ASF1 Proteins are Involved in UV-induced DNA Damage Repair and are Cell Cycle Regulated by E2F Transcription Factors in Arabidopsis thaliana

ASF1 is a key histone H3/H4 chaperone that participates in a variety of DNA and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly is of primaryrelevance. Information concerning the role of ASF1 proteins in post-UV response in higher plants is currently limited. In Arabidopsis thaliana, an initial analysis of in vivo localization of ASF1A andASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoteranalysis identified ASF1A and ASF1B as potential targets of E2F transcription factors. Theseobservations were experimentally validated, both in vitro by electrophoretic mobility shift assays, and in vivo by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B induced DNA damage response in A. thaliana. Transcript levels of ASF1A and ASF1B were increased following a UV-B-treatment. Consistent with a potential role in ultraviolet-B (UV-B) response, RNAi silenced plants of both genes showed increased sensitivity to UV-B compared to wild type plants. Finally, by coimmunoprecipitation analysis, we found that ASF1 physically interacts with N-terminal acetylated histones H3 and H4, and with acetyltransferases of the HAM subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, here we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation.Fil: Lario, Luciana Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Gutierrez, Crisanto. Universidad Autónoma de Madrid; EspañaFil: Ramirez Parra, Elena. Universidad Politecnica de Madrid; EspañaFil: Spampinato, Claudia Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Casati, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentin

In vitro PKA phosphorylation-mediated human PDE4A4 activation

AbstractThe PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg2+ dose–response (EC50 of ∼0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA-catalytic subunit increases the enzyme’s sensitivity to Mg2+, leading to 4-fold increased cAMP hydrolysis without affecting its Km. The phosphorylation also increases the potencies of (R)- and (S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity

CD38 Deficiency Ameliorates Chronic Graft-Versus-Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

© 2021 Martínez-Blanco, Domínguez-Pantoja, Botía-Sánchez, Pérez-Cabrera, Bello-Iglesias, Carrillo-Rodríguez, Martin-Morales, Lario-Simón, Pérez-Sánchez-Cañete, Montosa-Hidalgo, Guerrero-Fernández, Longobardo-Polanco, Redondo-Sánchez, Cornet-Gomez, Torres-Sáez, Fernández-Ibáñez, Terrón-Camero, Andrés-León, O’Valle, Merino, Zubiaur and Sancho.The absence of the mouse cell surface receptor CD38 in Cd38−/− mice suggests that this receptor acts as a positive regulator of inflammatory and autoimmune responses. Here, we report that, in the context of the chronic graft-versus-host disease (cGVHD) lupus inducible model, the transfer of B6.C-H2bm12/KhEg(bm12) spleen cells into co-isogenic Cd38−/− B6 mice causes milder lupus-like autoimmunity with lower levels of anti-ssDNA autoantibodies than the transfer of bm12 spleen cells into WT B6 mice. In addition, significantly lower percentages of Tfh cells, as well as GC B cells, plasma cells, and T-bet+CD11chi B cells, were observed in Cd38−/− mice than in WT mice, while the expansion of Treg cells and Tfr cells was normal, suggesting that the ability of Cd38−/− B cells to respond to allogeneic help from bm12 CD4+ T cells is greatly diminished. The frequencies of T-bet+CD11chi B cells, which are considered the precursors of the autoantibody-secreting cells, correlate with anti-ssDNA autoantibody serum levels, IL-27, and sCD40L. Proteomics profiling of the spleens from WT cGVHD mice reflects a STAT1-driven type I IFN signature, which is absent in Cd38−/− cGVHD mice. Kidney, spleen, and liver inflammation was mild and resolved faster in Cd38−/− cGVHD mice than in WT cGVHD mice. We conclude that CD38 in B cells functions as a modulator receptor that controls autoimmune responses.S and MZ received financial support through “Proyecto del Plan Estatal”: SAF2017–89801-R. The IPBLN-CSIC Proteomics Unit belonged to ProteoRed-ISCIII (PRB2; PRB3) and was supported by grants PT13/0001/0011 (IPBLN-CSIC) and PT17/0019/0010 (CIB-CSIC; IPBLN-CSIC). RM: Project: SAF2017-82905-R. FO'V: Cátedra MIS IMPLANT-UGR. The stay of AC-G in Sancho’s lab was supported by a fellowship-contract JAE-Intro (CSIC). The stay of MD-P in Sancho’s lab was supported by a 1-year post-doctoral fellowship (Reference No. 502492) from the Consejo Nacional de Ciencia y Tecnología (CONACYT) of México. EA-L was recipient of a postdoctoral fellowship from the regional Andalusian Government

Extracellular vesicles from pristane-treated CD38-deficient mice express an antiinflammatory neutrophil protein signature, which reflects the mild lupus severity elicited in these mice

In CD38-deficient (Cd38-/-) mice intraperitoneal injection of pristane induces a lupus-like disease, which is milder than that induced in WT mice, showing significant differences in the inflammatory and autoimmune processes triggered by pristane. Extracellular vesicles (EV) are present in all body fluids. Shed by cells, their molecular make-up reflects that of their cell of origin and/or tissue pathological situation. The aim of this study was to analyze the protein composition, protein abundance, and functional clustering of EV released by peritoneal exudate cells (PECs) in the pristane experimental lupus model, to identify predictive or diagnostic biomarkers that might discriminate the autoimmune process in lupus from inflammatory reactions and/or normal physiological processes. In this study, thanks to an extensive proteomic analysis and powerful bioinformatics software, distinct EV subtypes were identified in the peritoneal exudates of pristane-treated mice: 1) small EV enriched in the tetraspanin CD63 and CD9, which are likely of exosomal origin; 2) small EV enriched in CD47 and CD9, which are also enriched in plasma-membrane, membrane-associated proteins, with an ectosomal origin; 3) small EV enriched in keratins, ECM proteins, complement/coagulation proteins, fibrin clot formation proteins, and endopetidase inhibitor proteins. This enrichment may have an inflammation-mediated mesothelial-tomesenchymal transition origin, representing a protein corona on the surface of peritoneal exudate EV; 4) HDL-enriched lipoprotein particles. Quantitative proteomic analysis allowed us to identify an anti-inflammatory, Annexin A1- enriched pro-resolving, neutrophil protein signature, which was more prominent in EV from pristane-treated Cd38-/- mice, and quantitative differences in the protein cargo of the ECM-enriched EV from Cd38-/- vs WT mice. These differences are likely to be related with the distinct inflammatory outcome shown by Cd38-/- vs WT mice in response to pristane treatment. Our results demonstrate the power of a hypothesis-free and data-driven approach to transform the heterogeneity of the peritoneal exudate EV from pristanetreated mice in valuable information about the relative proportion of different EV in a given sample and to identify potential protein markers specific for the different small EV subtypes, in particular those proteins defining EV involved in the resolution phase of chronic inflammation.Proyecto del plan estatal, Ministerio de Ciencia e Innovacion PT13/0001/011CSIC PT17/0019/0010 PID2020-119567RB-I0

Organización del Campeonato Mundial de Gimnasia Artística

El evento se trata de un Mundial de Gimnasia Artística celebrado en Madrid en 2023. Se disputará en el IFEMA, Pabellón 9, entre el 6 y el 15 de Octubre. Acudirán 80 nacionalidades distintas. De las cuales serán 23 miembros de la FIG, 102 jueces, 459 gimnastas y 800 miembros de las delegaciones. Se seguirán los apartados principales indicados en el Handbook de la FIG para organizar un Mundial de esta disciplina, abordando todos los puntos necesarios para su realización. Este evento será viable económicamente a la vez que conseguirá el objetivo principal de promocionar la Gimnasia Artística en España y aumentar el número de practicantes y aficionados

Bioisosterism: a hydrogen bonding study of methanesulfonanilides

Bibliography: p. 186-193

Towards understanding flavin reactivity : a structural study of cholesterol oxidase

Flavoenzymes catalyze a wide variety of biochemical reactions and are commonly observed as electron transport proteins. The redox reactive portion of the enzymes is the isoalloxazine ring system of the flavin cofactor. It is known that the protein environment modulates the redox potential of the flavin, for example, "tuning" its redox potential to favor either a one-electron transfer (electron transfer proteins) or a two-electron transfer (oxidation reactions). This thesis presents an in depth structural study of the flavoenzyme, cholesterol oxidase (EC 1.1.3.6) from Streptomyces sp. SA-COO (SCOA) a multifunctional enzyme that oxidizes and isomerizes 3-beta-hydroxysteroids. This work was pursued in order to further our understanding of the mechanisms through which the protein interacts with the isoalloxazine system and modulates reactivity. Previous kinetic experiments have identified an active site asparagine (N485) and a histidine residue (H447) both of which are critical to the oxidative activity of the enzyme. On an atomic scale the role of the asparagine residue was unknown. Using mutagensis and crystallographic techniques we have characterized this novel N-H ··· pi protein-flavin interaction. SCOA crystals diffract to sub-atomic resolution providing us with a unique view of the protein bound isoalloxazine system. These atomic resolution maps have revealed unexpected structural features that were not previously apparent in the 1.5 A resolution of SCOA. For example, a second narrow pathway leading directly to the isoalloxazine system was discovered, which has provided a more complete mechanistic understanding of the reactions catalyzed by SCOA. Five atomic resolution structures of SCOA at varying pH values are reported. Differences among these structures provide insight into the affect of pH on protein structure and have revealed structural differences resulting from an inadvertent reduction of the cofactor. For example, these str

Role of AtMSH7 in UV-B-induced DNA damage recognition and recombination

The mismatch repair (MMR) system maintains genome integrity by correcting replication associated errors and inhibiting recombination between divergent DNA sequences. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in this DNA repair system vary among organisms. Plants have an extra mismatch recognition protein, named MutS. The protein is a heterodimer of MSH2?MSH7. To further understand the in vivo role of MSH7, we present data from this protein in Arabidopsis thaliana plants. First, we generated transgenic plants that express the β-glucuronidase (GUS) under the control of the MSH7 promoter. Histochemical staining of the transgenic plants indicated that MSH7 is preferentially expressed in proliferating tissues. Then, we identified msh7 T-DNA insertion mutants. Plants deficient in MSH7 show increased levels of UV-B-induced cyclobutane pyrimidine dimers (CPDs) relative to wild-type (WT) plants. Consistent with the patterns of MSH7 expression, we next analyzed the role of the protein during somatic and meiotic recombination. The frequency of somatic recombination between homologous or homeologous repeats (divergence level of 1.6%) was monitored using a previously described GUS recombination reporter assay. Disruption of MSH7 has no effect on the rates of somatic homologous or homeologous recombination under control conditions or after UV-B exposure. However, the rate of meiotic recombination between two genetically linked seed-specific fluorescent markers was 97% higher in msh7 than in WT plants. Taken together, these results suggest that MSH7 is involved in UV-B-induced DNA damage recognition and in controlling meiotic recombination.Fil: Lario, Luciana Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Botta, Pablo Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Casati, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Spampinato, Claudia Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentin