Development of Allosteric Small Molecule APOBEC3B Inhibitors from In Silico Screening.

APOBEC3B cytosine deaminase contributes to the mutational burdens of tumors, resulting in tumor progression and therapy resistance. Small molecule APOBEC3B inhibitors have potential to slow or mitigate these detrimental outcomes. Through molecular dynamics (MD) simulations and computational solvent mapping analysis, we identified a novel putative allosteric pocket on the C-terminal domain of APOBEC3B (A3Bctd), and virtually screened the ChemBridge Diversity Set (N~110,000) against both the active and potential allosteric sites. Selected high-scoring compounds were subsequently purchased, characterized for purity and composition, and tested in biochemical assays, which yielded 13 hit compounds. Orthogonal NMR assays verified binding to the target protein. Initial selectivity studies suggest these compounds preferentially target A3Bctd over related deaminase APOBEC3A (A3A), and MD simulations indicate this selectivity may be due to the steric repulsion from H56 that is unique to A3A. Taken together, our studies represent the first virtual screening effort against A3Bctd that has yielded candidate inhibitors suitable for further development.

Investigating the individual and combined effects of coenzyme Q10 and vitamin C on CLP-induced cardiac injury in rats.

Sepsis-induced cardiac injury represents a major clinical challenge, amplifying the urgency for effective therapeutic interventions. This study aimed to delve into the individual and combined prophylactic effects of Vitamin C (Vit C) and Coenzyme Q10 (CoQ10) against inflammatory heart injury in a cecal ligation and puncture (CLP) induced polymicrobial sepsis rat model. Thirty adult female Sprague-Dawley rats were randomly divided into five groups: Control, CLP, Vitamin C, CoQ10, and Vit C + CoQ10, each consisting of six rats. Treatments were administered orally via gavage for 10 days prior to the operation. Eighteen hours post-sepsis induction, the animals were euthanized, and specimens were collected for analysis. The study examined variations in oxidative (TOS, OSI, MDA, MPO) and antioxidative markers (TAS, SOD, CAT, GSH), histopathological changes, inflammatory cytokine concentrations (TNF-α, IL-1ß), nitric oxide (NO) dynamics, and cardiac indicators such as CK-MB. Impressively, the combined regimen markedly diminished oxidative stress, and antioxidative parameters reflected notable enhancements. Elevated NO levels, a central player in sepsis-driven inflammatory cascades, were effectively tempered by our intervention. Histological examinations corroborated the biochemical data, revealing diminished cardiac tissue damage in treated subjects. Furthermore, a marked suppression in pro-inflammatory cytokines was discerned, solidifying the therapeutic potential of our intervention. Interestingly, in certain evaluations, CoQ10 exhibited superior benefits over Vit C. Collectively, these findings underscore the potential therapeutic promise of Vit C and CoQ10 combination against septic cardiac injuries in rats.

Hispidulin exerts a protective effect against oleic acid induced-ARDS in the rat via inhibition of ACE activity and MAPK pathway.

This study investigates the protective role of Hispidulin on acute respiratory distress syndrome (ARDS) in rats. Rats were divided into three groups: control, ARDS, ARDS+ Hispidulin. The ARDS models were established by injecting rats with oleic acid. Hispidulin (100 mg/kg) was injected i.p. an hour before ARDS. Myeloperoxidase (MPO), Interleukin-8 (IL-8), Mitogen-activated protein kinases (MAPK), Lipid Peroxidation (LPO), Superoxide Dismutase (SOD), Glutathione (GSH), and Angiotensin-converting enzyme (ACE) were determined by ELISA. Tumor necrosis factor-alpha (TNF-α) expression was described by RT-qPCR. Caspase-3 immunostaining was performed to evaluate apoptosis. Compared with the model group, a significant decrease was observed in the MPO, IL-8, MAPK, ACE, LPO levels, and TNF-α expression in the ARDS+ Hispidulin group. Moreover, reduced caspase-3 immunoreactivity and activity of ACE were detected in the Hispidulin+ARDS group. The protective effect of Hispidulin treatment may act through inhibition of the ACE activity and then regulation of inflammatory cytokine level and alteration of apoptosis.

