Endogenous ADP-ribosylation of the G Protein β Subunit Prevents the Inhibition of Type 1 Adenylyl Cyclase

Mono-ADP-ribosylation is a post-translational modification of cellular proteins that has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking, and secretion. In several cell systems we have observed that the major substrate of endogenous mono-ADP-ribosylation is a 36-kDa protein. This ADP-ribosylated protein was both recognized in Western blotting experiments and selectively immunoprecipitated by a G protein beta subunit-specific polyclonal antibody, indicating that this protein is the G protein beta subunit. The ADP-ribosylation of the beta subunit was due to a plasma membrane-associated enzyme, was sensitive to treatment with hydroxylamine, and was inhibited by meta-iodobenzylguanidine, indicating that the involved enzyme is an arginine-specific mono-ADP-ribosyltransferase. By mutational analysis, the target arginine was located in position 129. The ADP-ribosylated beta subunit was also deribosylated by a cytosolic hydrolase. This ADP-ribosylation/deribosylation cycle might be an in vivo modulator of the interaction of betagamma with specific effectors. Indeed, we found that the ADP-ribosylated betagamma subunit is unable to inhibit calmodulin-stimulated type 1 adenylyl cyclase in cell membranes and that the endogenous ADP-ribosylation of the beta subunit occurs in intact Chinese hamster ovary cells, where the NAD(+) pool was labeled with [(3)H]adenine. These results show that the ADP-ribosylation of the betagamma subunit could represent a novel cellular mechanism in the regulation of G protein-mediated signal transduction

Constraints on the [C II] luminosity of a proto-globular cluster at z ∼ 6 obtained with ALMA

We report on ALMA observations of D1, a system at z 3c 6.15 with stellar mass M 17 3c 107M containing globular cluster (GC) precursors, strongly magnified by the galaxy cluster MACS J0416.1-2403. Since the discovery of GC progenitors at high redshift, ours is the first attempt to probe directly the physical properties of their neutral gas through infrared observations. A careful analysis of our dataset, performed with a suitable procedure designed to identify faint narrow lines and which can test various possible values for the unknown linewidth value, allowed us to identify a 4\u3c3 tentative detection of [CII] emission with intrinsic luminosity L[CII] = (2.9 \ub1 1.4) 106L, one of the lowest values ever detected at high redshift. This study offers a first insight on previously uncharted regions of the L[CII] 12 SF R relation. Despite large uncertainties affecting our measure of the star formation rate, if taken at face value our estimate lies more than 3c 1 dex below the values observed in local and high redshift systems. Our weak detection indicates a deficiency of [CII] emission, possibly ascribed to various explanations, such as a low-density gas and/or a strong radiation field caused by intense stellar feedback, and a low metal content. From the non-detection in the continuum we derive constraints on the dust mass, with 3 12 \u3c3 upper limit values as low as 3c a few 104 M, consistent with the values measured in local metal-poor galaxies

Endogenous mono-ADP-ribosylation of the free Gbetagamma prevents stimulation of phosphoinositide 3-kinase-gamma and phospholipase C-beta2 and is activated by G-protein-coupled receptors.

We have recently demonstrated that the beta subunit of the heterotrimeric G-proteins is endogenously mono-ADP-ribosylated in intact cells. The modified betagamma heterodimer loses its ability to inhibit calmodulin-stimulated type 1 adenylate cyclase and, remarkably, is de-ADP-ribosylated by a cytosolic hydrolase that completes an ADP-/de-ADP-ribosylation cycle of potential physiological relevance. In the present study, we show that this ADP-ribosylation might indeed be a general mechanism for termination of betagamma signalling, since the ADP-ribosylated betagamma subunit is also unable to activate both phosphoinositide 3-kinase-gamma and phospholipase C-beta2. Moreover, we show that beta subunit ADP-ribosylation is induced by G-protein-coupled receptor activation, since hormone stimulation of Chinese-hamster ovary plasma membranes leads to increases in beta subunit labelling. This occurs when betagamma is in its active heterodimeric conformation, since full inhibition of this modification can be achieved by binding of GDP-alphai3 to the betagamma heterodimer. Taken together, these findings delineate a pathway that arises from the activation of a G-protein-coupled receptor and leads to the inhibition of betagamma activity through its reversible mono-ADP-ribosylation

Comprehensive kinome NGS targeted expression profiling by KING-REX

Abstract Background Protein kinases are enzymes controlling different cellular functions. Genetic alterations often result in kinase dysregulation, making kinases a very attractive class of druggable targets in several human diseases. Existing approved drugs still target a very limited portion of the human ‘kinome’, demanding a broader functional knowledge of individual and co-expressed kinase patterns in physiologic and pathologic settings. The development of novel rapid and cost-effective methods for kinome screening is therefore highly desirable, potentially leading to the identification of novel kinase drug targets. Results In this work, we describe the development of KING-REX (KINase Gene RNA EXpression), a comprehensive kinome RNA targeted custom assay-based panel designed for Next Generation Sequencing analysis, coupled with a dedicated data analysis pipeline. We have conceived KING-REX for the gene expression analysis of 512 human kinases; for 319 kinases, paired assays and custom analysis pipeline features allow the evaluation of 3′- and 5′-end transcript imbalances as readout for the prediction of gene rearrangements. Validation tests on cell line models harboring known gene fusions demonstrated a comparable accuracy of KING-REX gene expression assessment as in whole transcriptome analyses, together with a robust detection of transcript portion imbalances in rearranged kinases, even in complex RNA mixtures or in degraded RNA. Conclusions These results support the use of KING-REX as a rapid and cost effective kinome investigation tool in the field of kinase target identification for applications in cancer biology and other human diseases

Discovery of 2-[1-(4,4-Difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118):a potent, orally available, and highly selective PARP-1 inhibitor for cancer therapy

The nuclear protein poly­(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology, as testified by the number of candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-[1-(4,4-difluorocyclohexyl)­piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1<i>H</i>-isoindole-4-carboxamide (NMS-P118, <b>20by</b>). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of <b>20by</b> with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity