Protein phosphatase 5 is required for ATR-mediated checkpoint activation

In response to DNA damage or replication stress, the protein kinase ATR is activated and subsequently transduces genotoxic signals to cell cycle control and DNA repair machinery through phosphorylation of a number of downstream substrates. Very little is known about the molecular mechanism by which ATR is activated in response to genotoxic insults. In this report, we demonstrate that protein phosphatase 5 (PP5) is required for the ATR-mediated checkpoint activation. PP5 forms a complex with ATR in a genotoxic stress-inducible manner. Interference with the expression or the activity of PP5 leads to impairment of the ATR-mediated phosphorylation of hRad17 and Chk1 after UV or hydroxyurea treatment. Similar results are obtained in ATM-deficient cells, suggesting that the observed defect in checkpoint signaling is the consequence of impaired functional interaction between ATR and PP5. In cells exposed to UV irradiation, PP5 is required to elicit an appropriate S-phase checkpoint response. In addition, loss of PP5 leads to premature mitosis after hydroxyurea treatment. Interestingly, reduced PP5 activity exerts differential effects on the formation of intranuclear foci by ATR and replication protein A, implicating a functional role for PP5 in a specific stage of the checkpoint signaling pathway. Taken together, our results suggest that PP5 plays a critical role in the ATR-mediated checkpoint activation.     

The COMPASS family of H3K4 methylases in Drosophila

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila.     

Mutations in the chloroplast inner envelope protein TIC100 impair and repair chloroplast protein import and impact retrograde signaling

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle’s envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100^cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100^cue8 identified a second mutation within the same gene, tic100^soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100^soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100^cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.

Complementary mutations in TIC100 of the chloroplast inner envelope membrane cause reductions or corrective improvements in chloroplast protein import, and highlight a signaling role.

IN A NUTSHELLBackground: Plants harvest energy from the sun and CO₂ from the air and convert them into the energy-rich molecules they, and eventually us, are made of. Plants do this, photosynthesis, in bodies called chloroplasts inside their cells. Chloroplasts, made of protein and membrane material, were, before plants evolved, free-living bacteria, but the synthesis of most of their proteins occurs outside them, using information carried by the cell’s nuclear DNA, so most proteins have to be brought into developing chloroplasts, across the double membrane surrounding them, through dedicated, selective channels, formed by TOC (outer) and TIC (inner envelope) proteins. The identity of those channels matters as it helps determine versions of chloroplasts suited for particular environments. Which TIC proteins constitute the inner envelope channel has been a matter of controversy.Question: A mutant Arabidopsis plant called cue8 is slow-to-green (young leaves begin almost white) and shows delayed chloroplast and plant development. We looked for the molecular identity of the CUE8 gene. We also caused further mutations in this mutant and searched whether any corrected the defects in cue8.Findings: We found the mutated gene causing the cue8 defects is the TIC100 gene. This is one essential component of the “TIC 1-MDa complex,” one of the two versions of the TIC import complex under debate. That complex is made of several proteins, all present at reduced levels in cue8. In laboratory assays in which proteins are imported into isolated chloroplasts, cue8 performed worse than normal plants for a photosynthetic and a housekeeping chloroplast protein. A corrective, “suppressor” mutant was identified, and it carried a second mutation in TIC100, one physically complementary to the first one. Both the single and the double (suppressed) mutant still were slow-to-green, which evidences a signaling role for import defects to the nucleus, making photosynthetic genes active or not.Next steps: Surprisingly the grasses, including the cereals, have one core protein of the TIC 1 MDa complex but not the rest (including TIC100). We don’t know how their TIC channels operate. We also need to learn how the information on the defect in protein import, which occurs at the chloroplast envelope, is relayed to the cell’s nucleus (but we do have some clues).     

Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.     

Preparation of photoluminescent nano-biocomposite nacre from graphene oxide and polylactic acid

Nano-biocomposites of inorganic and organic components wereprepared to produce long-persistent phosphorescent artificial nacre-like materials. Biodegradable polylactic acid (PLA), graphene oxide (GO), and nanoparticles (13–20 nm) of lanthanide-doped aluminate pigment (NLAP) were used in a simple production procedure of an organic/inorganic hybrid nano-biocomposite. Both polylactic acid and GO nanosheets were chemically modified to form covalent and hydrogen bonding. The high toughness, good tensile strength, and great endurance of those bonds were achieved by their interactions at the interfaces. Long-persistent and reversible photoluminescence was shown by the prepared nacre substrates. Upon excitation at 365 nm, the nacre substrates generated an emission peak at 517 nm. When ultraviolet light was shone on luminescent nacres, they displayed a bright green colour. The high superhydrophobicity of the generated nacres was obtained without altering their mechanical characteristics.     

Evaluating the Effects of Separate and Concomitant Use of MK-2206 and Salinomycin on Prostate Cancer Cell Line

Background:Prostate cancer is known as one of the most prevalent health disorders in the male population globally. The aim of the current study was to evaluate the effects of separate and concomitant use of MK-2206 and salinomycin on prostate cancer cell line.Methods:The antitumor potential of separate and concomitant use of MK-2206 and salinomycin was evaluated in a panel of prostate cancer cell line (PC-3). To get insights into the underlying mechanism of action, different assays including the rate of apoptosis, cell viability, and gene expression were performed in treated prostate cancer cells.Results:A significant reduction was detected in the viability percentage of prostate cancer cells (p< 0.001) and the rate of Akt expression (p< 0.001) in all salinomycin, MK-2206, and salinomycin+MK-2206 groups compared to the negative control group. Furthermore, in comparison with the negative control group, there was a notable increase in both the rate of Bad expression (p< 0.001) and prostate cancer cells apoptosis after salinomycin, MK-2206, and salinomycin+MK-2206 treatments. Moreover, the concomitant use of salinomycin+MK-2206 revealed synergistic improvements regarding the viability of prostate cancer cells and the rate of the Akt and Bad expressions compared to the separate administration of salinomycin and MK-2206 (all p< 0.05)Conclusion:The findings of the present study may contribute to improving the efficacy of the therapies regarding the management of prostate cancer and providing a beneficial strategy in clinical trials.Key Words: Apoptosis, Gene Expression, MK 2206, Prostatic Neoplasms, Salinomycin     

Novel pyrrolopyrimidine derivatives: design,synthesis, molecular docking,molecular simulations and biological evaluations as antioxidant and anti-inflammatory agents

Current medical approaches to control the Covid-19 pandemic are either to directly target the SARS-CoV-2 via innovate a defined drug and a safe vaccine or indirectly target the medical complications of the virus. One of the indirect strategies for fighting this virus has been mainly dependent on using anti‐inflammatory drugs to control cytokines storm responsible for severe health complications. We revealed the discovery of novel fused pyrrolopyrimidine derivatives as promising antioxidant and anti-inflammatory agents. The newly synthesised compounds were evaluated for their in vitro anti-inflammatory activity using RAW264.7 cells after stimulation with lipopolysaccharides (LPS). The results revealed that 3a, 4b, and 8e were the most potent analogues. Molecular docking and simulations of these compounds against COX-2, TLR-2 and TLR-4 respectively was performed. The former results were in line with the biological data and proved that 3a, 4b and 8e have potential antioxidant and anti-inflammatory effects.