ENHANCED EFFICACY OF RESVERATROL-LOADED SILVER NANOPARTICLE IN ATTENUATING SEPSIS-INDUCED ACUTE LIVER INJURY: MODULATION OF INFLAMMATION, OXIDATIVE STRESS, AND SIRT1 ACTIVATION.

ABSTRACT: Sepsis-induced acute liver injury is a life-threatening condition involving inflammation, oxidative stress, and endothelial dysfunction. In the present study, the preventive effects of resveratrol (RV) alone and RV-loaded silver nanoparticles (AgNPs + RV) against sepsis-induced damage were investigated and compared in a rat model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Rats were divided into four groups: Sham, CLP, RV, and AgNPs + RV. Pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, presepsin, procalcitonin (PCT), 8-hydroxy-2'-deoxyguanosine (8-OHDG), vascular endothelial growth factor (VEGF), and sirtuin-1 (SIRT1) levels were assessed to determine the treatments' effects. AgNPs + RV treatment significantly reduced pro-inflammatory cytokines, NF-κB activation, presepsin, PCT, 8-OHDG, and VEGF levels compared with the CLP group, indicating attenuation of sepsis-induced liver injury. Both RV and AgNPs + RV treatments increased SIRT1 levels, suggesting a potential role of SIRT1 activation in mediating the protective effects. In conclusion, AgNPs + RV treatment demonstrated extremely enhanced efficacy in alleviating sepsis-induced liver injury by modulating inflammation, oxidative stress, and endothelial dysfunction, potentially mediated through SIRT1 activation. In this study, the effect of AgNPs + RV on sepsis was evaluated for the first time, and these findings highlight AgNPs + RV as a promising therapeutic strategy for managing sepsis-induced liver injury, warranting further investigation.

Technology Trends for Massive MIMO towards 6G.

At the dawn of the next-generation wireless systems and networks, massive multiple-input multiple-output (MIMO) in combination with leading-edge technologies, methodologies, and architectures are poised to be a cornerstone technology. Capitalizing on its successful integration and scalability within 5G and beyond, massive MIMO has proven its merits and adaptability. Notably, a series of evolutionary advancements and revolutionary trends have begun to materialize in recent years, envisioned to redefine the landscape of future 6G wireless systems and networks. In particular, the capabilities and performance of future massive MIMO systems will be amplified through the incorporation of cutting-edge technologies, structures, and strategies. These include intelligent omni-surfaces (IOSs)/intelligent reflecting surfaces (IRSs), artificial intelligence (AI), Terahertz (THz) communications, and cell-free architectures. In addition, an array of diverse applications built on the foundation of massive MIMO will continue to proliferate and thrive. These encompass wireless localization and sensing, vehicular communications, non-terrestrial communications, remote sensing, and inter-planetary communications, among others.

Protective effect of astaxanthin and metformin in the liver of rats in which the polycystic ovary syndrome model was formed by giving letrozole.

Objectives: In this study, the effects of astaxanthin on liver tissue in rats with polycystic ovary syndrome (PCOS) were evaluated. Materials and Methods: Fifty-four Spraque-Dawley rats were divided into 9 groups: Groups: Control, PCOS, PCOS+Metformin (Met), PCOS+ Astaxanthin (ASX)10, PCOS+ASX20, PCOS+ASX40, PCOS+Met+ASX10, PCOS+Met+ASX20, and PCOS+Met+ASX40. PCOS was induced in female rats by oral administration of letrozole (1 mg/kg) for 21 days. Rats were treated with ASX (10 mg/kg), ASX (20 mg/kg), ASX (40 mg/kg), and metformin (20 mg/kg) for 7 days after PCOS induction. At the end of the experiment, malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured in the liver tissue. The liver was stained with hematoxylin/eosin for histological examination. Additionally, NF-kB and caspase 3 were analyzed immunohistochemically. Results: A remarkable abnormality was observed in the biochemical and histological parameters in the liver tissue of the PCOS model rats. Astaxanthin dose-dependently normalized the MDA level. Additionally, astaxanthin showed a protective effect by increasing the SOD level and increasing its antioxidant activities. We observed that administration of astaxanthin in addition to metformin applied in the standard was more effective. Caspase 3 and NF-kB immune positivity was lower in the groups given astaxanthin compared with PCOS. Histologically, it was observed that astaxanthin improved the deteriorated liver morphology in the letrozole-induced PCOS group. Conclusion: According to our results, it was observed that astaxanthin had antioxidant, anti-inflammatory and anti-apoptotic effects on PCOS in the treatment groups. Therefore, it was concluded that astaxanthin may have a protective effect against PCOS side effects.

A new treatment approach: Melatonin and ascorbic acid synergy shields against sepsis-induced heart and kidney damage in male rats.

AIM: To investigate the combined therapeutic potential of melatonin and ascorbic acid in mitigating sepsis-induced heart and kidney injury in male rats and assess the combination therapy's effects on inflammation, cellular damage, oxidative stress, and vascular function-related markers. MATERIALS AND METHODS: Cecal ligation and puncture (CLP) induced sepsis in male rats, which were divided into five groups: Sham, CLP, MEL (melatonin), ASA (ascorbic acid), and MEL+ASA (melatonin and ascorbic acid). Rats were treated, and heart and kidney tissues were collected for biochemical and histopathological analyses. Inflammatory markers (presepsin, procalcitonin, NF-κB, IL-1ß, IL-6, TNF-α), cellular damage marker (8-OHDG), oxidative status, nitric oxide (NO), vascular endothelial growth factor (VEGF), and sirtuin 1 (SIRT1) levels were assessed. KEY FINDINGS: Melatonin and ascorbic acid treatment reduced inflammatory and cellular damage markers compared to the CLP group. Combined treatment improved NO, VEGF levels, and increased SIRT1 expression, suggesting a synergistic effect in mitigating sepsis-induced inflammation, cellular damage, and oxidative stress. Histopathological analyses supported these findings, revealing reduced heart and kidney injury in the MEL+ASA group. SIGNIFICANCE: Our study highlights potential benefits of combining melatonin and ascorbic acid as a therapeutic strategy for alleviating sepsis-induced heart and kidney injury. The synergistic effects of these agents may provide stronger protection against inflammation, oxidative stress, and tissue damage, opening new avenues for future research and clinical applications in sepsis management.

Protective effect of melatonin and ascorbic acid combination on sepsis-induced lung injury: An Experimental study.

This study investigated the synergistic protective effects of melatonin (MEL) and ascorbic acid (vitamin C, ASA) in treating sepsis-induced lung injury in rats. Rats were divided into five groups: control, cecal ligation and puncture (CLP), CLP + MEL, CLP + ASA and CLP + MEL + ASA. The effects of MEL (10 mg/kg), ASA (100 mg/kg) and their combination on oxidative stress, inflammation and histopathology were evaluated in septic rats' lung tissues. Sepsis-induced oxidative stress and inflammation were evident through increased levels of malondialdehyde (MDA), myeloperoxidase (MPO), total oxidant status (TOS) and oxidative stress index (OSI); decreased levels of superoxide dismutase (SOD), glutathione (GSH), catalase (CAT) and glutathione peroxidase (GPx); and elevated levels of tumour necrosis factor-α (TNF-α) and interleukin-1 ß (IL-1ß) in the lung tissue. Treatment with MEL, ASA and their combination significantly improved antioxidant capacity and reduced oxidative stress, with the combination treatment being more effective. The combination treatment also significantly reduced TNF-α and IL-1ß levels and improved peroxisome proliferator-activated receptor (PPAR), arylesterase (ARE) and paraoxonase (PON) levels in the lung tissue. Histopathological examination showed reduced oedema and lymphocyte infiltration with a lung tissue appearance similar to the control group. Immunohistochemical staining for caspase 3 demonstrated reduced immune positivity in the treatment groups. In conclusion, this study supports the potential synergistic protective effects of MEL and ASA in treating sepsis-induced lung injury. The combination therapy could effectively reduce oxidative stress, inflammation and improve antioxidant capacity in septic rats, suggesting a promising strategy for treating sepsis-induced lung injury.

Inhibition of PRMT5 by market drugs as a novel cancer therapeutic avenue.

