ATP Incorporation Research Articles

Effect of Adenosine 5´ Triphosphate (ATP) and Incubation Time on Frozen Thawed Buffalo Bull Spermatozoa

The present study was aimed to determine the effect of ATP dose rate on longevity, motility, and viability of frozen-thawed buffalo bull’s sperm. For this purpose, 20 frozen semen straws of four different buffalo bulls were obtained from a private semen production unit (Al-Hiawan Sires, Sahiwal). The experiment was replicated four times. In each replicate, four frozen straws of the same bulls were thawed and pooled. After evaluating the motility, the pooled frozen-thawed sample was aliquoted into three separate tubes containing 0 (control), 1-, or 2-mM concentrations of ATP. Afterward, each aliquot was incubated at 37oC for 4 hrs. The sperm motility, viability, and membrane integrity were observed at various time points (10 min., 1hr, 2hr, and 4hr) during the incubation period of four hrs. It was observed that progressive motility and most of the motion kinematic parameters were greater (P<0.05) in control. A significant (P<0.05) interaction was observed between ATP dose and incubation time. The progressive motility, total motility, VCL, VAP, ALH, and BCF decreased (P<0.05) both by incorporation of ATP (1mM or 2mM) and increasing incubation time (from 10 min to 4hrs) which indicate the ATP and incubation time of 4 hours negatively affect the motion kinetics of buffalo bull sperm. In conclusion, it was witnessed that the motility and plasma membrane integrity of sperm declined by the supplementation of ATP in frozen-thawed buffalo bull semen.

Transient-State Kinetic Analysis of the RNA Polymerase II Nucleotide Incorporation Mechanism.

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed Saccharomyces cerevisiae Pol II incorporation of ATP or an ATP analog, Sp-ATP-α-S. Here we have measured the rate constants governing individual steps of the Pol II transcription cycle in the presence of ATP or Sp-ATP-α-S. These results suggest that Pol II catalyzes nucleotide incorporation by binding the next cognate nucleotide and immediately catalyzes bond formation and bond formation is either followed by a conformational change or pyrophosphate release. By comparing our previously published RNA polymerase I (Pol I) and Pol I lacking the A12 subunit (Pol I ΔA12) results that we collected under the same conditions with the identical technique, we show that Pol II and Pol I ΔA12 exhibit similar nucleotide addition mechanisms. This observation indicates that removal of the A12 subunit from Pol I results in a Pol II like enzyme. Taken together, these data further our collective understanding of Pol II's nucleotide incorporation mechanism and the evolutionary divergence of RNA polymerases across the three domains of life.

ATP-binding and hydrolysis of human NLRP3

The innate immune system uses inflammasomal proteins to recognize danger signals and fight invading pathogens. NLRP3, a multidomain protein belonging to the family of STAND ATPases, is characterized by its central nucleotide-binding NACHT domain. The incorporation of ATP is thought to correlate with large conformational changes in NLRP3, leading to an active state of the sensory protein. Here we analyze the intrinsic ATP hydrolysis activity of recombinant NLRP3 by reverse phase HPLC. Wild-type NLRP3 appears in two different conformational states that exhibit an approximately fourteen-fold different hydrolysis activity in accordance with an inactive, autoinhibited state and an open, active state. The impact of canonical residues in the nucleotide binding site as the Walker A and B motifs and sensor 1 and 2 is analyzed by site directed mutagenesis. Cellular experiments show that reduced NLRP3 hydrolysis activity correlates with higher ASC specking after inflammation stimulation. Addition of the kinase NEK7 does not change the hydrolysis activity of NLRP3. Our data provide a comprehensive view on the function of conserved residues in the nucleotide-binding site of NLRP3 and the correlation of ATP hydrolysis with inflammasome activity.

Atypical cyclic di-AMP signaling is essential for Porphyromonas gingivalis growth and regulation of cell envelope homeostasis and virulence

Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3′,5′)-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pdepg) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pdepg and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe–microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.

Engineering Aptamers with Selectively Enhanced Biostability in the Tumor Microenvironment.

