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1.
Immunol Cell Biol ; 101(3): 191-203, 2023 03.
Article in English | MEDLINE | ID: mdl-36529987

ABSTRACT

Type 1 diabetes (T1D) is a condition characterized by an absolute deficiency of insulin. Loss of insulin-producing pancreatic islet ß cells is one of the many causes of T1D. Viral infections have long been associated with new-onset T1D and the balance between virulence and host immunity determines whether the viral infection would lead to T1D. Herein, we detail the dynamic interaction of pancreatic ß cells with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the host immune system with respect to new-onset T1D. Importantly, ß cells express the crucial entry receptors and multiple studies confirmed that ß cells are infected by SARS-CoV-2. Innate immune system effectors, such as natural killer cells, can eliminate such infected ß cells. Although CD4+ CD25+ FoxP3+ regulatory T (TREG ) cells provide immune tolerance to prevent the destruction of the islet ß-cell population by autoantigen-specific CD8+ T cells, it can be speculated that SARS-CoV-2 infection may compromise self-tolerance by depleting TREG -cell numbers or diminishing TREG -cell functions by repressing Forkhead box P3 (FoxP3) expression. However, the expansion of ß cells by self-duplication, and regeneration from progenitor cells, could effectively replace lost ß cells. Appearance of islet autoantibodies following SARS-CoV-2 infection was reported in a few cases, which could imply a breakdown of immune tolerance in the pancreatic islets. However, many of the cases with newly diagnosed autoimmune response following SARS-CoV-2 infection also presented with significantly high HbA1c (glycated hemoglobin) levels that indicated progression of an already set diabetes, rather than new-onset T1D. Here we review the potential underlying mechanisms behind loss of functional ß-cell mass as a result of SARS-CoV-2 infection that can trigger new-onset T1D.


Subject(s)
COVID-19 , Diabetes Mellitus, Type 1 , Virus Diseases , Humans , CD8-Positive T-Lymphocytes/metabolism , T-Lymphocytes, Regulatory , SARS-CoV-2/metabolism , Insulin/metabolism , Forkhead Transcription Factors/metabolism
2.
Mol Divers ; 27(2): 931-938, 2023 Apr.
Article in English | MEDLINE | ID: mdl-35543797

ABSTRACT

The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA. Overexpression of ALKBH2 has been implicated in both tumorigenesis and chemotherapy resistance in some cancers, including glioblastoma and renal cancer rendering it a potential therapeutic target and a diagnostic marker. However, no inhibitor is available against these important DNA repair proteins. Intending to repurpose a drug as an inhibitor of ALKBH2, we performed in silico evaluation of HIV protease inhibitors and identified Ritonavir as an ALKBH2-interacting molecule. Using molecular dynamics simulation, we elucidated the molecular details of Ritonavir-ALKBH2 interaction. The present work highlights that Ritonavir might be used to target the ALKBH2-mediated DNA alkylation repair.


Subject(s)
HIV Protease Inhibitors , Ritonavir , Humans , Ritonavir/pharmacology , HIV Protease Inhibitors/pharmacology , Molecular Dynamics Simulation , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA Repair , AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics , AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism
3.
Org Biomol Chem ; 20(29): 5820-5835, 2022 07 27.
Article in English | MEDLINE | ID: mdl-35838243

ABSTRACT

Herein, we report a novel and unexpected metal-free oxygenation of 2,3-diphenyl-1-indenones, under an oxygen atmosphere (air), to either 2,3-epoxy-2,3-diphenyl-1-indenone or 2-hydroxy-2,3-diphenyl-1-indanone, depending on the conditions. Several bioactive epoxy indenones and one-pot α-hydroxy indanones (α-acyloin) were synthesized from 2,3-diaryl dihydroindanone and 2,3-diarylindenone, respectively. A plausible reaction mechanism is also proposed, where oxygenation would take place at the α-position and further proton abstraction from the ß-position leads to epoxy indenone derivatives. A one-pot cis-hydroxy indanone protocol is also achieved directly from biaryl indenone via reduction, epimerization, and oxygenation. The synthesized compounds were evaluated for inhibitory activity against the DNA repair protein AlkB. Among the screened (17 tested) compounds, one epoxide derivative was found to be a specific inhibitor of AlkB enzyme function.


Subject(s)
DNA Repair , Epoxy Compounds , Alkylation , DNA , Solvents
4.
Biochem Biophys Res Commun ; 534: 114-120, 2021 01 01.
Article in English | MEDLINE | ID: mdl-33321288

ABSTRACT

DNA integrity is challenged by both exogenous and endogenous alkylating agents. DNA repair proteins such as Escherichia coli AlkB family of enzymes can repair 1-methyladenine and 3-methylcytosine adducts by oxidative demethylation. Human AlkB homologue 5 (ALKBH5) is RNA N6-methyladenine demethylase and not known to be involved in DNA repair. Herein we show that ALKBH5 also has weak DNA repair activity and it can demethylate DNA 3-methylcytosine. The mutation of the amino acid residues involved in demethylation also abolishes the DNA repair activity of ALKBH5. Overexpression of ALKBH5 decreases the 3-methylcytosine level in genomic DNA and reduces the cytotoxic effects of the DNA damaging alkylating agent methyl methanesulfonate. Thus, demethylation by ALKBH5 might play a supporting role in maintaining genome integrity.


Subject(s)
AlkB Homolog 5, RNA Demethylase/metabolism , Alkylating Agents/toxicity , DNA Damage , DNA Repair/physiology , AlkB Homolog 5, RNA Demethylase/genetics , Cytosine/analogs & derivatives , Cytosine/metabolism , DNA Adducts , DNA Methylation , Demethylation , HEK293 Cells , Humans , Mesylates/toxicity
5.
Bioorg Med Chem Lett ; 39: 127883, 2021 05 01.
Article in English | MEDLINE | ID: mdl-33662536

ABSTRACT

Marine sponges of the genusSuberea produce variety of brominated tyrosine alkaloids which display diverse range of biological activities including antiproliferative, antimicrobial and antimalarial activities. In continuation of our search for biologically active marine natural products for antibacterial compounds, we report here the synthesis and evaluation of biological activity of panel of ianthelliformisamines and subereamine analogues using the literature known acid-amine coupling reaction. Several derivatives of Ianthelliformisamine were achieved by the coupling of Boc-protected polyamine chain with brominated aromatic acrylic acid derivatives by varying the bromine substituents on aromatic acid derivatives, amine spacer as well as geometry of the double bond, and then Boc-deprotection using TFA. Similarly, subereamine analogues were also synthesized employing coupling reaction between various brominated phenyl acrylic acids with commercially available chiral amino ester derivatives followed by ester hydrolysis. We screened these synthetic analogues for antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) strains. One of the compound 7c showed bactericidal activity against Staphylococcus aureus with an IC50 value of 3.8 µM (MIC = 25 µM).


Subject(s)
Anti-Bacterial Agents/pharmacology , Arginine/analogs & derivatives , Biological Products/pharmacology , Escherichia coli/drug effects , Hydrocarbons, Brominated/pharmacology , Staphylococcus aureus/drug effects , Tyrosine/analogs & derivatives , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Arginine/chemical synthesis , Arginine/chemistry , Arginine/pharmacology , Biological Products/chemical synthesis , Biological Products/chemistry , Dose-Response Relationship, Drug , Escherichia coli/growth & development , HEK293 Cells , Humans , Hydrocarbons, Brominated/chemical synthesis , Hydrocarbons, Brominated/chemistry , Microbial Sensitivity Tests , Molecular Structure , Staphylococcus aureus/growth & development , Structure-Activity Relationship , Tyrosine/chemical synthesis , Tyrosine/chemistry , Tyrosine/pharmacology
6.
Nucleic Acids Res ; 47(22): 11729-11745, 2019 12 16.
Article in English | MEDLINE | ID: mdl-31642493

