End-binding protein 1 controls signal propagation from the T cell receptor

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome.     

The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress

The p57(Kip2) cyclin-dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem-cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57(Kip2) is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57(Kip2) at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell-cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell-cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38-mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.     

BMP8B increases brown adipose tissue thermogenesis through both central and peripheral actions

Thermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b(-/-) mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain despite hypophagia. BMP8B is also expressed in the hypothalamus, and Bmp8b(-/-) mice display altered neuropeptide levels and reduced phosphorylation of AMP-activated protein kinase (AMPK), indicating an anorexigenic state. Central BMP8B treatment increased sympathetic activation of BAT, dependent on the status of AMPK in key hypothalamic nuclei. Our results indicate that BMP8B is a thermogenic protein that regulates energy balance in partnership with hypothalamic AMPK. BMP8B may offer a mechanism to specifically increase energy dissipation by BAT.     

Novel triazolopyridylbenzamides as potent and selective p38α inhibitors

A new class of p38α inhibitors based on a biaryl-triazolopyridine scaffold was investigated. X-ray crystallographic data of the initial lead compound cocrystallised with p38α was crucial in order to uncover a unique binding mode of the inhibitor to the hinge region via a pair of water molecules. Synthesis and SAR was directed towards the improvement of binding affinity, as well as ADME properties for this new class of p38α inhibitors and ultimately afforded compounds showing good in vivo efficacy.     

Pyrazine-based Syk kinase inhibitors

A series of aminopyrazines as inhibitors of Syk kinase activity and showing inhibition of LAD2 cells degranulation is described. Optimization of the carboxamide motif with aminomethylpiperidines provided high potency inhibiting Syk but low cellular activity. Amides of cis and trans adamantanol showed good inhibitory activity against Syk as well as remarkable activity in LAD2 cells degranulation assay.     

Characterization of the proteolytic system present in Vasconcellea quercifolia latex

Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia (“oak leaved papaya”) latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman’s degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.     

Statins and Selective Inhibition of Rho Kinase Protect Small Conductance Calcium-Activated Potassium Channel Function (KCa2.3) in Cerebral Arteries

Background

In rat middle cerebral and mesenteric arteries the K_Ca2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, K_Ca2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain K_Ca2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA).

Methods

MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence.

Results

Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of K_Ca2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. K_Ca2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation.

Conclusions

Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell K_Ca2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of K_Ca2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.