Aptamers are emerging as promising molecular tools in cancer-targeted theranostics. Improving their in vivo stability has been a critical issue in promoting clinical translation, but such efforts could lead to more serious side effects resulting from prolonged retention in healthy organs. To address this problem, we developed an environment-responsive stabilization strategy for the selective enhancement of aptamer biostability in the tumor microenvironment (TME). Briefly, by means of the end extension of an ATP-responsive protection (ARP) module, the designed aptamer could be protected from nuclease degradation through the specific incorporation of ATP. Based on our in vivo results, this ARP-aptamer probe was effectively accumulated in tumors via aptamer-based molecular recognition. It showed selectively prolonged tumor retention time, but rapid digestion in healthy organs. Our strategy should provide a new paradigm for the development of organ-specific nucleic acid-based imaging and therapeutic agents.

Engineering Aptamers with Selectively Enhanced Biostability in the Tumor Microenvironment

AbstractAptamers are emerging as promising molecular tools in cancer‐targeted theranostics. Improving their in vivo stability has been a critical issue in promoting clinical translation, but such efforts could lead to more serious side effects resulting from prolonged retention in healthy organs. To address this problem, we developed an environment‐responsive stabilization strategy for the selective enhancement of aptamer biostability in the tumor microenvironment (TME). Briefly, by means of the end extension of an ATP‐responsive protection (ARP) module, the designed aptamer could be protected from nuclease degradation through the specific incorporation of ATP. Based on our in vivo results, this ARP‐aptamer probe was effectively accumulated in tumors via aptamer‐based molecular recognition. It showed selectively prolonged tumor retention time, but rapid digestion in healthy organs. Our strategy should provide a new paradigm for the development of organ‐specific nucleic acid‐based imaging and therapeutic agents.

Synergistic effects of nanoattapulgite and hydroxyapatite on vascularization and bone formation in a rabbit tibia bone defect model.

Hydroxyapatite (HA) is a promising scaffold material for the treatment of bone defects. However, the lack of angiogenic properties and undesirable mechanical properties (such as fragility) limits the application of HA. Nanoattapulgite (ATP) is a nature-derived clay mineral and has been proven to be a promising bioactive material for bone regeneration due to its ability to induce osteogenesis. In this study, polyvinyl alcohol/collagen/ATP/HA (PVA/COL/ATP/HA) scaffolds were printed. Mouse bone marrow mesenchymal stem/stromal cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) were used in vitro to assess the biocompatibility and the osteogenesis and vascularization induction potentials of the scaffolds. Subsequently, in vivo micro-CT and histological staining were carried out to evaluate new bone formation in a rabbit tibial defect model. The in vitro results showed that the incorporation of ATP increased the printing fidelity and mechanical properties, with values of compressive strengths up to 200% over raw PC-H scaffolds. Simultaneously, the expression levels of osteogenic-related genes and vascularization-related genes were significantly increased after the incorporation of ATP. The in vivo results showed that the PVA/COL/ATP/HA scaffolds exhibited synergistic effects on promoting vascularization and bone formation. The combination of ATP and HA provides a promising strategy for vascularized bone tissue engineering.

Transient-state kinetic analysis of multi-nucleotide addition catalyzed by RNA polymerase I

RNA polymerases execute the first step in gene expression: transcription of DNA into RNA. Eukaryotes, unlike prokaryotes, express at least three specialized nuclear multisubunit RNA polymerases (Pol I, Pol II, and Pol III). RNA polymerase I (Pol I) synthesizes the most abundant RNA, ribosomal RNA. Nearly 60% of total transcription is devoted to ribosomal RNA synthesis, making it one of the cell’s most energy consuming tasks. While a kinetic mechanism for nucleotide addition catalyzed by Pol I has been reported, it remains unclear to what degree different nucleotide sequences impact the incorporation rate constants. Furthermore, it is currently unknown if the previous investigation of a single-nucleotide incorporation was sensitive to the translocation step. Here, we show that Pol I exhibits considerable variability in both kmax and K1/2values using an in vitro multi-NTP incorporation assay measuring AMP and GMP incorporations. We found the first two observed nucleotide incorporations exhibited faster kmax-values (∼200 s−1) compared with the remaining seven positions (∼60 s−1). Additionally, the average K1/2 for ATP incorporation was found to be approximately threefold higher compared with GTP, suggesting Pol I has a tighter affinity for GTP compared with ATP. Our results demonstrate that Pol I exhibits significant variability in the observed rate constant describing each nucleotide incorporation. Understanding of the differences between the Pol enzymes will provide insight on the evolutionary pressures that led to their specialized roles. Therefore, the findings resulting from this work are critically important for comparisons with other polymerases across all domains of life.

Selective and stoichiometric incorporation of ATP by self-assembling amyloid fibrils.

ATP acts as a biological hydrotrope preventing protein aggregation. Here, we report a novel chimeric peptide, ACC1-13K8, with an unusual capacity to bind and incorporate ATP while self-assembling into amyloid fibrils. The amino acid sequence combines a highly amyloidogenic segment of insulin's A-chain (ACC1-13) and octalysine (K8). Fibrillization requires binding 2 ATP molecules per ACC1-13K8 monomer and is not triggered by adenosine di- and monophosphates (ADP, AMP). Infrared and CD spectra and AFM-based morphological analysis reveal tight and orderly entrapment of ATP within superstructural hybrid peptide-ATP fibrils. The incorporation of ATP is an emergent property of ACC1-13K8 not observed for ACC1-13 and K8 segments separately. We demonstrate how new functionalities (e.g. ATP storage) emerge from synergistic coupling of amyloidogenic segments with non-amyloidogenic peptide ligands, and suggest that ATP's role in protein misfolding is more nuanced than previously assumed.

Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein.

BackgroundThe Crimean-Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFVas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. Here we adopted a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity, which was shown to interfere with innate immune responses and viral replication. We report on the expression, characterization and inhibition of the CCHFV full-length L-protein and studied both RNA synthesis and DUB activity.Methodology/Principle findingsRecombinant full-length CCHFV L protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. RdRp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. We observed a 14-mer full length RNA product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. The D2517N mutation of the putative active site rendered the enzyme inactive. Inhibition of RNA synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. The triphosphate form of these compounds act like ATP or GTP; however, incorporation of ATP or GTP is markedly favored over the inhibitors. We also studied the effects of bona fide nucleotide analogues 2’-deoxy-2’-fluoro-CTP (FdC) and 2’-deoxy-2’-amino-CTP and demonstrate increased inhibitory effects due to higher rates of incorporation. We further show that the CCHFV L full-length protein and the isolated OTU domain cleave Lys48- and Lys63-linked polyubiqutin chains. Moreover, the ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. Inhibition of DUB activity does not affect elongation of RNA synthesis, and inhibition of RNA synthesis does not affect DUB activity. Both domains are functionally independent under these conditions.Conclusions/SignificanceThe requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. The availability of full-length CCHFV L-protein provides an important tool in this regard. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be employed to identify novel polymerase and DUB inhibitors.

RNA polymerase II stalls on oxidative DNA damage via a torsion-latch mechanism involving lone pair–π and CH–π interactions

Oxidation of guanine generates several types of DNA lesions, such as 8-oxoguanine (8OG), 5-guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp). These guanine-derived oxidative DNA lesions interfere with both replication and transcription. However, the molecular mechanism of transcription processing of Gh and Sp remains unknown. In this study, by combining biochemical and structural analysis, we revealed distinct transcriptional processing of these chemically related oxidized lesions: 8OG allows both error-free and error-prone bypass, whereas Gh or Sp causes strong stalling and only allows slow error-prone incorporation of purines. Our structural studies provide snapshots of how polymerase II (Pol II) is stalled by a nonbulky Gh lesion in a stepwise manner, including the initial lesion encounter, ATP binding, ATP incorporation, jammed translocation, and arrested states. We show that while Gh can form hydrogen bonds with adenosine monophosphate (AMP) during incorporation, this base pair hydrogen bonding is not sufficient to hold an ATP substrate in the addition site and is not stable during Pol II translocation after the chemistry step. Intriguingly, we reveal a unique structural reconfiguration of the Gh lesion in which the hydantoin ring rotates ∼90° and is perpendicular to the upstream base pair planes. The perpendicular hydantoin ring of Gh is stabilized by noncanonical lone pair-π and CH-π interactions, as well as hydrogen bonds. As a result, the Gh lesion, as a functional mimic of a 1,2-intrastrand crosslink, occupies canonical -1 and +1 template positions and compromises the loading of the downstream template base. Furthermore, we suggest Gh and Sp lesions are potential targets of transcription-coupled repair.

UvrB protein of Corynebacterium pseudotuberculosis complements the phenotype of knockout Escherichia coli and recognizes DNA damage caused by UV radiation but not 8-oxoguanine in vitro

In prokaryotic cells, the UvrB protein plays a central role in nucleotide excision repair, which is involved in the recognition of bulky DNA lesions generated by chemical or physical agents. The present investigation aimed to characterize the uvrB gene of Corynebacterium pseudotuberculosis (CpuvrB) and evaluate its involvement in the DNA repair system of this pathogenic organism. In computational analysis, the alignment of the UvrB protein sequences of Escherichia coli, Mycobacterium tuberculosis, Bacillus caldotenax and Corynebacterium pseudotuberculosis showed high similarity and the catalytic amino acid residues and functional domains are preserved. A CpUvrB model was constructed by comparative modeling and presented structural similarity with the UvrB of E. coli. Moreover, in molecular docking analysis CpUvrB showed favorable interaction with EcUvrA and revealed a preserved ATP incorporation site. Heterologous functional complementation assays using E. coli uvrB-deficient cells exposed to UV irradiation showed that the CpUvrB protein contributed to an increased survival rate in relation to those in the absence of CpUvrB. Damaged oligonucleotides containing thymine dimer or 8-oxoguanine lesion were synthesized and incubated with CpUvrB protein, which was able to recognize and excise UV irradiation damage but not 8-oxoguanine. These results suggest that CpUvrB is involved in repairing lesions derived from UV light and encodes a protein orthologous to EcUvrB.

Increased Expression of Myc in Hairy Cell Leukemia, and Cell-Sensitivity to JQ1

Classic hairy cell leukemia (HCL), comprising 2% of leukemias, is an indolent B-cell malignancy with malignant lymphocytes expressing B-cell antigens CD20 and CD22, CD103, CD11c, CD25, Annexin A1, BRAF V600E mutation, and monoclonal immunoglobulin (Ig) rearrangement. HCL variant (HCLv), which is ~10% as common as HCL, has much poorer response to therapy and more aggressive course, lacks CD25 and Annexin A1, and is wild-type for BRAF. HCLv is considered separate from HCL by the World Health Organization in the unclassifiable splenic B-cell leukemia/lymphomas. Another poor-prognosis group overlaps HCL and HCLv in which unmutated IGHV4-34 Ig rearrangement is expressed. IGHV4-34+ leukemic cells can resemble classic HCL with CD25 and Annexin A1 expression, but are BRAF wild-type. No uniform mutation has been identified for HCLv and IGHV4-34+ HCL, although MAP2K1 (MEK1) mutations have recently been identified in half of cases. Thus HCLv and IGHV4-34+ HCL are less indolent leukemias with few therapy options and no known molecular target.To study HCLv and IGHV4-34+ HCL, leukemic samples were purified by negative B-cell isolation followed by positive CD11c sorting. Following extraction (Qiagene, AllPrep DNA/RNA Kit), RNA samples from patients were analyzed in microarray studies (Human HT-12 v4 BeadChips, Illumina, Inc.). Expression data were compared by unpaired nonparametric analysis using Mann-Whitney. MYC expression using one probe (log2 values, mean +/- standard deviation) was 7.30 +/- 1.51 for 37 HCL vs 9.77 +/- 1.15 for 32 HCLv or IGHV4-34+ HCL (2-sided p<0.0001). For the other probe, expression was 7.07 +/- 1.51 vs 9.44 +/- 1.17 (p<0.0001). Expression data for MYC had previously been submitted for 31 chronic lymphocytic leukemia (CLL) and 16 HCL samples (Dataset GSE2350, Basso et al, Nat Genet, 37:382, 2005). By 1 probe for MYC, expression was 8.07 +/- 0.55 for CLL vs 9.31 +/- 1.19 for HCL (p=0.0023). By another MYC probe, expression was 9.28 +/- 0.47 for CLL vs 10.22 +/- 0.98 for HCL (p=0.0032).To investigate potential therapeutic relevance of aberrant MYC expression in HCL, HCLv and IGHV4-34+ HCL, the bromodomain and extra terminal (BET) protein inhibitor JQ1, which has been associated with down-regulation of c-Myc via Brd4, was incubated with primary leukemic cells and ATP incorporation was measured. JQ1 inhibited 12 samples of HCL (IC50s 214 +/- 217 nM) more potently than 14 samples of CLL (IC50s 1.77uM +/-2.62 uM, p=0.020), and also inhibited 14 samples of HCLv or IGHV4-34+ HCL (IC50s 221 +/- 234 nM) more potently than the 14 CLL samples (p=0.0079). However, JQ1 inhibition was similar comparing HCL and HCLv or IGHV4-34+ HCL (p=0.89). To exclude non-specific inhibition of the cells, the inactive control molecule JQ1R was tested and was only 6.0% +/- 4.0% as active as JQ1 toward HCL or HCLv or IGHV4-34+ HCL samples. Normal peripheral blood mononuclear cells were resistant (IC50 > 20 uM).In conclusion, our results show that MYC expression is higher in HCLv and IGHV4-34+ HCL than in classic HCL and higher in classic HCL than CLL. Moreover, JQ1 inhibits HCL or its variants more potently than CLL, although the inhibition assay used does not detect a difference between the variants and classic HCL. Further experiments with other inhibitors will be needed to determine if the increased expression of MYC in HCL and its poor-prognosis variants can be exploited for treatment. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Hepatitis C Virus Replication by GS-6620, a Potent C -Nucleoside Monophosphate Prodrug

As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 μM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

CRGT Peptide In Cytoplasm Regulates Thrombus Formation Via Dissociating c-Src-β3 Interaction

The RGT sequences of the integrin β3 tail directly and constitutively bind the inactive c-Src, regulating integrin αIIbβ3 signaling and platelet function. Previous work has shown that disrupting the interaction of c-Src with β3 via myristoylated RGT peptide or deletion of the RGT sequences in β3 selectively inhibits integrin αIIbβ3 outside-in signaling in platelets. However, the precise molecular mechanisms by which the Src-β3 association regulates integrin αIIbβ3 signaling need to be clarified. We found that active c-Src phosphoylated the Y747 and Y759 residues of β3 directly at the in vitro protein/protein level or in CHO cell models bearing Tac-β3 chimeras, which were devoid of the intact β3 signal transduction. Furthermore, data from mass spectrometry, [γ-32P] ATP incorporation assays and CHO cell/Tac-β3 chimeras demonstrated that the direct phosphorylation of Y747 and Y759 by active c-Src did not depend on the binding of c-Src to the RGT sequences of the β3 tail. To further investigate the biological functions of Src-β3 association in signal transduction we employed a cell-permeable and reduction-sensitive peptide (myr-AC∼CRGT), which disrupted the Src-β3 association in platelets independent of membrane-anchorage, and found that when platelets were stimulated by thrombin the c-Src activation and the phosphorylation of the tyrosine residues of the β3 tail were substantially inhibited by the presence of the peptide. These results suggest that one of the crucial biological functions of Src-β3 association is to serve as a “bridge” linking integrin signaling with the c-Src full activation and phosphorylation of the tyrosines of the β3 tail. To answer whether the RGT peptide binding to Src is able to alter the enzymatic activity of c-Src, we examined the Src-Csk association, the phosphorylation status of Y416 and Y527 of c-Src and the c-Src kinase catalytic activity. Results showed that myr-AC∼CRGT did not dissociate Csk from c-Src in resting platelets and the phosphorylation level of Y416 and Y527 of c-Src remained unaltered. Consistent data were also obtained from in vitro analysis of the c-Src kinase catalytic activity in the presence of CRGT peptide. These results suggest that myr-AC∼CRGT peptide per se does not fully activate c-Src. Myr-AC∼CRGT was also found to inhibit integrin αIIbβ3 outside-in signaling in human platelets. To examine the effect of the myr-AC∼CRGT on platelet adhesion and aggregation under flow conditions, we measured the platelet thrombus formation under different shear rates. Myr-AC∼CRGT did not affect the platelet adhesion at a wall shear rate of 125 s-1. The inability of myr-AC∼CRGT to affect platelet adhesion and aggregation remained at 500 s-1 shear rates. At 1,500 s-1, or 5,000 s-1 rates, myr-AC∼CRGT partially inhibited platelet adhesion and aggregation. These observations indicate that the Src-regulated outside-in signaling plays a pivotal role in the stable thrombus formation and the thrombus growth under flow conditions. The present study reveals novel insights into the molecular mechanisms by which c-Src regulates integrin αIIbβ3 signaling, particularly the phorsphorylation of the β3 cytoplasmic tyrosines, and provides first evidence in human platelets that the RGT peptide or derivatives regulate thrombus formation through dissociating the Src-β3 interaction. The data of this work allow us to anticipate that intracellular delivery of the RGT peptide or its analogues may have potential in the development of a new antithrombotic strategy where only the Src-β3 interaction is specifically interrupted so as to provide an effective inhibition on thrombosis together with a decent hemostasis. Disclosures:No relevant conflicts of interest to declare.