ABSTRACT

The integrity of our DNA is challenged daily by a variety of chemicals that cause DNA base alkylation. DNA alkylation repair is an essential cellular defence mechanism to prevent the cytotoxicity or mutagenesis from DNA alkylating chemicals. Human oxidative demethylase ALKBH3 is a central component of alkylation repair, especially from single-stranded DNA. However, the molecular mechanism of ALKBH3-mediated damage recognition and repair is less understood. We report that ALKBH3 has a direct protein-protein interaction with human RAD51 paralogue RAD51C. We also provide evidence that RAD51C-ALKBH3 interaction stimulates ALKBH3-mediated repair of methyl-adduct located within 3'-tailed DNA, which serves as a substrate for the RAD51 recombinase. We further show that the lack of RAD51C-ALKBH3 interaction affects ALKBH3 function in vitro and in vivo. Our data provide a molecular mechanism underlying upstream events of alkyl adduct recognition and repair by ALKBH3.


Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , DNA Repair , DNA-Binding Proteins/physiology , Transferases/metabolism , Alkylation , Cells, Cultured , DNA Adducts/metabolism , DNA Damage/genetics , DNA Repair/genetics , DNA-Binding Proteins/metabolism , HEK293 Cells , Humans , PC-3 Cells , Protein Binding , Rad51 Recombinase/metabolism
7.
J Biol Chem ; 294(26): 10336-10348, 2019 06 28.
Article in English | MEDLINE | ID: mdl-31113867

ABSTRACT

Parkinson's disease (PD) is a major human disease associated with degeneration of the central nervous system. Evidence suggests that several endogenously formed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mimicking chemicals that are metabolic conversion products, especially ß-carbolines and isoquinolines, act as neurotoxins that induce PD or enhance progression of the disease. We have demonstrated previously that mitochondrially targeted human cytochrome P450 2D6 (CYP2D6), supported by mitochondrial adrenodoxin and adrenodoxin reductase, can efficiently catalyze the conversion of MPTP to the toxic 1-methyl-4-phenylpyridinium ion. In this study, we show that the mitochondrially targeted CYP2D6 can efficiently catalyze MPTP-mimicking compounds, i.e. 2-methyl-1,2,3,4-tetrahydroisoquinoline, 2-methyl-1,2,3,4-tetrahydro-ß-carboline, and 9-methyl-norharmon, suspected to induce PD in humans. Our results reveal that activity and respiration in mouse brain mitochondrial complex I are significantly affected by these toxins in WT mice but remain unchanged in Cyp2d6 locus knockout mice, indicating a possible role of CYP2D6 in the metabolism of these compounds both in vivo and in vitro These metabolic effects were minimized in the presence of two CYP2D6 inhibitors, quinidine and ajmalicine. Neuro-2a cells stably expressing predominantly mitochondrially targeted CYP2D6 were more sensitive to toxin-mediated respiratory dysfunction and complex I inhibition than cells expressing predominantly endoplasmic reticulum-targeted CYP2D6. Exposure to these toxins also induced the autophagic marker Parkin and the mitochondrial fission marker Dynamin-related protein 1 (Drp1) in differentiated neurons expressing mitochondrial CYP2D6. Our results show that monomethylamines are converted to their toxic cationic form by mitochondrially directed CYP2D6 and result in neuronal degradation in mice.


Subject(s)
Cytochrome P-450 CYP2D6/physiology , Disease Models, Animal , Methylamines/toxicity , Mitochondria/pathology , Neuroblastoma/pathology , Neurons/pathology , Parkinson Disease/pathology , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/adverse effects , Animals , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/drug effects , Mitochondria/metabolism , Neuroblastoma/etiology , Neuroblastoma/metabolism , Neurons/drug effects , Neurons/metabolism , Neurotoxins/toxicity , Parkinson Disease/etiology , Parkinson Disease/metabolism , Tumor Cells, Cultured
8.
Biochem Biophys Res Commun ; 509(3): 779-783, 2019 02 12.
Article in English | MEDLINE | ID: mdl-30616886

ABSTRACT

Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.


Subject(s)
Bacterial Proteins/genetics , Escherichia coli/drug effects , Methyl Methanesulfonate/toxicity , Mutagens/toxicity , Mycobacterium leprae/genetics , Saccharomyces cerevisiae/drug effects , Alkylation/drug effects , DNA Damage/drug effects , Escherichia coli/genetics , Genes, Bacterial , Genome, Bacterial/drug effects , Humans , Leprosy/microbiology , Models, Molecular , Mycobacterium leprae/drug effects , Saccharomyces cerevisiae/genetics
9.
Biochem Biophys Res Commun ; 496(2): 274-279, 2018 02 05.
Article in English | MEDLINE | ID: mdl-29326044

ABSTRACT

Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coli (E.coli) protein AlkB removes various alkyl DNA adducts including N1-methyladenine (N1meA) and N3-methylcytosine (N3meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N1meA and N3meC from single-stranded DNA (ssDNA). It can also remove N1meA and N3meC from double-stranded DNA by base-flipping. Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AlkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AlkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AlkB could efficiently remove N3meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N3meC-containing oligonuleotides were used as the substrate, efficiency of AlkB catalyzed reaction was abated compared to SSB-bound DNA substrate of identical length. Truncated SSB containing only the DNA binding domain could also support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-mer oligonucleotide containing single N3meC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts.


Subject(s)
DNA Repair , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Mixed Function Oxygenases/genetics , Alkylation , DNA/genetics , DNA/metabolism , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA Damage , DNA Methylation , DNA, Bacterial/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Kinetics , Mixed Function Oxygenases/metabolism , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Oxidation-Reduction , Protein Binding , Substrate Specificity
10.
Microb Pathog ; 124: 316-321, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30172902

ABSTRACT

Mycobacterium leprae is an unculturable obligate pathogen and causative agent for debilitating human disease leprosy. Due to reductive genome evolution M leprae genome harbours large number of pseudogenes and small number of genes (∼1600 genes and ∼1300 pseudogenes). How M leprae remained a successful human parasite with small set of genes remains poorly understood and provided us the impetus to investigate the intergenic regions of M leprae genome for the presence of possible open reading frames (ORFs). In this work, we have manually scanned all the intergenic regions of M leprae genome and identified 106 potential ORFs. Among these, 12 are large ORFs: encoding hypothetical proteins (HP) of more than 100 amino acids. We have also found 67 ORFs encoding 50-100 amino acids proteins and another 27 ORFs for 30-50 amino acids peptides. We have validated the presence of transcripts for large HPs by quantitative reverse transcriptase PCR (qRT-PCR). Our results suggest that some of the M leprae large HPs are indeed expressed at low level in leprosy patients. The present results will shed light on the intergenic ORFs of M leprae and further our understanding of the pathogenesis of leprosy.


Subject(s)
Bacterial Proteins/genetics , DNA, Intergenic/genetics , Genome, Bacterial , Leprosy/microbiology , Mycobacterium leprae/genetics , Open Reading Frames , Humans , Mycobacterium leprae/metabolism , Pseudogenes , Reverse Transcriptase Polymerase Chain Reaction
11.
FEMS Yeast Res ; 18(2)2018 03 01.
Article in English | MEDLINE | ID: mdl-29438506

ABSTRACT

Iron deprivation induces transcription of genes required for iron uptake, and transcription factor Aft1 and Aft2 mediate this by regulating transcriptional program in Saccharomyces cerevisiae. Iron-dependent Fe(II) and 2-oxoglutarate-dependent dioxygenase family proteins are involved in various cellular pathways including DNA alkylation damage repair. Whether Aft1/Aft2 are required for DNA alkylation repair is currently unknown. In this report, we have analyzed DNA alkylation repair under iron-deprived condition. Saccharomyces cerevisiae Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that deletion of AFT1 and AFT2 genes affects Tpa1 function resulting in sensitivity to alkylating agent methyl methane sulfonate (MMS). Deletion of AFT1 and AFT2 along with base excision repair pathway DNA glycosylase MAG1 renders the aft1Δaft2Δmag1Δ mutant highly sensitive to MMS. We have further studied effect of iron depletion by replacing S. cerevisiae Tpa1 with Escherichia coli AlkB and human AlkBH3. We observed that the activity of AlkB and AlkBH3 is also diminished similarly when present in aft1Δaft2Δ background as evident by sensitivity to MMS.


Subject(s)
DNA Repair , Dioxygenases/metabolism , Ferrous Compounds/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Enzyme Activation , Iron Deficiencies , Microbial Viability , Mutation , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
12.
Analyst ; 143(14): 3366-3373, 2018 Jul 09.
Article in English | MEDLINE | ID: mdl-29893758

ABSTRACT

The clinical diagnosis of traumatic brain injury (TBI) is based on neurological examination and neuro-imaging tools such as CT scanning and MRI. However, neurological examination at times may be confounded by consumption of alcohol or drugs and neuroimaging facilities may not be available at all centers. Human ubiquitin C-terminal hydrolase (UCHL1) is a well-accepted serum biomarker for severe TBI and can be used to detect the severity of a head injury. A reliable, rapid, cost effective, bedside and easy to perform method for the detection of UCHL1 is a pre-requisite for wide clinical applications of UCHL1 as a TBI biomarker. We developed a rapid detection method for UCHL1 using surface plasmon resonance of gold nanoparticles with a limit of detection (LOD) of 0.5 ng mL-1. It has a sensitivity and specificity of 100% each and meets an analytical precision similar to that of conventional sandwich ELISA but can be performed rapidly. Using this method we successfully detected UCHL1 in a cohort of 66 patients with TBI and were reliably able to distinguish mild TBI from moderate to severe TBI.


Subject(s)
Biomarkers/blood , Brain Injuries/diagnosis , Metal Nanoparticles , Ubiquitin Thiolesterase/blood , Brain Injuries/blood , Gold , Humans
13.
Bioorg Med Chem ; 26(14): 4100-4112, 2018 08 07.
Article in English | MEDLINE | ID: mdl-30041948

ABSTRACT

The mammalian AlkB homologue-3 (AlkBH3) is a member of the dioxygenase family of enzymes that in humans is involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkBH3 DNA repair activity. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Using a robust quantitative assay, we have obtained an AlkBH3 inhibitor that display specific binding and competitive mode of inhibition against DNA substrate. Finally, we established that this compound could prevent the proliferation of lung cancer cell line and enhance sensitivity to DNA damaging alkylating agent.


Subject(s)
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/antagonists & inhibitors , Indenes/pharmacology , AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase/metabolism , Calorimetry , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Indenes/chemical synthesis , Indenes/chemistry , Molecular Docking Simulation , Molecular Structure , Structure-Activity Relationship
14.
Mol Biol Rep ; 45(5): 865-870, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29974396

ABSTRACT

Intrinsically disordered regions (IDRs) of proteins often regulate function through interactions with folded domains. Escherichia coli single-stranded DNA binding protein SSB binds and stabilizes single-stranded DNA (ssDNA). The N-terminal of SSB contains characteristic OB (oligonucleotide/oligosaccharide-binding) fold which binds ssDNA tightly but non-specifically. SSB also forms complexes with a large number proteins via the C-terminal interaction domain consisting mostly of acidic amino acid residues. The amino acid residues located between the OB-fold and C-terminal acidic domain are known to constitute an IDR and no functional significance has been attributed to this region. Although SSB is known to bind many DNA repair protein, it is not known whether it binds to DNA dealkylation repair protein AlkB. Here, we characterize AlkB SSB interaction and demonstrate that SSB binds to AlkB via the IDR. We have established that AlkB-SSB interaction by in vitro pull-down and yeast two-hybrid analysis. We mapped the site of contact to be the residues 152-169 of SSB. Unlike most of the SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of SSB is necessary and sufficient for AlkB interaction.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Mixed Function Oxygenases/metabolism , Binding Sites , DNA, Bacterial/metabolism , DNA, Single-Stranded/metabolism , Escherichia coli/chemistry , Models, Molecular , Protein Binding , Protein Domains
15.
Mol Cell ; 38(5): 637-48, 2010 Jun 11.
Article in English | MEDLINE | ID: mdl-20541997

ABSTRACT

Transcription-coupled nucleotide excision repair (TC-NER) allows RNA polymerase II (RNAPII)-blocking lesions to be rapidly removed from the transcribed strand of active genes. Defective TCR in humans is associated with Cockayne syndrome (CS), typically caused by defects in either CSA or CSB. Here, we show that CSB contains a ubiquitin-binding domain (UBD). Cells expressing UBD-less CSB (CSB(del)) have phenotypes similar to those of cells lacking CSB, but these can be suppressed by appending a heterologous UBD, so ubiquitin binding is essential for CSB function. Surprisingly, CSB(del) remains capable of assembling nucleotide excision repair factors and repair synthesis proteins around damage-stalled RNAPII, but such repair complexes fail to excise the lesion. Together, our results indicate an essential role for protein ubiquitylation and CSB's UBD in triggering damage incision during TC-NER and allow us to integrate the function of CSA and CSB in a model for the process.


Subject(s)
DNA Helicases , DNA Repair Enzymes , DNA Repair , Ubiquitin/metabolism , Amino Acid Sequence , Cell Line/radiation effects , Cell Nucleus/metabolism , Cockayne Syndrome/genetics , Cockayne Syndrome/metabolism , DNA Damage , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Humans , Molecular Sequence Data , Mutation , Poly-ADP-Ribose Binding Proteins , Promoter Regions, Genetic , Protein Structure, Tertiary , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Tetrahydrofolate Dehydrogenase/genetics , Ubiquitin/genetics , Ultraviolet Rays
16.
Nucleic Acids Res ; 44(18): 8754-8763, 2016 Oct 14.
Article in English | MEDLINE | ID: mdl-27378775

ABSTRACT

The Escherichia coli AlkB protein is a 2-oxoglutarate/Fe(II)-dependent demethylase that repairs alkylated single stranded and double stranded DNA. Immunoaffinity chromatography coupled with mass spectrometry identified RecA, a key factor in homologous recombination, as an AlkB-associated protein. The interaction between AlkB and RecA was validated by yeast two-hybrid assay; size-exclusion chromatography and standard pull down experiment and was shown to be direct and mediated by the N-terminal domain of RecA. RecA binding results AlkB-RecA heterodimer formation and RecA-AlkB repairs alkylated DNA with higher efficiency than AlkB alone.


Subject(s)
AlkB Enzymes/metabolism , DNA Adducts , DNA Repair , Rec A Recombinases/metabolism , AlkB Enzymes/chemistry , Carrier Proteins/chemistry , Carrier Proteins/metabolism , DNA Methylation , Escherichia coli/genetics , Escherichia coli/metabolism , Models, Molecular , Molecular Conformation , Oxidation-Reduction , Protein Binding , Protein Interaction Domains and Motifs , Rec A Recombinases/chemistry
17.
J Biol Chem ; 289(52): 35939-52, 2014 Dec 26.
Article in English | MEDLINE | ID: mdl-25381260

ABSTRACT

Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.


Subject(s)
Carrier Proteins/physiology , DNA Repair , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Alkylation , DNA Damage , DNA Glycosylases/metabolism , DNA Methylation , DNA, Fungal/metabolism , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism
18.
J Biol Chem ; 289(14): 9936-51, 2014 Apr 04.
Article in English | MEDLINE | ID: mdl-24497629

ABSTRACT

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41-48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.


Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Benzo(a)pyrene/adverse effects , Mitochondria/enzymology , Oxygen Consumption/drug effects , Polychlorinated Dibenzodioxins/adverse effects , Protein Sorting Signals , Teratogens , Adrenodoxin/genetics , Adrenodoxin/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Benzo(a)pyrene/pharmacology , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Cytochrome P-450 CYP1B1 , Female , Humans , Male , Mice , Mice, Knockout , Mitochondria/genetics , Mitochondria/pathology , Mutagenesis , Oxidation-Reduction/drug effects , Polychlorinated Dibenzodioxins/pharmacology , Protein Transport/drug effects
19.
Analyst ; 140(4): 1166-73, 2015 Feb 21.
Article in English | MEDLINE | ID: mdl-25516911

ABSTRACT

Ubiquitin C-terminal hydrolase-1 (UCH-L1) is a specific neuronal endoprotease that cleaves the specific peptide bond between ubiquitin molecules. UCH-L1 is released in serum and cerebrospinal fluid after severe brain injury and is considered to be an important biomarker of brain injury. A common polymorphism of UCH-L1 (S18Y) is also linked to a reduced risk of Parkinson's disease. In addition to its function in neuronal tissues, UCH-L1 may also play a part in the progression of certain non-neuronal cancers. UCH-L1 is highly expressed in primary lung tumors and colo-rectal cancers, suggesting a role in tumorigenesis. We report here the development of a sensitive and accurate UCH-L1 assay based on the surface plasmon resonance (SPR) absorbance of gold nanoparticles. We created a unique UCH-L1 substrate containing a ubiquitin molecule with two terminal thiol groups. This UCH-L1 substrate interacted with gold nanoparticles via the terminal thiol groups and induced clustering of the nanoparticles, which was detected by SPR absorbance at 650 nm. UCH-L1 proteolytically cleaved the substrate and the clustered gold nanoparticles were dispersed and could be detected by a shift in the SPR absorbance to 530 nm. This change in absorbance was proportional to the concentration of UCH-L1 and can be used for the quantification of functional UCH-L1. The currently available fluorescence-based UCH-L1 assay is affected by a high background signal and a poor detection limit, especially in the presence of serum. The assay reported here can detect concentrations of UCH-L1 as low as 20 ng ml(-1) (0.8 nM) and the presence of serum had no effect on the detection limit. This assay could be adapted for the rapid determination of the severity of brain injury and could also be applied to high-throughput screening of inhibitors of UCH-L1 enzymatic activity in Parkinson's disease and cancer.


Subject(s)
Citric Acid/chemistry , Gold/chemistry , Nanoparticles/chemistry , Surface Plasmon Resonance/methods , Ubiquitin Thiolesterase/blood , Amino Acid Sequence , Enzyme Assays/methods , Humans , Limit of Detection , Models, Molecular , Molecular Sequence Data , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitin Thiolesterase/analysis , Ubiquitin Thiolesterase/metabolism
20.
Anal Biochem ; 465: 35-7, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25062850

ABSTRACT

The Escherichia coli DNA repair enzyme AlkB belongs to the Fe(II)/2-oxoglutarate-dependent dioxygenase family. It removes methyl groups from 1-methyl adenine (1-meA) and 3-methyl cytosine (3-meC) lesions present in single-stranded DNA by oxidative decarboxylation. In the current article, we describe an in vitro assay that permits rapid detection of AlkB activity. To achieve this, we generated methylated oligonucleotide using methyl methanesulfonate and then monitored DNA repair using a methylation-sensitive restriction enzyme and novel agarose gel electrophoresis system capable of resolving small oligonucleotides. Our approach overcomes several drawbacks of NAD(+)-dependent formaldehyde dehydrogenase-coupled assay and radioisotope-based assay for determining AlkB DNA repair activity.


Subject(s)
DNA Repair , Escherichia coli Proteins , Escherichia coli , Mixed Function Oxygenases , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism
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