Biochemical characterization of the PARP domain-containing proteins AtRCD1 and TaSRO1

Quick and finely tuned response to the different abiotic stresses are an essential feature for the survival of of plants as sessile organisms. The signalling pathways that determine tailored responses at cellular and systemic level need to be strictly regulated, as well as coordinated, in order to allow a fast rise of defensive mechanisms. A pivotal role in the integration and regulation of different molecular pathways is played by proteins defined as “cellular hubs”: an example of these proteins is A. thaliana RADICAL-INDUCED CELL DEATH1 (RCD1), the best characterized member of the plant-specific SIMILAR TO RCD1 (SRO) protein family. It was first described to be involved in apoplastic ROS tolerance and development, as well as in response to heat and chloroplastic ROS stress, via interaction with different transcription factors through its RST domain. However, the mechanism, by which the interaction between RCD1 and its targets is regulated, has not been uncovered yet. In this work, the biochemical characterization of the function of the WWE domain, located at the N-terminus of RCD1, was carried out, in order to uncover its functions in the regulation of RCD1 activity in the plant cell nucleus. The localization study performed with different RCD1 constructs showed how the WWE domain has a major influence on the characteristic subnuclear localization of RCD1 in nuclear bodies, present in different sizes and number. The Co immunoprecipitation assays confirmed the role of the N-terminus of RCD1 and its closest homologue, SIMILAR TO RCD1 1 (SRO1), including the WWE domain, in the oligomerisation process, that could be involved in the formation of the nuclear bodies. Alteration of previously identified phophosites on the N-terminal portion of RCD1 were generated to test whether the phosphorylation state of the considered phosphorylatable residues might play a role in the regulation of RCD1’s activity in the plant cell. In this work it was shown that mutations on phosphosites produced noticeable effects both on the macroscopic and on the molecular level. Additionally, in this work another member of the SRO protein family, Triticum aestivum SRO1 was studied, in relation to the previously described catalytic activity of its Poly(ADP)-ribose Polymerase (PARP) domain, that would make this the only known member of the SRO family to present PARP enzymatic activity. However, with the assays performed in this work, the previously published findings could not be confirmed

Nuclear Import of Arabidopsis Poly(ADP-Ribose) Polymerase 2 Is Mediated by Importin-α and a Nuclear Localization Sequence Located Between the Predicted SAP Domains

Proteins of the Poly(ADP-Ribose) Polymerase (PARP) family modify target proteins by covalent attachment of ADP-ribose moieties onto amino acid side chains. In Arabidopsis, PARP proteins contribute to repair of DNA lesions and modulate plant responses to various abiotic and biotic stressors. Arabidopsis PARP1 and PARP2 are nuclear proteins and given that their molecular weights exceed the diffusion limit of nuclear pore complexes, an active import mechanism into the nucleus is likely. Here we use confocal microscopy of fluorescent protein-tagged Arabidopsis PARP2 and PARP2 deletion constructs in combination with site-directed mutagenesis to identify a nuclear localization sequence in PARP2 that is required for nuclear import. We report that in co-immunoprecipitation assays PARP2 interacts with several isoforms of the importin-α group of nuclear transport adapters and that PARP2 binding to IMPORTIN-α2 is mediated by the identified nuclear localization sequence. Our results demonstrate that PARP2 is a cargo protein of the canonical importin-α/β nuclear import pathway

The superior salinity tolerance of bread wheat cultivar Shanrong No. 3 is unlikely to be caused by elevated Ta-sro1 poly-(ADP-ribose) polymerase activity

Structural and biochemical analyses demonstrate that the elevated salinity tolerance of bread wheat cultivar Shanrong No. 3 is unlikely to be caused by elevated Ta-sro1 poly(ADP-ribose) polymerase activity

Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs

Five-year follow-up of children with perinatal HIV-1 infection receiving early highly active antiretroviral therapy

<p>Abstract</p> <p>Background</p> <p>Early highly active antiretroviral therapy (HAART), started within the first months of age, has been proven to be the optimal strategy to prevent immunological and clinical deterioration in perinatally HIV-infected children. Nevertheless, data about long-term follow-up of early treated children are lacking.</p> <p>Methods</p> <p>We report data from 40 perinatally HIV-infected-children receiving early HAART, with a median follow-up period of 5.96 years (interquartile range [IQR]:4.21–7.62). Children were enrolled at birth in the Italian Register for HIV Infection in Children. Comparison with 91 infected children born in the same period, followed-up from birth, and receiving deferred treatment was also provided.</p> <p>Results</p> <p>Nineteen children (47.5%) were still receiving their first HAART regimen at last follow-up. In the remaining children the first regimen was discontinued, after a median period of 3.77 years (IQR: 1.71–5.71) because of viral failure (8 cases), liver toxicity (1 case), structured therapy interruption (3 cases), or simplification/switch to a PI-sparing regimen (9 cases). Thirty-nine (97.5%) children showed CD4⁺T-lymphocyte values>25%, and undetectable viral load was reached in 31 (77.5%) children at last visit. Early treated children displayed significantly lower viral load than not-early treated children, until 6 years of age, and higher median CD4⁺T-lymphocyte percentages until 4 years of age. Twenty-seven (29.7%) not-early treated vs. 0/40 early treated children were in clinical category C at last follow-up (P < 0.0001).</p> <p>Conclusion</p> <p>Our findings suggest that clinical, virologic and immunological advantages from early-HAART are long-lasting. Recommendations indicating the long-term management of early treated children are needed.</p

The importance of microvascular inflammation in ageing and age-related diseases: a position paper from the ESH working group on small arteries, section of microvascular inflammation

Microcirculation is pervasive and orchestrates a profound regulatory cross-talk with the surrounding tissue and organs. Similarly, it is one of the earliest biological systems targeted by environmental stressors and consequently involved in the development and progression of ageing and age-related disease. Microvascular dysfunction, if not targeted, leads to a steady derangement of the phenotype, which cumulates comorbidities and eventually results in a nonrescuable, very high-cardiovascular risk. Along the broad spectrum of pathologies, both shared and distinct molecular pathways and pathophysiological alteration are involved in the disruption of microvascular homeostasis, all pointing to microvascular inflammation as the putative primary culprit. This position paper explores the presence and the detrimental contribution of microvascular inflammation across the whole spectrum of chronic age-related diseases, which characterise the 21st-century healthcare landscape. The manuscript aims to strongly affirm the centrality of microvascular inflammation by recapitulating the current evidence and providing a clear synoptic view of the whole cardiometabolic derangement. Indeed, there is an urgent need for further mechanistic exploration to identify clear, very early or disease-specific molecular targets to provide an effective therapeutic strategy against the otherwise unstoppable rising prevalence of age-related diseases

Photodisintegration of 4^4He into p+t

The two-body photodisintegration of $^4$He into a proton and a triton has been studied using the CEBAF Large-Acceptance Spectrometer (CLAS) at Jefferson Laboratory. Real photons produced with the Hall-B bremsstrahlung-tagging system in the energy range from 0.35 to 1.55 GeV were incident on a liquid $^4$He target. This is the first measurement of the photodisintegration of $^4$He above 0.4 GeV. The differential cross sections for the $\gamma$$^4$He$\to pt$ reaction have been measured as a function of photon-beam energy and proton-scattering angle, and are compared with the latest model calculations by J.-M. Laget. At 0.6-1.2 GeV, our data are in good agreement only with the calculations that include three-body mechanisms, thus confirming their importance. These results reinforce the conclusion of our previous study of the three-body breakup of $^3$He that demonstrated the great importance of three-body mechanisms in the energy region 0.5-0.8 GeV .Comment: 13 pages submitted in one tgz file containing 2 tex file and 22 postscrip figure