RESUMO
In spite of 150 years of studying malaria, the unique features of the malarial parasite, Plasmodium, still perplex researchers. One of the methods by which the parasite manages its gene expression is epigenetic regulation, the champion of which is PfGCN5, an essential enzyme responsible for acetylating histone proteins. PfGCN5 is a â¼170 kDa chromatin-remodeling enzyme that harbors the conserved bromodomain and acetyltransferase domain situated in its C-terminus domain. Although the PfGCN5 proteolytic processing is essential for its activity, the specific protease involved in this process still remains elusive. Identification of PfGCN5 interacting proteins through immunoprecipitation (IP) followed by LC-tandem mass spectrometry analysis revealed the presence of food vacuolar proteins, such as the cysteine protease Falcipain 3 (FP3), in addition to the typical members of the PfGCN5 complex. The direct interaction between FP3 and PfGCN5 was further validated by in vitro pull-down assay as well as IP assay. Subsequently, use of cysteine protease inhibitor E64d led to the inhibition of protease-specific processing of PfGCN5 with concomitant enrichment and co-localization of PfGCN5 and FP3 around the food vacuole as evidenced by confocal microscopy as well as electron microscopy. Remarkably, the proteolytic cleavage of the nuclear protein PfGCN5 by food vacuolar protease FP3 is exceptional and atypical in eukaryotic organisms. Targeting the proteolytic processing of GCN5 and the associated protease FP3 could provide a novel approach for drug development aimed at addressing the growing resistance of parasites to current antimalarial drugs.
RESUMO
The glycolytic enzyme phosphoglycerate mutase (PGM) is of utmost importance for overall cellular metabolism and has emerged as a novel therapeutic target in cancer cells. This enzyme is also conserved in the rapidly proliferating malarial parasite Plasmodium falciparum, which have a similar metabolic framework as cancer cells and rely on glycolysis as the sole energy-yielding process during intraerythrocytic development. There is no redundancy among the annotated PGM enzymes in Plasmodium, and PfPGM1 is absolutely required for the parasite survival as evidenced by conditional knockdown in our study. A detailed comparison of PfPGM1 with its counterparts followed by in-depth structure-function analysis revealed unique attributes of this parasitic protein. Here, we report for the first time the importance of oligomerization for the optimal functioning of the enzyme in vivo, as earlier studies in eukaryotes only focused on the effects in vitro. We show that single point mutation of the amino acid residue W68 led to complete loss of tetramerization and diminished catalytic activity in vitro. Additionally, ectopic expression of the WT PfPGM1 protein enhanced parasite growth, whereas the monomeric form of PfPGM1 failed to provide growth advantage. Furthermore, mutation of the evolutionarily conserved residue K100 led to a drastic reduction in enzymatic activity. The indispensable nature of this parasite enzyme highlights the potential of PfPGM1 as a therapeutic target against malaria, and targeting the interfacial residues critical for oligomerization can serve as a focal point for promising drug development strategies that may not be restricted to malaria only.
Assuntos
Fosfoglicerato Mutase , Plasmodium falciparum , Humanos , Malária/parasitologia , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Plasmodium falciparum/enzimologiaRESUMO
Developing an efficient photobioreactor (PBR) and reducing freshwater dependence are among the significant challenges for generating 3rd generation biomass feedstock. Addressing these, the present study focused on developing a modified airlift (MoAL) PBR. Its performance was further evaluated and compared with the traditional airlift PBR by cultivating microalgae in dark fermentation spent wash. Lower mixing time and higher interfacial mass transfer coefficient was observed in the MoAL PBR having a perforated draft tube. Experimentally, the MoAL exhibited the maximum biomass concentration of 3.18 g L-1, which was 30% higher than that of the conventional airlift PBR. The semi-continuous operation of the MoAL (with water recycling) achieved the maximum biomass productivity of 0.83 g L-1 d-1, two folds superior to that of batch culture. The comprehensive biomass characterization (proximate, ultimate, and thermochemical) further confirmed its potential for bioenergy application. Considering that, hydrothermal liquefaction of the biomass resulted in a maximum biocrude yield of 31% w/w with a higher heating value (HHV) of 36.6 MJ kg-1. In addition, the biocrude comprised 66.6% w/w lighter fraction (<343 °C), including 21.5% w/w of heavy naphtha, 20.5% w/w of kerosene, and 24.6% w/w of diesel. The results can help develop sustainable technology for simultaneous wastewater remediation and biocrude production.