Market drugs, such as Food and Drug Administration (FDA) or European Medicines Agency (EMA)-approved drugs for specific indications provide opportunities for repurposing for newer therapeutics. This potentially saves resources invested in clinical trials that verify drug safety and tolerance in humans prior to alternative indication approval. Protein arginine methyltransferase 5 (PRMT5) overexpression has been linked to promoting the tumor phenotype in several cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and breast cancer (BC), making PRMT5 an important target for cancer therapy. Previously, we showed that PRMT5-mediated methylation of the nuclear factor (NF)-κB, partially contributes to its constitutive activation observed in cancers. In this study, we utilized an AlphaLISA-based high-throughput screening method adapted in our lab, and identified one FDA-approved drug, Candesartan cilexetil (Can, used in hypertension treatment) and one EMA-approved drug, Cloperastine hydrochloride (Clo, used in cough treatment) that had significant PRMT5-inhibitory activity, and their anti-tumor properties were validated using cancer phenotypic assays in vitro. Furthermore, PRMT5 selective inhibition of methyltransferase activity was confirmed by reduction of both NF-κB methylation and its subsequent activation upon drug treatment. Using in silico prediction, we identified critical residues on PRMT5 targeted by these drugs that may interfere with its enzymatic activity. Finally, Clo and Can treatment have exhibited marked reduction in tumor growth in vivo. Overall, we provide basis for pursuing repurposing Clo and Can as anti-PRMT5 cancer therapies. Our study offers potential safe and fast repurposing of previously unknown PRMT5 inhibitors into clinical practice.

Impact of temperature and biomass augmentation on biosulfur-driven autotrophic denitrification in membrane bioreactors treating real nitrate-contaminated groundwater.

Nitrate (NO3-) contamination of groundwater is a major health concern worldwide as it can lead to serious illnesses such as methemoglobinemia and cancer. Autotrophic denitrification is a smart approach for treating groundwater, being typically organic-deficient. Lately, biogenic sulfur (S0bio) has emerged as a sustainable, free, and high-efficiency substrate to fuel membrane bioreactors (MBRs) treating contaminated groundwater. However, the effects of moderate temperature and biomass concentration on the performance and fouling of the S0bio-fed MBR were not investigated previously. This study shows that biomass levels of ~1 g MLVSS/L limit membrane fouling but also denitrification efficiency. Biomass augmentation up to 3 g MLVSS/L enhanced denitrification but worsened fouling due to increase of extracellular polymeric substance (EPS) levels in the bulk liquid. Temperature decrease from 30 °C to 20 °C halved denitrification efficiency, which could be partially recovered through bioaugmentation. The mechanisms affected by temperature decrease, practical applications, and future research needs were discussed.

Discovery of compounds that reactivate p53 mutants in vitro and in vivo.

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

Potential protective effect of astaxanthin on ovary ischemia-reperfusion injury.

Objectives: We thought that astaxanthin (ASX) might be a protective agent in oxidative stress damage that develops against ischemia and reperfusion injury in the rat ovary. Materials and Methods: The experimental groups consisted of healthy, I (Ischemia), I+ASX50, I+ASX100, I/R (Ischemia/Reperfusion), I/R+ ASX50, and I/R+ ASX100. Vascular clamps were applied to the ovaries for 3 hr to induce ischemia. For the reperfusion groups, the clamps were opened and blood flow was restored to the ovaries for 3 hr. At the end of the experiment, biochemical, histopathological, and immunohistochemical analyses were made from the tissue samples taken. Results: While MDA levels increased significantly in I and I/R groups, SOD levels decreased. It was found that ASX significantly decreased MDA levels and increased SOD activity in treatment groups depending on the dose. Caspase 3, IL-1 ß, and IL-6 expressions were severely increased in ischemia and I/R groups, while the severity of I+ASX50 and I/R+ASX100 immunoreactivity was decreased. While severe hemorrhage areas were observed in I and IR groups, minimal hemorrhage areas were observed in the treatment groups, especially in I/R+ASX100 groups. In addition, inflammatory cells and necrotic cells in the I/R group were not observed in I/R+ASX50 and I/R+ASX100 groups. Conclusion: As a result, it was determined that ASX has a strong protective role against oxidative damage in the treatment of ovarian ischemia-reperfusion injury.

The effects of dragon fruit (Hylocereus polyrhizus) extract on indomethacin-induced stomach ulcer in rats.

