Interferon-γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein.

The ORF6 protein of the SARS-CoV-2 virus plays a crucial role in blocking the innate immune response of the infected cells by inhibiting interferon pathways. Additionally, it binds to and immobilises the RAE1 protein on the cytoplasmic membranes, thereby blocking mRNA transport from the nucleus to the cytoplasm. In all these cases, the host cell proteins are tethered by the flexible C-terminus of ORF6. A possible strategy to inhibit the biological activity of ORF6 is to bind its C-terminus with suitable ligands. Our in silico experiments suggest that hIFNγ binds the ORF6 protein with high affinity, thus impairing its interactions with RAE1 and, consequently, its activity in viral invasion. The in vitro studies reported here reveal a shift of the localisation of RAE1 in ORF6 overexpressing cells upon treatment with hIFNγ from predominantly cytoplasmic to mainly nuclear, resulting in the restoration of the export of mRNA from the nucleus. We also explored the expression of GFP in transfected-with-ORF6 cells by means of fluorescence microscopy and qRT-PCR, finding that treatment with hIFNγ unblocks the mRNA trafficking and reinstates the GFP expression level. The ability of the cytokine to block ORF6 is also reflected in minimising its negative effects on DNA replication by reducing accumulated RNA-DNA hybrids. Our results, therefore, suggest hIFNγ as a promising inhibitor of the most toxic SARS-CoV-2 protein.

Insights into the SARS-CoV-2 ORF6 Mechanism of Action.

ORF6 is responsible for suppressing the immune response of cells infected by the SARS-CoV-2 virus. It is also the most toxic protein of SARS-CoV-2, and its actions are associated with the viral pathogenicity. Here, we study in silico and in vitro the structure of the protein, its interaction with RAE1 and the mechanism of action behind its high toxicity. We show both computationally and experimentally that SARS-CoV-2 ORF6, embedded in the cytoplasmic membranes, binds to RAE1 and sequesters it in the cytoplasm, thus depleting its availability in the nucleus and impairing nucleocytoplasmic mRNA transport. This negatively affects the cellular genome stability by compromising the cell cycle progression into the S-phase and by promoting the accumulation of RNA-DNA hybrids. Understanding the multiple ways in which ORF6 affects DNA replication may also have important implications for elucidating the pathogenicity of SARS-CoV-2 and developing therapeutic strategies to mitigate its deleterious effects on host cells.

Antitumor Activity of Bioactive Compounds from Rapana venosa against Human Breast Cell Lines.

This study is the first report describing the promising antitumor activity of biologically active compounds isolated from the hemolymph of marine snail Rapana venosa-a fraction with Mw between 50 and 100 kDa and two structural subunits (RvH1 and RvH2), tested on a panel of human breast cell lines-six lines of different molecular subtypes of breast cancer MDA-MB-231, MDA-MB-468, BT-474, BT-549, SK-BR-3, and MCF-7 and the non-cancerous MCF-10A. The fraction with Mw 50-100 kDa (HRv 50-100) showed good antitumor activity manifested by a significant decrease in cell viability, altered morphology, autophagy, and p53 activation in treated cancer cells. An apparent synergistic effect was observed for the combination of HRv 50-100 with cis-platin for all tested cell lines. The combination of HRv 50-100 with cisplatin and/or tamoxifen is three times more effective compared to treatment with classical chemotherapeutics alone. The main proteins in the active fraction, with Mw at ~50 kDa, ~65 kDa, ~100 kDa, were identified by MALDI-MS, MS/MS analyses, and bioinformatics. Homology was established with known proteins with antitumor potential detected in different mollusc species: peroxidase-like protein, glycoproteins Aplysianin A, L-amino acid oxidase (LAAO), and the functional unit with Mw 50 kDa of RvH. Our study reveals new perspectives for application of HRv 50-100 as an antitumor agent used alone or as a booster in combination with different chemotherapies.

Chromatin regulators in DNA replication and genome stability maintenance during S-phase.

