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1.
Science ; 385(6704): 105-112, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38963841

ABSTRACT

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ. We show that gp210 targets a specific sequence in ΦKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions.


Subject(s)
Endonucleases , Introns , Pseudomonas Phages , Pseudomonas aeruginosa , Viral Interference , Viral Proteins , Endonucleases/metabolism , Endonucleases/genetics , Viral Interference/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Assembly , Virus Replication , Pseudomonas Phages/enzymology , Pseudomonas Phages/genetics , Pseudomonas aeruginosa/virology
2.
Proc Natl Acad Sci U S A ; 120(43): e2308600120, 2023 10 24.
Article in English | MEDLINE | ID: mdl-37862384

ABSTRACT

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCA's binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.


Subject(s)
Carbonic Anhydrases , Ribulose-Bisphosphate Carboxylase , Ribulose-Bisphosphate Carboxylase/metabolism , Carbonic Anhydrases/metabolism , Carbon Dioxide/metabolism , Cryoelectron Microscopy , Organelles/metabolism , Bacterial Proteins/metabolism
3.
bioRxiv ; 2023 Sep 30.
Article in English | MEDLINE | ID: mdl-37808663

ABSTRACT

Mobile introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. Here we studied a mobile intron in bacteriophage ΦPA3 and found its homing endonuclease gp210 contributes to viral competition by interfering with the virogenesis of co-infecting phage ΦKZ. We show that gp210 targets a specific sequence in its competitor ΦKZ, preventing the assembly of progeny viruses. This work reports the first demonstration of how a mobile intron can be deployed to engage in interference competition and provide a reproductive advantage. Given the ubiquity of introns, this selective advantage likely has widespread evolutionary implications in nature.

4.
Cell Rep ; 42(5): 112432, 2023 05 30.
Article in English | MEDLINE | ID: mdl-37120812

ABSTRACT

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.


Subject(s)
Bacteriophages , Erwinia , Bacteriophages/genetics , Bacteriophages/metabolism , Erwinia/genetics , Erwinia/metabolism , Phylogeny , Genome, Viral , DNA, Viral/genetics , DNA, Viral/metabolism
5.
bioRxiv ; 2023 Feb 25.
Article in English | MEDLINE | ID: mdl-36865095

ABSTRACT

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were unknown. By studying phages that encode the major phage nucleus protein chimallin, including previously sequenced yet uncharacterized phages, we discovered that chimallin-encoding phages share a set of 72 highly conserved genes encoded within seven distinct gene blocks. Of these, 21 core genes are unique to this group, and all but one of these unique genes encode proteins of unknown function. We propose that phages with this core genome comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryo-electron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication encoded in the core genome are conserved among diverse chimalliviruses, and reveal that non-core components can confer intriguing variations on this replication mechanism. For instance, unlike previously studied nucleus-forming phages, RAY doesn't degrade the host genome, and its PhuZ homolog appears to form a five-stranded filament with a lumen. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

6.
Acad Med ; 98(2): 188-198, 2023 02 01.
Article in English | MEDLINE | ID: mdl-35671407

ABSTRACT

The growing international adoption of competency-based medical education has created a desire for descriptions of innovative assessment approaches that generate appropriate and sufficient information to allow for informed, defensible decisions about learner progress. In this article, the authors provide an overview of the development and implementation of the approach to programmatic assessment in postgraduate family medicine training programs in Canada, called Continuous Reflective Assessment for Training (CRAFT). CRAFT is a principles-guided, high-level approach to workplace-based assessment that was intentionally designed to be adaptable to local contexts, including size of program, resources available, and structural enablers and barriers. CRAFT has been implemented in all 17 Canadian family medicine residency programs, with each program taking advantage of the high-level nature of the CRAFT guidelines to create bespoke assessment processes and tools appropriate for their local contexts. Similarities and differences in CRAFT implementation between 5 different family medicine residency training programs, representing both English- and French-language programs from both Western and Eastern Canada, are described. Despite the intentional flexibility of the CRAFT guidelines, notable similarities in assessment processes and procedures across the 5 programs were seen. A meta-evaluation of findings from programs that have published evaluation information supports the value of CRAFT as an effective approach to programmatic assessment. While CRAFT is currently in place in family medicine residency programs in Canada, given its adaptability to different contexts as well as promising evaluation data, the CRAFT approach shows promise for application in other training environments.