Assuntos
Microalgas , Fotobiorreatores , Biomassa , Fermentação , Água , Água DoceRESUMO
The pathogenesis of human malarial parasite Plasmodium falciparum is interlinked with its timely control of gene expression during its complex life cycle. In this organism, gene expression is partially controlled through epigenetic mechanisms, the regulation of which is, hence, of paramount importance to the parasite. The P. falciparum (Pf)-GCN5 histone acetyltransferase (HAT), an essential enzyme, acetylates histone 3 and regulates global gene expression in the parasite. Here, we show the existence of a novel proteolytic processing for PfGCN5 that is crucial for its activity in vivo We find that a cysteine protease-like enzyme is required for the processing of PfGCN5 protein. Immunofluorescence and immuno-electron microscopy analysis suggest that the processing event occurs in the vicinity of the digestive vacuole of the parasite following its trafficking through the classical ER-Golgi secretory pathway, before it subsequently reaches the nucleus. Furthermore, blocking of PfGCN5 processing leads to the concomitant reduction of its occupancy at the gene promoters and a reduced H3K9 acetylation level at these promoters, highlighting the important correlation between the processing event and PfGCN5 activity. Altogether, our study reveals a unique processing event for a nuclear protein PfGCN5 with unforeseen role of a food vacuolar cysteine protease. This leads to a possibility of the development of new antimalarials against these targets.This article has an associated First Person interview with the first author of the paper.
Assuntos
Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Animais , HumanosRESUMO
Chromatin modification through histone acetylation/deacetylation is important for the regulation of transcription as well as DNA replication in eukaryotes. PfGCN5 and PfMYST are two well-studied histone acetyltransferases in Plasmodium. PfMYST containing the MYST domain, zinc finger domain, and the chromodomain primarily acetylates histone 4. Here, we show that PfMYST is expressed in two isoforms, a long version (â¼72 kDa) and a short version (â¼45 kDa) of the protein, while the shorter version is predominantly present in the nucleus. Further, the association of PfMYST with the putative Plasmodium autonomously replicating sequences (PfARS) was found to be much stronger than the binding of PfGCN5 in these regions with concomitant enrichment of the H4 acetylation level. The binding of PfMYST at these sites was also correlated with another replication protein PfORC1 as well as with the replicating stage (trophozoite) of the parasite. Collectively these results show for the first time the potential role of PfMYST in parasite DNA replication through chromatin modification that may be found useful for the intervention of parasite growth.
Assuntos
Plasmodium falciparum , Proteínas de Protozoários , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Histonas/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Cromatina , Replicação do DNARESUMO
BACKGROUND: Endoscopy has long been widely used to screen for esophageal varices (EV) in cirrhotic patients. Recurrent endoscopy is a significant burden for the healthcare system of the endoscopic unit as well as uncomfortable and high costs for patients. This study intended to prognosticate Right Liver Lobe Diameter/Serum Albumin Ratio (RLLD/Alb) as a non-invasive approach in the early diagnosis of EV among chronic liver disease (CLD) Bangladeshi patients enrolled in a specific hospital. PARTICIPANTS AND METHODS: A total of 150 admitted patients with CLD were included in the study. Patients were subjected through a comprehensive biochemical checkup and upper digestive endoscopic or ultrasonographic inspection. The correlation was evaluated between the RLLD/Alb ratio and esophageal varices grades. RESULTS: The upper digestive endoscopy demonstration among 150 patients resulted in no EV in 18%, while 24% of patients was identified as EV grade I, 20% as grade II, 20% as grade III, and 18% patients as grade IV. The mean value of the RLLD/Alb ratio was 4.89 ± 1.49 (range from 2.30 to 8.45). The RLLD/Alb ratio diagnosed the EV employing the cut-off value of 4.01 with 85.3% sensitivity and 68.8% specificity. Furthermore, it was positively correlated with the grading of EV, when this ratio increased the grading of EV increases and vice versa (r = 0.630, p < 0.001). CONCLUSION: The RLLD/Alb ratio is a non-invasive parameter giving exact guidance relevant to the ascertainment of the existence of EV and their grading in chronic liver disease patients.