Mechanism of Action of T-705 Ribosyl Triphosphate against Influenza Virus RNA Polymerase

T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) selectively and strongly inhibits replication of the influenza virus in vitro and in vivo. T-705 has been shown to be converted to T-705-4-ribofuranosyl-5-triphosphate (T-705RTP) by intracellular enzymes and then functions as a nucleotide analog to selectively inhibit RNA-dependent RNA polymerase (RdRp) of the influenza virus. To elucidate these inhibitory mechanisms, we analyzed the enzyme kinetics of inhibition using Lineweaver-Burk plots of four natural nucleoside triphosphates and conducted polyacrylamide gel electrophoresis of the primer extension products initiated from (32)P-radiolabeled 5'Cap1 RNA. Enzyme kinetic analysis demonstrated that T-705RTP inhibited the incorporation of ATP and GTP in a competitive manner, which suggests that T-705RTP is recognized as a purine nucleotide by influenza virus RdRp and inhibited the incorporation of UTP and CTP in noncompetitive and mixed-type manners, respectively. Primer extension analysis demonstrated that a single molecule of T-705RTP was incorporated into the nascent RNA strand of the influenza virus and inhibited the subsequent incorporation of nucleotides. These results suggest that a single molecule of T-705RTP is incorporated into the nascent RNA strand as a purine nucleotide analog and inhibits strand extension, even though the natural ribose of T-705RTP has a 3'-OH group, which is essential for forming a covalent bond with the phosphate group.

Crystal structures of the Cid1 poly (U) polymerase reveal the mechanism for UTP selectivity

Polyuridylation is emerging as a ubiquitous post-translational modification with important roles in multiple aspects of RNA metabolism. These poly (U) tails are added by poly (U) polymerases with homology to poly (A) polymerases; nevertheless, the selection for UTP over ATP remains enigmatic. We report the structures of poly (U) polymerase Cid1 from Schizoscaccharomyces pombe alone and in complex with UTP, CTP, GTP and 3′-dATP. These structures reveal that each of the 4 nt can be accommodated at the active site; however, differences exist that suggest how the polymerase selects UTP over the other nucleotides. Furthermore, we find that Cid1 shares a number of common UTP recognition features with the kinetoplastid terminal uridyltransferases. Kinetic analysis of Cid1’s activity for its preferred substrates, UTP and ATP, reveal a clear preference for UTP over ATP. Ultimately, we show that a single histidine in the active site plays a pivotal role for poly (U) activity. Notably, this residue is typically replaced by an asparagine residue in Cid1-family poly (A) polymerases. By mutating this histidine to an asparagine residue in Cid1, we diminished Cid1’s activity for UTP addition and improved ATP incorporation, supporting that this residue is important for UTP selectivity.