The aim of this study was to investigate the effects of red dragon fruit (Hylocereus polyrhizus) extract (DFE) on the stomach in ulcer model induced by indomethacin in rats. Effects of DFE were evaluated in indomethacin-induced gastric damage model on Sprague-Dawley rats. Experimental model: all rats were fasted for 24 h. At the end of this period, DFE was administered to the ulcer-induced groups. One hour after this application, a dose of 25 mg/kg of indomethacin was applied by oral gavage to all groups except the HEALTHY and DFE1000 groups. Six hours after indomethacin administration, the rats were euthanized with high-dose anesthesia and the experiment was terminated. Macroscopic and microscopic analyses for investigating ulcerative area, molecular and biochemical analyses for oxidative damages investigation and molecular analyses for the effect mechanism of indomethacin and DFE were conducted on stomach tissues in the study. While oxidative stress-associated markers such as MDA, BAX, and Caspase 3 increased dramatically in the indomethacin group, GSH antioxidant levels decreased. It was observed that these parameters were significantly improved in DFE 500 mg/kg and DFE 1000 mg/kg groups compared to ulcer group, and the results of especially DFE 1000 mg/kg group were similar to famotidine group. We observed that our histopathological findings also supported all our other findings. Dragon fruit extract was protected against indomethacin-induced ulcer damage by decreased MDA levels, increased GSH levels, and inhibition of Caspase 3, BAX, and Cox-2, and activation of Cox-1. PRACTICAL APPLICATIONS: People of all ages around the world suffer from gastric ulcer which is one of the most common gastrointestinal ailments. The etiological factors of the disease are using of cigarette and alcohol, nutritional deficiencies, infections, and using non-steroidal anti-inflammatory drugs which use frequent and indiscriminate. Indomethacin is one of the NSAIDs and is commonly preferred to induce ulcer modeling in rats due to its gastric toxicity potential. Current anti-ulcer drugs have many serious side effects. Patients who suffered from gastric ulcer tend to discontinue the drug because of side effects. Therefore, patients need new agents that are non-toxic, have few side effects, and are easily accessible anti-ulcer drugs. Dragon fruit, as a medicinal herb, is highly valuable and widely used in traditional medicine, and may provide gastroprotective activity. Studies have shown that H. polyrhizus has antioxidant activities. We consider the effects of dragon fruit extract (DFE) to be a therapeutic drug for an indomethacin-induced ulcer model.

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-Å resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

Structural Characterization of a Minimal Antibody against Human APOBEC3B.

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.

An integrated view of p53 dynamics, function, and reactivation.

The tumor suppressor p53 plays a vital role in responding to cell stressors such as DNA damage, hypoxia, and tumor formation by inducing cell-cycle arrest, senescence, or apoptosis. Expression level alterations and mutational frequency implicates p53 in most human cancers. In this review, we show how both computational and experimental methods have been used to provide an integrated view of p53 dynamics, function, and reactivation potential. We argue that p53 serves as an exceptional case study for developing methods in modeling intrinsically disordered proteins. We describe how these methods can be leveraged to improve p53 reactivation molecule design and other novel therapeutic modalities, such as PROteolysis TARgeting Chimeras (PROTACs).

RNA Metabolism Guided by RNA Modifications: The Role of SMUG1 in rRNA Quality Control.

RNA modifications are essential for proper RNA processing, quality control, and maturation steps. In the last decade, some eukaryotic DNA repair enzymes have been shown to have an ability to recognize and process modified RNA substrates and thereby contribute to RNA surveillance. Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that not only recognizes and removes uracil and oxidized pyrimidines from DNA but is also able to process modified RNA substrates. SMUG1 interacts with the pseudouridine synthase dyskerin (DKC1), an enzyme essential for the correct assembly of small nucleolar ribonucleoproteins (snRNPs) and ribosomal RNA (rRNA) processing. Here, we review rRNA modifications and RNA quality control mechanisms in general and discuss the specific function of SMUG1 in rRNA metabolism. Cells lacking SMUG1 have elevated levels of immature rRNA molecules and accumulation of 5-hydroxymethyluridine (5hmU) in mature rRNA. SMUG1 may be required for post-transcriptional regulation and quality control of rRNAs, partly by regulating rRNA and stability.

Functional and Structural Insights into a Vif/PPP2R5 Complex Elucidated Using Patient HIV-1 Isolates and Computational Modeling.

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G2/M cell cycle arrest. Vif initiates G2/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-ß/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis.IMPORTANCE A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G2/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G2/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B.

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.