Subject(s)
Internship and Residency , Humans , Family Practice/education , Canada , Competency-Based Education/methods , Curriculum
7.
Cell Rep ; 40(7): 111179, 2022 08 16.
Article in English | MEDLINE | ID: mdl-35977483

ABSTRACT

Nucleus-forming jumbo phages establish an intricate subcellular organization, enclosing phage genomes within a proteinaceous shell called the phage nucleus. During infection in Pseudomonas, some jumbo phages assemble a bipolar spindle of tubulin-like PhuZ filaments that positions the phage nucleus at midcell and drives its intracellular rotation. This facilitates the distribution of capsids on its surface for genome packaging. Here we show that the Escherichia coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of a mutant PhuZ protein strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes for cytoplasmic streaming and nucleus alignment, this work identifies a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.


Subject(s)
Bacteriophages , Bacteriophages/genetics , DNA, Viral/genetics , Escherichia coli/genetics , Genome, Viral , Viral Proteins/metabolism
8.
Nature ; 608(7922): 429-435, 2022 08.
Article in English | MEDLINE | ID: mdl-35922510

ABSTRACT

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems1. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors2-4. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.


Subject(s)
Bacteria , Bacteriophages , Cell Compartmentation , Viral Proteins , Virus Assembly , Bacteria/cytology , Bacteria/immunology , Bacteria/metabolism , Bacteria/virology , Bacteriophages/chemistry , Bacteriophages/immunology , Bacteriophages/physiology , Bacteriophages/ultrastructure , Cryoelectron Microscopy , Viral Proteins/chemistry , Viral Proteins/metabolism , Viral Proteins/ultrastructure
9.
Nat Commun ; 13(1): 4863, 2022 08 18.
Article in English | MEDLINE | ID: mdl-35982043

ABSTRACT

Despite the importance of microcompartments in prokaryotic biology and bioengineering, structural heterogeneity has prevented a complete understanding of their architecture, ultrastructure, and spatial organization. Here, we employ cryo-electron tomography to image α-carboxysomes, a pseudo-icosahedral microcompartment responsible for carbon fixation. We have solved a high-resolution subtomogram average of the Rubisco cargo inside the carboxysome, and determined the arrangement of the enzyme. We find that the H. neapolitanus Rubisco polymerizes in vivo, mediated by the small Rubisco subunit. These fibrils can further pack to form a lattice with six-fold pseudo-symmetry. This arrangement preserves freedom of motion and accessibility around the Rubisco active site and the binding sites for two other carboxysome proteins, CsoSCA (a carbonic anhydrase) and the disordered CsoS2, even at Rubisco concentrations exceeding 800 µM. This characterization of Rubisco cargo inside the α-carboxysome provides insight into the balance between order and disorder in microcompartment organization.


Subject(s)
Carbonic Anhydrases , Ribulose-Bisphosphate Carboxylase , Bacterial Proteins/metabolism , Carbon Cycle , Carbon Dioxide/metabolism , Carbonic Anhydrases/metabolism , Catalytic Domain , Organelles/metabolism , Ribulose-Bisphosphate Carboxylase/metabolism
10.
Nat Commun ; 12(1): 5664, 2021 09 27.
Article in English | MEDLINE | ID: mdl-34580310

ABSTRACT

Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.


Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , Protein Interaction Domains and Motifs/genetics , RNA, Guide, Kinetoplastida/metabolism , CRISPR-Associated Protein 9/metabolism , CRISPR-Associated Protein 9/ultrastructure , Cell Line, Tumor , Cryoelectron Microscopy , DNA/metabolism , Gene Editing/methods , Humans , Single Molecule Imaging
11.
Biochim Biophys Acta Bioenerg ; 1861(11): 148254, 2020 11 01.
Article in English | MEDLINE | ID: mdl-32645407

ABSTRACT

Photosynthetic NADH dehydrogenase-like complex type-1 (a.k.a, NDH, NDH-1, or NDH-1L) is a multi-subunit, membrane-bound oxidoreductase related to the respiratory complex I. Although originally discovered 30 years ago, a number of recent advances have revealed significant insight into the structure, function, and physiology of NDH-1. Here, we highlight progress in understanding the function of NDH-1 in the photosynthetic light reactions of both cyanobacteria and chloroplasts from biochemical and structural perspectives. We further examine the cyanobacterial-specific forms of NDH-1 that possess vectorial carbonic anhydrase (vCA) activity and function in the CO2-concentrating mechanism (CCM). We compare the proposed mechanism for the cyanobacterial NDH-1 vCA-activity to that of the DAB (DABs accumulates bicarbonate) complex, another putative vCA. Finally, we discuss both new and remaining questions pertaining to the mechanisms of NDH-1 complexes in light of these recent advances.


Subject(s)
Chloroplasts/metabolism , Cyanobacteria/metabolism , Electron Transport Complex I/chemistry , Electron Transport Complex I/metabolism , Oxygen/metabolism , Photosynthesis , Electron Transport
12.
Plant Physiol ; 181(3): 1050-1058, 2019 11.
Article in English | MEDLINE | ID: mdl-31501298

ABSTRACT

Carboxysomes are capsid-like, CO2-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary production. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO2-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbon-concentrating mechanism, by packaging enzymes that collectively enhance catalysis, the carboxysome shell enables the generation of a locally elevated concentration of substrate CO2 and the prevention of CO2 escape. A functional carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO2 concentrations. Using cryo-electron microscopy, we have determined the structure of a recombinantly produced simplified ß-carboxysome shell. The structure reveals the sidedness and the specific interactions between the carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochemical properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concentrating mechanism in metabolic engineering. Moreover, we find striking similarity between the carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells.


Subject(s)
Cryoelectron Microscopy/methods , Organelles/ultrastructure , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbonic Anhydrases/metabolism , Chromatography, Ion Exchange , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/ultrastructure , Organelles/metabolism , Ribulose-Bisphosphate Carboxylase/metabolism , Ribulose-Bisphosphate Carboxylase/ultrastructure , Synechococcus/metabolism , Synechococcus/ultrastructure
13.
Nat Microbiol ; 4(12): 2204-2215, 2019 12.
Article in English | MEDLINE | ID: mdl-31406332

ABSTRACT

Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative ß-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.


Subject(s)
Bacterial Proteins/metabolism , Carbon/metabolism , Carbonic Anhydrases/metabolism , Carrier Proteins/metabolism , Prokaryotic Cells/metabolism , Archaea/enzymology , Archaea/genetics , Archaea/metabolism , Bacillus anthracis/metabolism , Bacteria/enzymology , Bacteria/genetics , Bacteria/metabolism , Bacterial Proteins/genetics , Carbon Dioxide/metabolism , Carbonic Anhydrases/genetics , DNA Transposable Elements/genetics , Diazonium Compounds , Genes, Bacterial/genetics , Genes, Essential , Halothiobacillus/genetics , Halothiobacillus/metabolism , Mutagenesis , Operon , Sulfanilic Acids , Vibrio cholerae/metabolism
14.
Viruses ; 11(7)2019 07 17.
Article in English | MEDLINE | ID: mdl-31319455

ABSTRACT

Moloney leukemia virus 10 (MOV10) is an RNA helicase that has been shown to affect the replication of several viruses. The effect of MOV10 on Hepatitis B virus (HBV) infection is not known and its role on the replication of this virus is poorly understood. We investigated the effect of MOV10 down-regulation and MOV10 over-expression on HBV in a variety of cell lines, as well as in an infection system using a replication competent virus. We report that MOV10 down-regulation, using siRNA, shRNA, and CRISPR/Cas9 gene editing technology, resulted in increased levels of HBV DNA, HBV pre-genomic RNA, and HBV core protein. In contrast, MOV10 over-expression reduced HBV DNA, HBV pre-genomic RNA, and HBV core protein. These effects were consistent in all tested cell lines, providing strong evidence for the involvement of MOV10 in the HBV life cycle. We demonstrated that MOV10 does not interact with HBV-core. However, MOV10 binds HBV pgRNA and this interaction does not affect HBV pgRNA decay rate. We conclude that the restriction of HBV by MOV10 is mediated through effects at the level of viral RNA.


Subject(s)
Hepatitis B virus/physiology , Hepatitis B/virology , Host-Pathogen Interactions , Microbial Interactions , Moloney murine leukemia virus/physiology , Virus Replication , Animals , Cell Line , Cells, Cultured , Gene Expression Regulation, Viral , Humans , Mice , Protein Binding , RNA , RNA Helicases/metabolism , RNA, Viral , Viral Proteins/metabolism
15.
Nature ; 566(7744): 411-414, 2019 02.
Article in English | MEDLINE | ID: mdl-30742075

ABSTRACT

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits-specifically NdhS-enable NDH to accept electrons from its electron donor, ferredoxin3-5,7. Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical.


Subject(s)
Cryoelectron Microscopy , Cyanobacteria/chemistry , Electron Transport Complex I/chemistry , Electron Transport Complex I/ultrastructure , NADPH Dehydrogenase/chemistry , NADPH Dehydrogenase/ultrastructure , Oxygen/metabolism , Photosynthesis , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Coenzymes/chemistry , Coenzymes/metabolism , Cyanobacteria/enzymology , Electron Transport , Electron Transport Complex I/metabolism , Ferredoxins/metabolism , Models, Biological , Models, Molecular , NADPH Dehydrogenase/metabolism , Oxidation-Reduction , Photosystem I Protein Complex/metabolism , Plastoquinone/metabolism , Protein Subunits/chemistry , Protein Subunits/metabolism
16.
ACS Infect Dis ; 5(5): 750-758, 2019 05 10.
Article in English | MEDLINE | ID: mdl-30582687

ABSTRACT

An estimated 240 million are chronically infected with hepatitis B virus (HBV), which can lead to liver disease, cirrhosis, and hepatocellular carcinoma. Currently, HBV treatment options include only nucleoside reverse transcriptase inhibitors and the immunomodulatory agent interferon alpha, and these treatments are generally not curative. New treatments with novel mechanisms of action, therefore, are highly desired for HBV therapy. The viral core protein (Cp) has gained attention as a possible therapeutic target because of its vital roles in the HBV life cycle. Several classes of capsid assembly effectors (CAEs) have been described in detail, and these compounds all increase capsid assembly rate but inhibit HBV replication by different mechanisms. In this study, we have developed a thermal shift-based screening method for CAE discovery and characterization, filling a much-needed gap in high-throughput screening methods for capsid-targeting molecules. Using this approach followed by cell-based screening, we identified the compound HF9C6 as a CAE with low micromolar potency against HBV replication. HF9C6 caused large multicapsid aggregates when capsids were assembled in vitro and analyzed by transmission electron microscopy. Interestingly, when HBV-expressing cells were treated with HF9C6, Cp was excluded from cell nuclei, suggesting that this compound may inhibit nuclear entry of Cp and capsids. Furthermore, mutational scanning of Cp suggested that HF9C6 binds the known CAE binding pocket, indicating that key Cp-compound interactions within this pocket have a role in determining the CAE mechanism of action.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Viral Core Proteins/antagonists & inhibitors , Virus Internalization/drug effects , Hep G2 Cells , Hepatitis B virus/physiology , Hepatocytes/drug effects , Hepatocytes/virology , Humans , Virus Assembly/drug effects , Virus Replication/drug effects
17.
Biomed J Sci Tech Res ; 2(2): 1-4, 2018 Jan 07.
Article in English | MEDLINE | ID: mdl-30370423