The duplication of genetic information is central to life. The replication of genetic information is strictly controlled to ensure that each piece of genomic DNA is copied only once during a cell cycle. Factors that slow or stop replication forks cause replication stress. Replication stress is a major source of genome instability in cancer cells. Multiple control mechanisms facilitate the unimpeded fork progression, prevent fork collapse and coordinate fork repair. Chromatin alterations, caused by histone post-translational modifications and chromatin remodeling, have critical roles in normal replication and in avoiding replication stress and its consequences. This text reviews the chromatin regulators that ensure DNA replication and the proper response to replication stress. We also briefly touch on exploiting replication stress in therapeutic strategies. As chromatin regulators are frequently mutated in cancer, manipulating their activity could provide many possibilities for personalized treatment.

Molecular Mechanism of the Anti-Inflammatory Action of Heparin.

Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecular-weight heparin (LMWH) based on its influence on the activity of two key cytokines, IFNγ and IL-6. The mechanism of heparin binding to IFNγ and IL-6 and the resulting inhibition of their activity were studied by means of extensive molecular-dynamics simulations. The effect of LMWH on IFNγ signalling inside stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. We found that LMWH binds with high affinity to IFNγ and is able to fully inhibit the interaction with its cellular receptor. It also influences the biological activity of IL-6 by binding to either IL-6 or IL-6/IL-6Rα, thus preventing the formation of the IL-6/IL-6Rα/gp130 signalling complex. These findings shed light on the molecular mechanism of the anti-inflammatory action of LMWH and underpin its ability to influence favourably conditions characterised by overexpression of these two cytokines. Such conditions are not only associated with autoimmune diseases, but also with inflammatory processes, in particular with COVID-19. Our results put forward heparin as a promising means for the prevention and suppression of severe CRS and encourage further investigations on its applicability as an anti-inflammatory agent.

Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability.

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.

Deregulated levels of RUVBL1 induce transcription-dependent replication stress.

Chromatin regulators control transcription and replication, however if and how they might influence the coordination of these processes still is largely unknown. RUVBL1 and the related ATPase RUVBL2 participate in multiple nuclear processes and are implicated in cancer. Here, we report that both the excess and the deficit of the chromatin regulator RUVBL1 impede DNA replication as a consequence of altered transcription. Surprisingly, cells that either overexpressed or were silenced for RUVBL1 had slower replication fork rates and accumulated phosphorylated H2AX, dependent on active transcription. However, the mechanisms of transcription-dependent replication stress were different when RUVBL1 was overexpressed and when depleted. RUVBL1 overexpression led to increased c-Myc-dependent pause release of RNAPII, as evidenced by higher overall transcription, much stronger Ser2 phosphorylation of Rpb1- C-terminal domain, and enhanced colocalization of Rpb1 and c-Myc. RUVBL1 deficiency resulted in increased ubiquitination of Rpb1 and reduced mobility of an RNAP subunit, suggesting accumulation of stalled RNAPIIs on chromatin. Overall, our data show that by modulating the state of RNAPII complexes, RUVBL1 deregulation induces replication-transcription interference and compromises genome integrity during S-phase.

The Chromatin Response to Double-Strand DNA Breaks and Their Repair.

Cellular DNA is constantly being damaged by numerous internal and external mutagenic factors. Probably the most severe type of insults DNA could suffer are the double-strand DNA breaks (DSBs). They sever both DNA strands and compromise genomic stability, causing deleterious chromosomal aberrations that are implicated in numerous maladies, including cancer. Not surprisingly, cells have evolved several DSB repair pathways encompassing hundreds of different DNA repair proteins to cope with this challenge. In eukaryotic cells, DSB repair is fulfilled in the immensely complex environment of the chromatin. The chromatin is not just a passive background that accommodates the multitude of DNA repair proteins, but it is a highly dynamic and active participant in the repair process. Chromatin alterations, such as changing patterns of histone modifications shaped by numerous histone-modifying enzymes and chromatin remodeling, are pivotal for proficient DSB repair. Dynamic chromatin changes ensure accessibility to the damaged region, recruit DNA repair proteins, and regulate their association and activity, contributing to DSB repair pathway choice and coordination. Given the paramount importance of DSB repair in tumorigenesis and cancer progression, DSB repair has turned into an attractive target for the development of novel anticancer therapies, some of which have already entered the clinic.

Universal Codons with Enrichment from GC to AU Nucleotide Composition Reveal a Chronological Assignment from Early to Late Along with LUCA Formation.

The emergence of a primitive genetic code should be considered the most essential event during the origin of life. Almost a complete set of codons (as we know them) should have been established relatively early during the evolution of the last universal common ancestor (LUCA) from which all known organisms descended. Many hypotheses have been proposed to explain the driving forces and chronology of the evolution of the genetic code; however, none is commonly accepted. In the current paper, we explore the features of the genetic code that, in our view, reflect the mechanism and the chronological order of the origin of the genetic code. Our hypothesis postulates that the primordial RNA was mostly GC-rich, and this bias was reflected in the order of amino acid codon assignment. If we arrange the codons and their corresponding amino acids from GC-rich to AU-rich, we find that: 1. The amino acids encoded by GC-rich codons (Ala, Gly, Arg, and Pro) are those that contribute the most to the interactions with RNA (if incorporated into short peptides). 2. This order correlates with the addition of novel functions necessary for the evolution from simple to longer folded peptides. 3. The overlay of aminoacyl-tRNA synthetases (aaRS) to the amino acid order produces a distinctive zonal distribution for class I and class II suggesting an interdependent origin. These correlations could be explained by the active role of the bridge peptide (BP), which we proposed earlier in the evolution of the genetic code.

Chromatin control in double strand break repair.

DNA double strand breaks (DSB) are the most deleterious type of damage inflicted on DNA by various environmental factors and as consequences of normal cellular metabolism. The multistep nature of DSB repair and the need to assemble large protein complexes at repair sites necessitate multiple chromatin changes there. This review focuses on the key findings of how chromatin regulators exert temporal and spatial control on DSB repair. These mechanisms coordinate repair with cell cycle progression, lead to DSB repair pathway choice, provide accessibility of repair machinery to damaged sites and move the lesions to nuclear environments permissive for repair.

Mammalian INO80 chromatin remodeler cooperates with FANCM to mediate DNA interstrand crosslink-induced checkpoint activation and repair.

Chromatin regulators play crucial roles in the DNA damage response. While the chromatin changes needed for double-strand break repair and nucleotide excision repair have been intensely studied, the chromatin requirements of interstrand crosslink (ICL) repair have remained largely unexplored. Here, we studied the effect of silencing the INO80 chromatin remodeler subunits on the cellular response to ICLs. Cells depleted of Ino80 ATPase were more sensitive to mitomycin C (MMC) and defective in FANCD2 chromatin recruitment. Ino80-deficient cells displayed strongly reduced Chk1 phosphorylation after MMC treatment indicating impaired ATR-dependent DNA damage signaling, likely due to the significantly slower RPA foci formation which we observed in these cells. MMC treatment of cells silenced for FANCM - a protein required for ICL-induced checkpoint signaling, Ino80 or both genes simultaneously led to similar decreases in RPA phosphorylation suggesting that the two proteins were involved in the same checkpoint pathway. Co-immunoprecipitation data indicated that Ino80 and FANCM interact physically. Taken together our data demonstrate for the first time that the INO80 chromatin remodeler cooperates with FANCM to mediate ICL-induced checkpoint activation by promoting accumulation of RPA at the lesion sites. This constitutes a novel mechanism by which the INO80 chromatin remodeler participates in the repair of ICLs and genome integrity maintenance.

Possible Emergence of Sequence Specific RNA Aminoacylation via Peptide Intermediary to Initiate Darwinian Evolution and Code Through Origin of Life.

One of the most intriguing questions in biological science is how life originated on Earth. A large number of hypotheses have been proposed to explain it, each putting an emphasis on different events leading to functional translation and self-sustained system. Here, we propose a set of interactions that could have taken place in the prebiotic environment. According to our hypothesis, hybridization-induced proximity of short aminoacylated RNAs led to the synthesis of peptides of random sequence. We postulate that among these emerged a type of peptide(s) capable of stimulating the interaction between specific RNAs and specific amino acids, which we call "bridge peptide" (BP). We conclude that translation should have emerged at the same time when the standard genetic code begun to evolve due to the stabilizing effect on RNA-peptide complexes with the help of BPs. Ribosomes, ribozymes, and the enzyme-directed RNA replication could co-evolve within the same period, as logical outcome of RNA-peptide world without the need of RNA only self-sustained step.

The repair capacity of lung cancer cell lines A549 and H1299 depends on HMGB1 expression level and the p53 status.

Elucidation of the cellular components responsive to chemotherapeutic agents as cisplatin rationalizes the strategy for anticancer chemotherapy. The removal of the cisplatin/DNA lesions gives the chance to the cancer cells to survive and compromises the chemotherapeutical treatment. Therefore, the cell repair efficiency is substantial for the clinical outcome. High mobility group box 1 (HMGB1) protein is considered to be involved in the removal of the lesions as it binds with high affinity to cisplatin/DNA adducts. We demonstrated that overexpression of HMGB1 protein inhibited cis-platinated DNA repair in vivo and the effect strongly depended on its C-terminus. We registered increased levels of DNA repair after HMGB1 silencing only in p53 defective H1299 lung cancer cells. Next, introduction of functional p53 resulted in DNA repair inhibition. H1299 cells overexpressing HMGB1 were significantly sensitized to treatment with cisplatin demonstrating the close relation between the role of HMGB1 in repair of cis-platinated DNA and the efficiency of the anticancer drug, the process being modulated by the C-terminus. In A549 cells with functional p53, the repair of cisplatin/DNA adducts is determined by а complex action of HMGB1 and p53 as an increase of DNA repair capacity was registered only after silencing of both proteins.

The mammalian INO80 chromatin remodeling complex is required for replication stress recovery.

A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart.

Chromatin structure in double strand break repair.

Cells are under constant assault by endogenous and environmental DNA damaging agents. DNA double strand breaks (DSBs) sever entire chromosomes and pose a major threat to genome integrity as a result of chromosomal fragment loss or chromosomal rearrangements. Exogenous factors such as ionizing radiation, crosslinking agents, and topoisomerase poisons, contribute to break formation. DSBs are associated with oxidative metabolism, form during the normal S phase, when replication forks collapse and are generated during physiological processes such as V(D)J recombination, yeast mating type switching and meiosis. It is estimated that in mammalian cells ∼10 DSBs per cell are formed daily. If left unrepaired DSBs can lead to cell death or deregulated growth, and cancer development. Cellular response to DSB damage includes mechanisms to halt the progression of the cell cycle and to restore the structure of the broken chromosome. Changes in chromatin adjacent to DNA break sites are instrumental to the DNA damage response (DDR) with two apparent ends: to control compaction and to bind repair and signaling molecules to the lesion. Here, we review the key findings related to each of these functions and examine their cross-talk.

Shaping chromatin for repair.

