia de SequênciaRESUMO
With his discovery of the ABO blood group system, Karl Landsteiner laid the foundation for modern day transfusion medicine. This discovery represents the basic knowledge for every blood transfusion. In recent years, certain blood groups have been linked to an increased risk for certain diseases. Hence, teaching the blood group serology is a relevant issue in medical education. In this review, we report about the history of the discovery of the blood groups by Landsteiner, the link of different blood groups to certain diseases and our experiences regarding teaching to medical students.
Assuntos
Sistema ABO de Grupos Sanguíneos/história , Educação Médica , Hematologia/educação , Estudantes de Medicina , Áustria , Transfusão de Sangue , Hematologia/história , História do Século XIX , História do Século XX , História do Século XXI , HumanosRESUMO
It is generally accepted that the endothelium regulates vascular tone independent of the activity of the sympathetic nervous system. Here, we tested the hypothesis that the activation of sympathetic nerves engages the endothelium to oppose vasoconstriction. Local inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) signals ("pulsars") in or near endothelial projections to vascular smooth muscle (VSM) were measured in an en face mouse mesenteric artery preparation. Electrical field stimulation of sympathetic nerves induced an increase in endothelial cell (EC) Ca(2+) pulsars, recruiting new pulsar sites without affecting activity at existing sites. This increase in Ca(2+) pulsars was blocked by bath application of the α-adrenergic receptor antagonist prazosin or by TTX but was unaffected by directly picospritzing the α-adrenergic receptor agonist phenylephrine onto the vascular endothelium, indicating that nerve-derived norepinephrine acted through α-adrenergic receptors on smooth muscle cells. Moreover, EC Ca(2+) signaling was not blocked by inhibitors of purinergic receptors, ryanodine receptors, or voltage-dependent Ca(2+) channels, suggesting a role for IP(3), rather than Ca(2+), in VSM-to-endothelium communication. Block of intermediate-conductance Ca(2+)-sensitive K(+) channels, which have been shown to colocalize with IP(3) receptors in endothelial projections to VSM, enhanced nerve-evoked constriction. Collectively, our results support the concept of a transcellular negative feedback module whereby sympathetic nerve stimulation elevates EC Ca(2+) signals to oppose vasoconstriction.
Assuntos
Sinalização do Cálcio/fisiologia , Artérias Mesentéricas/inervação , Artérias Mesentéricas/fisiologia , Sistema Nervoso Simpático/fisiologia , Vasoconstrição/fisiologia , Animais , Cálcio/metabolismo , Conexinas/genética , Endotélio Vascular/metabolismo , Retroalimentação Fisiológica/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Proteína alfa-5 de Junções ComunicantesRESUMO
Here, we report the design of unprecedented, non-FRET based cGMP-biosensors, named FlincGs, to assess the dynamics of nitric oxide (NO) and atrial natriuretic peptide (ANP) induced synthesis of intracellular cGMP, [cGMP](i). Regulatory fragments of PKG I alpha, PKG I beta, and an N-terminal deletion mutant of PKG I alpha were fused to circular permutated EGFP to generate alpha-, beta-, and delta-FlincG, with high dynamic ranges and apparent K(D,cGMP) values of 35 nM, 1.1 microM, and 170 nM, respectively. All indicators displayed significant selectivity for cGMP over cAMP, and 1.5- to 2.1-fold increases in fluorescence intensity at 510 nm when excited at 480 nm. Surprisingly, FlincGs displayed an additional excitation peak at 410 nm. delta-FlincG permitted ratiometric (480/410 nm) measurements, with a cGMP-specific 3.5-fold ratio change. In addition, delta-FlincG presented cGMP association and dissociation kinetics sufficiently fast to monitor rapid changes of [cGMP](i) in intact cells. In unpassaged, adenoviral transfected vascular smooth muscle (VSM) cells, delta-FlincG had an EC(50,cGMP) of 150 nM, and revealed transient global cGMP elevations to sustained physiological NO (EC(50,DEA/NO) = 4 nM), and the decay phase depended on the activity of PDE-5. In contrast, ANP elicited sustained submembrane elevations in [cGMP](i), which were converted to global cGMP elevations by inhibition of PDE-5 by sildenafil. These results indicate that FlincG is an innovative tool to elucidate the dynamics of a central biological signal, cGMP, and that NO and natriuretic peptides induce distinct cGMP patterning under the regulation of PDE-5, and therefore likely differentially engage cGMP targets.
Assuntos
Técnicas Biossensoriais , GMP Cíclico/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Animais , Aorta/patologia , Calibragem , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Cinética , Microscopia Confocal/métodos , Peptídeos Natriuréticos/química , Óxido Nítrico/química , RatosRESUMO
In eukaryotes, the poly(A) tail added at the 3' end of an mRNA precursor is essential for the regulation of mRNA stability and the initiation of translation. Poly(A) polymerase (PAP) is the enzyme that catalyzes the poly(A) addition reaction. Multiple isoforms of PAP have been identified in vertebrates, which originate from gene duplication, alternative splicing or post-translational modifications. The complexity of PAP isoforms suggests that they might play different roles in the cell. Phylogenetic studies indicate that vertebrate PAPs are grouped into three clades termed α, ß and γ, which originated from two gene duplication events. To date, all the available PAP structures are from the PAPα clade. Here, we present the crystal structure of the first representative of the PAPγ clade, human PAPγ bound to cordycepin triphosphate (3'dATP) and Ca(2+). The structure revealed that PAPγ closely resembles its PAPα ortholog. An analysis of residue conservation reveals a conserved catalytic binding pocket, whereas residues at the surface of the polymerase are more divergent.