ABSTRACT

It is universally agreed that dopamine is a major neurotransmitter in terms of reward dependence, however, there remains controversy regarding how to modulate its role clinically to treat and prevent relapse for both substance and non-substance-related addictive behaviors. It is also agreed by most that there is a need to provide early genetic identification possibly through a novel researched technology referred to Genetic Addiction Risk Score(GARS).™ The existing FDA-approved medications promote blocking dopamine, however, we argue that a more prudent paradigm shift should be biphasic-short-term blockade and long-term upregulation, enhancing functional connectivity of brain reward. It is critical to understand that the real phenotype is not any specific drug or non -drug addictive behavior, but instead is Reward Deficiency Syndrome (RDS). Thus the true phenotype of all addictive behaviors is indeed RDS. Finally, we are suggesting that one way to combat the current out of control Opioid /Alcohol crisis worldwide is to seriously reconsider treating RDS by simply supplying powerful narcotic agents (e.g. Buprenorphine). This type of treatment will only keep people addicted. A more reasonable solution involving genetic testing, urine drug screens using Comprehensive Analysis of Reported Drugs (CARD) and dopamine homeostasis we call " Precision Addiction Management" ™ seems parsonomiuos.

18.
mSphere ; 3(2)2018 04 25.
Article in English | MEDLINE | ID: mdl-29669885

ABSTRACT

Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly in vitro have been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp in vitro and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level.IMPORTANCE Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and stability, capsid formation, and host immune modulators, with the ultimate goal of an HBV cure. Understanding the mechanisms by which novel antiviral agents act will be imperative for the development of curative HBV therapies.


Subject(s)
Antiviral Agents/pharmacology , Capsid Proteins/chemistry , Hepatitis B virus/drug effects , Inclusion Bodies, Viral/chemistry , Protein Aggregates/drug effects , Pyridines/pharmacology , Pyrimidines/pharmacology , Capsid/chemistry , Capsid/drug effects , Capsid Proteins/genetics , Fluorescent Antibody Technique , Hep G2 Cells , Hepatitis B/drug therapy , Hepatitis B virus/physiology , Humans , Recombinant Proteins/chemistry , Virus Assembly/drug effects , Virus Replication/drug effects
19.
Int J Biol Macromol ; 87: 246-51, 2016 Jun.
Article in English | MEDLINE | ID: mdl-26930579

ABSTRACT

Venom peptides are known to have strong antimicrobial activity and anticancer properties. King cobra cathelicidin or OH-CATH (KF-34), banded krait cathelicidin (BF-30), wolf spider lycotoxin I (IL-25), and wolf spider lycotoxin II (KE-27) venom peptides were found to strongly inhibit Escherichia coli membrane bound F1Fo ATP synthase. The potent inhibition of wild-type E. coli in comparison to the partial inhibition of null E. coli by KF-34, BF-30, Il-25, or KE-27 clearly links the bactericidal properties of these venom peptides to the binding and inhibition of ATP synthase along with the possibility of other inhibitory targets. The four venom peptides KF-34, BF-30, IL-25, and KE-27, caused ≥85% inhibition of wild-type membrane bound E.coli ATP synthase. Venom peptide induced inhibition of ATP synthase and the strong abrogation of wild-type E. coli cell growth in the presence of venom peptides demonstrates that ATP synthase is a potent membrane bound molecular target for venom peptides. Furthermore, the process of inhibition was found to be fully reversible.


Subject(s)
ATP Synthetase Complexes/antagonists & inhibitors , Antimicrobial Cationic Peptides/pharmacology , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Spider Venoms/pharmacology , Amino Acid Sequence , Antimicrobial Cationic Peptides/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Proliferation/drug effects , Enzyme Inhibitors/chemistry , Escherichia coli/cytology , Spider Venoms/chemistry , Cathelicidins
20.
PLoS One ; 10(5): e0127802, 2015.
Article in English | MEDLINE | ID: mdl-25996607

ABSTRACT

We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.


Subject(s)
ATP Synthetase Complexes/antagonists & inhibitors , Benzoquinones/pharmacology , Enzyme Inhibitors/pharmacology , Escherichia coli/drug effects , Escherichia coli/physiology , Culture Media , Dose-Response Relationship, Drug , Enzyme Activation/drug effects
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