To counteract the adverse effects of various DNA lesions, cells have evolved an array of diverse repair pathways to restore DNA structure and to coordinate repair with cell cycle regulation. Chromatin changes are an integral part of the DNA damage response, particularly with regard to the types of repair that involve assembly of large multiprotein complexes such as those involved in double strand break (DSB) repair and nucleotide excision repair (NER). A number of phosphorylation, acetylation, methylation, ubiquitylation and chromatin remodeling events modulate chromatin structure at the lesion site. These changes demarcate chromatin neighboring the lesion, afford accessibility and binding surfaces to repair factors and provide on-the-spot means to coordinate repair and damage signaling. Thus, the hierarchical assembly of repair factors at a double strand break is mostly due to their regulated interactions with posttranslational modifications of histones. A large number of chromatin remodelers are required at different stages of DSB repair and NER. Remodelers physically interact with proteins involved in repair processes, suggesting that chromatin remodeling is a requisite for repair factors to access the damaged site. Together, recent findings define the roles of histone post-translational modifications and chromatin remodeling in the DNA damage response and underscore possible differences in the requirements for these events in relation to the chromatin context.

The inhibitor of histone deacetylases sodium butyrate enhances the cytotoxicity of mitomycin C.

The use of histone deacetylase inhibitors has been proposed as a promising approach to increase the cell killing effect of DNA damage-inducing drugs in chemotherapy. However, the molecular mechanism of their action remains understudied. In the present article, we have assessed the effect of the histone deacetylase inhibitor sodium butyrate on the DNA damage response induced by the crosslinking agent mitomycin C. Sodium butyrate increased mitomycin C cytotoxicity, but did not impair the repair pathways required to remove mitomycin C-induced lesions as neither the rate of nucleotide excision repair nor the homologous recombination repair rate were diminished. Sodium butyrate treatment abrogated the S-phase cell-cycle checkpoint in mitomycin C-treated cells and induced the G(2)-M checkpoint. However, sodium butyrate treatment alone resulted in accumulation of reactive oxygen species, double-strand breaks in DNA, and apoptosis. These results imply that the accumulation of reactive oxygen species-mediated increase in DNA lesion burden may be the major mechanism by which sodium butyrate enhances the cytotoxicity of mitomycin C.

Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection.

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.

Lack of effect of RuvB-like proteins on DNA damage signaling activation.

Ataxia telangiectasia mutated (ATM) kinase is a central player in cellular response to DNA damage. Phosphorylation of the histone H2AX by ATM is required for the accumulation of repair proteins at the sites of double-strand breaks. Recently, it was reported that the histone acetyltransferase Tat interactive protein-60 (IPP60) is required to acetylate ATM prior to its activation. The RuvB-like proteins TIP48 and TIP49 are known to be necessary for the assembly and functional activity of the TIP60 acetyltransferase complex. In the present communication, we investigated the requirements of IIP48 and IIP49 for ATM activation by monitoring the cell cycle distribution and H2AX phosphorylation after irradiation of IIP48- and IIP49-depleted cells. We found that neither the cell cycle norgammay-H2AX were affected in IIP48- and IIP49-silenced cells, suggesting that the IIP60 chromatin modification complex is not engaged in DNA damage signaling upstream of ATM.

RAD51 foci formation in response to DNA damage is modulated by TIP49.

Chromatin modification plays an important role in modulating the access of homologous recombination proteins to the sites of DNA damage. TIP49 is highly conserved component of chromatin modification/remodeling complexes, but its involvement in homologous recombination repair in mammalian cells has not been examined in details. In the present communication we studied the role of TIP49 in recruitment of the key homologous recombination protein RAD51 to sites of DNA damage. RAD51 redistribution to chromatin and nuclear foci formation induced by double-strand breaks and interstrand crosslinks were followed under conditions of TIP49 depletion by RNA interference. TIP49 silencing reduced RAD51 recruitment to chromatin and nuclear foci formation to about 50% of that of the control. Silencing of TIP48, which is closely related to TIP49, induced a similar reduction in RAD51 foci formation. RAD51 foci reduction in TIP49-silenced cells was not a result of defective DNA damage checkpoint signaling as judged by the normal histone H2AX phosphorylation and cell cycle distribution. Treatment with the histone deacetylase inhibitor sodium butyrate restored RAD51 foci formation in the TIP49-depleted cells. The results suggest that as a constituent of chromatin modification complexes TIP49 may facilitate the access of the repair machinery to the sites of DNA damage.