Understanding the molecular mechanism of sequence dependent Tenofovir removal by HIV-1 reverse transcriptase: Differences in primer binding site versus polypurine tract

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor (NtRTI) that is often administered as first-line therapy against human immunodeficiency virus type-1 (HIV-1) infection and acts as a chain terminator when incorporated into viral DNA. However, HIV-1 reverse transcriptase (RT) excises TFV in the presence of either ATP or pyrophosphate, which is an important drug resistance mechanism that would interfere with the effective treatment. Previous studies have shown conflicting results on excision efficiencies for TFV-terminated primer-templates derived from either primer binding site (PBS) or polypurine tract (PPT) sequences. To provide mechanistic insight into the variation in TFV removal from both sequences that are vital for the HIV-1 life cycle, we compared the efficiencies of removal reaction in response to sequence dependence via utilizing blocked PBS and PPT primer-templates. We found an enhanced TFV excision with PPT sequence over PBS sequence through ATP-mediated removal and a subsequent incorporation of ATP into the unblocked primers. Furthermore, the rate of pyrophosphorolytic excision of TFV from PPT sequence was 21-fold higher than that for the PBS sequence. However, the addition of efavirenz, nonnucleoside reverse transcriptase inhibitor (NNRTI), to the removal reaction effectively inhibits the TFV excision from both primers by forming a stable complex that would leave TFV inaccessible for excision. These results illuminate the degree of primer-template sequence contribution on TFV removal as well as increase our understanding of the molecular mechanism for the beneficial effects of widely used combinations of antiretroviral regimens in the context of synergistic antiviral activity and drug resistance.

Nonpolar nucleosides alter RNA Polymerase II NTP specificity by disrupting hydrogen bonding and base stacking

RNA Polymerase II (RNAPII) catalyzes the synthesis of mRNA during transcription. RNAPII nucleotide discrimination is governed by the relative specificity constants (kcat/Km) for correct versus incorrect nucleotide substrates. We have investigated the mechanisms governing nucleotide specificity by substituting the DNA templates with nonpolar nucleosides containing C2 and C4 halogen substitutions of increasing size. These substitutions disrupt hydrogen bonding by removing charged groups that interact with the incoming correct ATP during traditional Watson‐Crick base pairing. Our findings reveal nonpolar analogs greatly decreases the specificity constant for correct ATP incorporation. This decrease in specificity also abolishes the polymerases ability to discriminate against incorrect UTP incorporation. We further determine the contribution of hydrogen bonding on next nucleotide incorporation. The results indicate that the absence of 3′ hydrogen bonding decreases mismatch recognition.

Effect of the Y955C Mutation on Mitochondrial DNA Polymerase Nucleotide Incorporation Efficiency and Fidelity

The human mitochondrial DNA polymerase (pol γ) is responsible for the replication of the mitochondrial genome. Mutation Y955C in the active site of pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson's disease. In single-turnover kinetic studies, we show that the Y955C mutation results in a decrease in the maximal rate of polymerization and an increase in the K(m) for correct incorporation. The mutation decreased the specificity constant for correct incorporation of dGTP, TTP, and ATP to values of 1.5, 0.35, and 0.044 μM(-1) s(-1), respectively, representing reductions of 30-, 110-, and 1300-fold, respectively, relative to the value for the wild-type enzyme. The fidelity of incorporation was reduced 6-130-fold, largely because of the significant decrease in the specificity constant for correct dATP:T incorporation. For example, k(cat)/K(m) for forming a TTP:T mismatch was decreased 10-fold from 0.0002 to 0.00002 μM(-1) s(-1) by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to that of the wild type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, the level of incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role for Y955 in stabilizing A:T base pairs at the active site of pol γ and suggest that the severe clinical symptoms of patients with this mutation may be due, in part, to the reduced efficiency of incorporation of dATP opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency.