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1.
Nature ; 523(7560): 333-6, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26061769

RESUMO

Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals, where it can be both a commensal and a pathogen. Intricate regulatory mechanisms ensure that bacteria have the right complement of ß-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat. Yet no mechanism is known for replacing OMPs in the outer membrane, an issue that is further confounded by the lack of an energy source and the high stability and abundance of OMPs. Here we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature, in which old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form ∼0.5-µm diameter islands, where their diffusion is restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the outer membrane. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence, the outer membrane of a Gram-negative bacterium is a spatially and temporally organized structure, and this organization lies at the heart of how OMPs are turned over in the membrane.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Polaridade Celular , Difusão , Escherichia coli/química , Escherichia coli/genética , Proteínas Ligadas a Lipídeos/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transporte Proteico
2.
J Cell Sci ; 128(8): 1518-27, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25736292

RESUMO

CIZ1 is a nuclear matrix protein that cooperates with cyclin A2 (encoded by CCNA2) and CDK2 to promote mammalian DNA replication. We show here that cyclin-A-CDK2 also negatively regulates CIZ1 activity by phosphorylation at threonines 144, 192 and 293. Phosphomimetic mutants do not promote DNA replication in cell-free and cell-based assays, and also have a dominant-negative effect on replisome formation at the level of PCNA recruitment. Phosphorylation blocks direct interaction with cyclin-A-CDK2 and recruitment of endogenous cyclin A to the nuclear matrix. In contrast, phosphomimetic CIZ1 retains the ability to bind to the nuclear matrix, and its interaction with CDC6 is not affected. Phospho-T192-specific antibodies confirm that CIZ1 is phosphorylated during S phase and G2, and show that phosphorylation at this site occurs at post-initiation concentrations of cyclin-A-CDK2. Taken together, the data suggest that CIZ1 is a kinase sensor that promotes initiation of DNA replication at low kinase levels, when in a hypophosphorylated state that is permissive for cyclin-A-CDK2 interaction and delivery to licensed origins, but blocks delivery at higher kinase levels when it is phosphorylated.


Assuntos
Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Replicação do DNA , Proteínas Nucleares/metabolismo , Células 3T3 , Animais , Células Cultivadas , Cromatina/genética , Fase G2 , Células HeLa , Humanos , Camundongos , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fase S
3.
J Cell Sci ; 125(Pt 10): 2466-77, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22366453

RESUMO

CIZ1 is a nuclear-matrix-associated DNA replication factor unique to higher eukaryotes, for which alternatively spliced isoforms have been associated with a range of disorders. In vitro, the CIZ1 N-terminus interacts with cyclin E and cyclin A at distinct sites, enabling functional cooperation with cyclin-A-Cdk2 to promote replication initiation. C-terminal sequences anchor CIZ1 to fixed sites on the nuclear matrix, imposing spatial constraint on cyclin-dependent kinase activity. Here we demonstrate that CIZ1 is predominantly expressed as a predicted full-length product throughout mouse development, consistent with a ubiquitous role in cell and tissue renewal. CIZ1 is expressed in proliferating stem cells of the testis, but is notably downregulated following commitment to differentiation. Significantly, CIZ1 is re-expressed at high levels in non-proliferative spermatocytes before meiotic division. Sequence analysis identifies at least seven alternatively spliced variants, including a dominant cancer-associated form and a set of novel isoforms. Furthermore, we show that in these post-replicative cells, CIZ1 interacts with germ-cell-specific cyclin A1, which has been implicated in the repair of DNA double-strand breaks. Consistent with this role, antibody depletion of CIZ1 reduces the capacity for testis extract to repair digested plasmid DNA in vitro. Together, the data imply post-replicative roles for CIZ1 in germ cell differentiation that might include meiotic recombination - a process intrinsic to genome stability and diversification.


Assuntos
Ciclina A1/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Espermatogênese , Espermatogônias/citologia , Processamento Alternativo , Animais , Proliferação de Células , Ciclina A1/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/fisiopatologia , Proteínas Nucleares/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Espermatogônias/crescimento & desenvolvimento , Espermatogônias/metabolismo
4.
Nucleic Acids Res ; 39(7): 2671-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21109536

RESUMO

Cyclin E supports pre-replication complex (pre-RC) assembly, while cyclin A-associated kinase activates DNA synthesis. We show that cyclin E, but not A, is mounted upon the nuclear matrix in sub-nuclear foci in differentiated vertebrate cells, but not in undifferentiated cells or cancer cells. In murine embryonic stem cells, Xenopus embryos and human urothelial cells, cyclin E is recruited to the nuclear matrix as cells differentiate and this can be manipulated in vitro. This suggests that pre-RC assembly becomes spatially restricted as template usage is defined. Furthermore, failure to become restricted may contribute to the plasticity of cancer cells.


Assuntos
Ciclina E/metabolismo , Neoplasias/metabolismo , Matriz Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Camundongos , Transporte Proteico , Xenopus laevis
5.
Drug Deliv Transl Res ; 13(8): 2159-2182, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-35751001

RESUMO

Light-responsive biomaterials can be used for the delivery of therapeutic drugs and nucleic acids, where the tunable/precise delivery of payload highlights the potential of such biomaterials for treating a variety of conditions. The translucency of eyes and advances of laser technology in ophthalmology make light-responsive delivery of drugs feasible. Importantly, light can be applied in a non-invasive fashion; therefore, light-triggered drug delivery systems have great potential for clinical impact. This review will examine various types of light-responsive polymers and the chemistry that underpins their application as ophthalmic drug delivery systems.


Assuntos
Materiais Biocompatíveis , Sistemas de Liberação de Medicamentos , Polímeros
6.
J Cell Sci ; 123(Pt 7): 1108-15, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20215406

RESUMO

Initiation of mammalian DNA replication can be reconstituted from isolated G1-phase nuclei and cell extracts, supplemented with cyclin-dependent protein kinases (CDKs). Under these conditions, cyclin E supports pre-replication complex assembly, whereas cyclin-A-associated kinase acts later to terminate assembly and activate DNA replication. The mechanism by which these events are coordinated is unknown. Here, we show that the replication factor Ciz1 interacts with cyclins E and A sequentially through distinct cyclin-binding motifs. Cyclin A displaces cyclin E from Ciz1 in a manner that is dependent on functional domains that are essential for its role in DNA replication. Furthermore, in cell-free assays, recombinant cyclin-A-CDK2 complexes and recombinant Ciz1 cooperate to promote initiation of DNA replication in late G1-phase nuclei. In addition, Ciz1 supports immobilization of cyclin A in isolated nuclei and depletion of Ciz1 by RNAi impairs immobilization, suggesting that Ciz1 promotes initiation by helping to target the kinase to a specific subnuclear compartment. We propose that Ciz1 acts to coordinate the functions of cyclins E and A in the nucleus, by delivering cyclin-A-associated kinase to sites that are specified by cyclin E, helping to ensure that they execute their functions in the same place and in the correct order.


Assuntos
Núcleo Celular/metabolismo , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Replicação do DNA , Proteínas Nucleares/metabolismo , Animais , Células 3T3 BALB , Sistema Livre de Células , Clonagem Molecular , Ciclina E/metabolismo , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , RNA Interferente Pequeno/genética
7.
Biomedicines ; 10(8)2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-36009559

RESUMO

Precise regulation of DNA replication complex assembly requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) activities to activate the replicative helicase complex and initiate DNA replication. Chemical probes have been essential in the molecular analysis of DDK-mediated regulation of MCM2-7 activation and the initiation phase of DNA replication. Here, the inhibitory activity of two distinct DDK inhibitor chemotypes, PHA-767491 and XL-413, were assessed in cell-free and cell-based proliferation assays. PHA-767491 and XL-413 show distinct effects at the level of cellular proliferation, initiation of DNA replication and replisome activity. XL-413 and PHA-767491 both reduce DDK-specific phosphorylation of MCM2 but show differential potency in prevention of S-phase entry. DNA combing and DNA replication assays show that PHA-767491 is a potent inhibitor of the initiation phase of DNA replication but XL413 has weak activity. Importantly, PHA-767491 decreased E2F-mediated transcription of the G1/S regulators cyclin A2, cyclin E1 and cyclin E2, and this effect was independent of CDK9 inhibition. Significantly, the enhanced inhibitory profile of PHA-767491 is mediated by potent inhibition of both DDK and the CDK2-Rb-E2F transcriptional network, that provides the molecular basis for its increased anti-proliferative effects in RB+ cancer cell lines.

8.
Biosensors (Basel) ; 11(1)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33429883

RESUMO

The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5'-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes.


Assuntos
Técnicas Biossensoriais/instrumentação , Engenharia Genética/instrumentação , Nanotubos de Carbono/química , Proteínas Nucleares/genética , Mutação Puntual , Células 3T3 , Animais , Sistemas CRISPR-Cas , Carbodi-Imidas/química , Eletrodos , Grafite/química , Limite de Detecção , Camundongos , Mutagênese Sítio-Dirigida
9.
Front Mol Biosci ; 7: 606807, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33282915

RESUMO

Cellular homeostasis requires adaption to environmental stress. In response to various environmental and genotoxic stresses, all cells produce dinucleoside polyphosphates (NpnNs), the best studied of which is diadenosine tetraphosphate (Ap4A). Despite intensive investigation, the precise biological roles of these molecules have remained elusive. However, recent studies have elucidated distinct and specific signaling mechanisms for these nucleotides in prokaryotes and eukaryotes. This review summarizes these key discoveries and describes the mechanisms of Ap4A and Ap4N synthesis, the mediators of the cellular responses to increased intracellular levels of these molecules and the hydrolytic mechanisms required to maintain low levels in the absence of stress. The intracellular responses to dinucleotide accumulation are evaluated in the context of the "friend" and "foe" scenarios. The "friend (or alarmone) hypothesis" suggests that ApnN act as bona fide secondary messengers mediating responses to stress. In contrast, the "foe" hypothesis proposes that ApnN and other NpnN are produced by non-canonical enzymatic synthesis as a result of physiological and environmental stress in critically damaged cells but do not actively regulate mitigating signaling pathways. In addition, we will discuss potential target proteins, and critically assess new evidence supporting roles for ApnN in the regulation of gene expression, immune responses, DNA replication and DNA repair. The recent advances in the field have generated great interest as they have for the first time revealed some of the molecular mechanisms that mediate cellular responses to ApnN. Finally, areas for future research are discussed with possible but unproven roles for intracellular ApnN to encourage further research into the signaling networks that are regulated by these nucleotides.

10.
Sci Rep ; 10(1): 18103, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093612

RESUMO

CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action.


Assuntos
Ciclo Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas , RNA Helicases DEAD-box/genética , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética
11.
Biomolecules ; 7(1)2016 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-28036012

RESUMO

Precise duplication of the genome is a prerequisite for the health and longevity of multicellular organisms. The temporal regulation of origin specification, replication licensing, and firing at replication origins is mediated by the cyclin-dependent kinases. Here the role of Cip1 interacting Zinc finger protein 1 (Ciz1) in regulation of cell cycle progression is discussed. Ciz1 contributes to regulation of the G1/S transition in mammalian cells. Ciz1 contacts the pre-replication complex (pre-RC) through cell division cycle 6 (Cdc6) interactions and aids localization of cyclin A- cyclin-dependent kinase 2 (CDK2) activity to chromatin and the nuclear matrix during initiation of DNA replication. We discuss evidence that Ciz1 serves as a kinase sensor that regulates both initiation of DNA replication and prevention of re-replication. Finally, the emerging role for Ciz1 in cancer biology is discussed. Ciz1 is overexpressed in common tumors and tumor growth is dependent on Ciz1 expression, suggesting that Ciz1 is a driver of tumor growth. We present evidence that Ciz1 may contribute to deregulation of the cell cycle due to its ability to alter the CDK activity thresholds that are permissive for initiation of DNA replication. We propose that Ciz1 may contribute to oncogenesis by induction of DNA replication stress and that Ciz1 may be a multifaceted target in cancer therapy.


Assuntos
Carcinogênese/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Replicação do DNA , Proteínas Nucleares/metabolismo , Animais , Antineoplásicos/uso terapêutico , Ciclo Celular , Transformação Celular Neoplásica , Cromatina/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Camundongos
12.
PLoS One ; 11(5): e0154674, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27144453

RESUMO

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Monoéster Fosfórico Hidrolases/deficiência , Linhagem Celular Tumoral , Regulação para Baixo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Monoéster Fosfórico Hidrolases/genética
13.
DNA Repair (Amst) ; 33: 90-100, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26204256

RESUMO

The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 µM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 µM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA.


Assuntos
Dano ao DNA , Replicação do DNA , Fosfatos de Dinucleosídeos/biossíntese , Células 3T3 , Adenosina Difosfato Ribose/metabolismo , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Cromatografia por Troca Iônica , Cricetinae , Cricetulus , Fosfatos de Dinucleosídeos/química , Compostos de Epóxi/farmacologia , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Camundongos , Mitomicina/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo
14.
J Biol Chem ; 280(1): 112-7, 2005 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-15501819

RESUMO

Activation of latent proteinases ensures that the timing of proteolysis is regulated precisely, a process that generally involves proteolytic excision of a pro-region or a tightly bound inhibitor. Here we define the activation mechanism for Lit, a dormant suicide proteinase in Escherichia coli K-12. Previous work has shown that Gol, a short sequence within the major capsid protein gp23, activates Lit during the latter stages of T4 phage infection. This results in cell death and exclusion of the phage from the culture. The Lit site specifically cleaves the host translation factor EF-Tu (elongation factor Tu) after it has formed a weak complex with Gol, which can be supplied as a 29-residue peptide. Gol is absolutely required for Lit activation. but its role in proteolysis is unknown. Using a purified three-component system and kinetic analysis, we demonstrate that under physiological conditions Lit hydrolyzes its substrate very slowly (k(cat) of approximately 1 s(-1)). Given the abundance of EF-Tu in the cell, this finding is consistent with a cell-killing mechanism in which a few cleaved EF-Tu proteins are able block translating ribosomes from functioning. We also demonstrate that less than half of the 29 Gol residues are needed for Lit activation and that the role of the peptide is not to provide catalytic groups but to influence catalysis indirectly through stabilization of the ternary Lit.Gol.EF-Tu complex. Hence, phage-elicited suicide of E. coli K-12 by Lit is a variant form of "cofactor-induced activation," a mechanism of protease activation that has only been documented previously in pathogen subversion of mammalian hemostasis cascades.


Assuntos
Endopeptidases/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Bacteriófago T4/metabolismo , Endopeptidases/fisiologia , Escherichia coli/fisiologia , Escherichia coli/virologia , Proteínas de Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Hidrólise , Cinética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Mutagênese , Fator Tu de Elongação de Peptídeos/química , Especificidade por Substrato
15.
Biochemistry ; 43(24): 7948-53, 2004 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-15196039

RESUMO

Bacteriophage exclusion is a suicide response to viral infection. In strains of Escherichia coli K-12 infected with T4 phage this process is mediated by the host-encoded Lit peptidase. Lit is activated by a unique sequence in the major head protein of the T4 phage (the Gol sequence) which then cleaves site-specifically the host translation factor EF-Tu, ultimately leading to cell death. Lit has very low sequence identity with other peptidases, with only a putative metallopeptidase motif, H(160)EXXH, giving an indication of its catalytic activity. The aim of the present study was to ascertain if Lit is a metallopeptidase, identify residues essential for Lit activity, and probe the involvement of the Gol sequence in the activation of enzymatic activity. Lit activity was inhibited by the zinc chelator 1,10-phenanthroline, consistent with the suggestion that it is a metallopeptidase. Preliminary covalent modification experiments found that Lit was susceptible to inactivation by diethyl pyrocarbonate, with about three histidines reversibly modified, one of which was found to be essential for proteolytic activity. Subsequently, 13 mutants of the Lit enzyme were constructed that included all 10 histidines as well as other residues within the metallopeptidase motif. This demonstrated that the residues within the HEXXH motif are required for Lit activity and further defined the essential catalytic core as H(160)EXXHX(67)H, with additional residues such as His169 being important but not essential for activity. Kinetic analysis of Lit activation by a synthetic Gol peptide highlighted that elevated concentrations of the peptide (>10-fold above activation K(M)) are inhibitory to Lit, with this effect also seen in partially active Lit mutants. The susceptibility of Lit to inhibition by its own activating peptide suggests that the Gol sequence may be able to bind nonproductively to the enzyme at high concentration. We discuss these data in the context of the currently understood models for Gol-mediated activation of the Lit peptidase and its mechanism of action.


Assuntos
Apoptose , Endopeptidases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Membrana/química , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Ativação Enzimática , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Cinética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Mutagênese , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
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