Assuntos
Varizes Esofágicas e Gástricas , Endoscopia Gastrointestinal , Varizes Esofágicas e Gástricas/diagnóstico , Varizes Esofágicas e Gástricas/etiologia , Humanos , Fígado/anatomia & histologia , Fígado/patologia , Cirrose Hepática/complicações , Cirrose Hepática/diagnóstico , Albumina Sérica/análiseRESUMO
Helicase loaders are required for the loading of helicases at the vicinity of replication origins. In Helicobacter pylori, Hp0897 has been shown to be a potential helicase loader for replicative helicase (HpDnaB) although it does not show any sequence homology with conventional DnaC like helicase loader proteins. Therefore, it is important to investigate the in vivo role of Hp0897 and structure-function analysis with respect to domain mapping of Hp0897 and HpDnaB. Although HporiC is divided into oriC1 and oriC2, the latter has been assigned as functional origin based on loading of initiator protein HpDnaA. Using chromatin immunoprecipitation (ChIP) experiment, we show preferential binding of Hp0897 at oriC2 over oriC1 like HpDnaA highlighting its helicase loader function in vivo. Furthermore, we generated series of deletion mutants for HpDnaB and Hp0897 that enabled us to map the domains of interaction between these two proteins. Interestingly, the C-terminal domain of Hp0897 (Hp0897CTD) shows stronger interaction with HpDnaB over the N-terminal region of Hp0897 (Hp0897NTD). Similar to the full-length protein, Hp0897CTD also stimulates the DNA binding activity of HpDnaB. Furthermore, overexpression of Hp0897 full-length protein in H. pylori leads to an elongated cell phenotype. While the overexpression of Hp0897CTD does not show a phenotype of cell elongation, overexpression of Hp0897NTD shows extensive cell elongation. These results highlight the possible role of Hp0897CTD in helicase loading and Hp0897NTD's unique function linked to cell division that make Hp0897 as a potential drug target against H. pylori.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Helicobacter pylori/enzimologia , Proteínas de Bactérias/genética , DNA Helicases/genética , DnaB Helicases/química , DnaB Helicases/genética , DnaB Helicases/metabolismo , Helicobacter pylori/química , Helicobacter pylori/genética , Ligação Proteica , Domínios ProteicosRESUMO
Malaria parasites use an extensive secretory pathway to traffic a number of proteins within itself and beyond. In higher eukaryotes, Endoplasmic Reticulum (ER) membrane bound transcription factors such as SREBP are reported to get processed en route and migrate to nucleus under the influence of specific cues. However, a protein constitutively trafficked to the nucleus via classical secretory pathway has not been reported. Herein, we report the presence of a novel trafficking pathway in an apicomplexan, Plasmodium falciparum where a homologue of an Origin Recognition Complex 2 (Orc2) goes to the nucleus following its association with the ER. Our work highlights the unconventional role of ER in protein trafficking and reports for the first time an ORC homologue getting trafficked through such a pathway to the nucleus where it may be involved in DNA replication and other ancillary functions. Such trafficking pathways may have a profound impact on the cell biology of a malaria parasite and have significant implications in strategizing new antimalarials.
Assuntos
Malária Falciparum/genética , Complexo de Reconhecimento de Origem/genética , Plasmodium falciparum/genética , Transporte Proteico/genética , Animais , Núcleo Celular/genética , Replicação do DNA/genética , Retículo Endoplasmático/genética , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidade , Transdução de Sinais/genéticaRESUMO
Proliferative cell nuclear antigen (PCNA) is the processivity factor for various DNA polymerases and it functions in response to DNA damage in eukaryotic system. Plasmodium falciparum contains two PCNAs, while PCNA1 has been attributed to DNA replication, the role of PCNA2 has been assigned to DNA damage response in erythrocytic developmental stages. Although a recent transposon mediated knockout strategy qualified pcna2 as a nonessential gene in Plasmodium berghei, a conventional homologous recombination-based knockout strategy has not been employed for this gene yet. Moreover, the cellular dynamics of PCNA2 in extraerythrocytic stages still remain elusive in Plasmodium. We attempted multiple times to knock out PbPCNA2 from the parasite genome using homologous recombination strategy without much success. However, we were able to generate PbPCNA2-GFP tagged transgenic parasites confirming that the pcna2 locus is amenable to genetic manipulation. The GFP-tagged parasites showed similar growth phenotype, compared to wild-type parasites, in both erythrocytic and sporogonic cycle, suggesting that tagging had no effect on parasite physiology. PbPCNA2 expression was also observed during the sporogonic cycle in midgut oocyst and salivary gland sporozoites. The PbPCNA2 expression was upregulated in the presence of DNA damaging agents like hydroxyurea and methyl methanesulphonate. Our inability to knock out PCNA2 suggested its essentiality in the parasite development and elevated expression during DNA damaging condition hint at a critical role of the protein in parasite physiology. © 2019 IUBMB Life, 71(9):1293-1301, 2019.
Assuntos
Dano ao DNA/genética , Plasmodium berghei/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Protozoários/genética , Animais , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Genoma/genética , Humanos , Plasmodium berghei/patogenicidade , Plasmodium falciparum/genéticaRESUMO
Nucleosome assembly in P. falciparum could be the key process in maintaining its genomic integrity as DNA replicates more than once per cell cycle during several stages of its life cycle. Here, we report the functional characterization of P. falciparum chromatin assembly factor 1 (CAF1), which interacts with several proteins namely PfCAF2, Histones, PfHP1 and others. Consistent with the above findings, we demonstrate the presence of PfCAF1 at the telomeric repeat regions, central and subtelomeric var genes of multiple var gene family along with PfHP1. Further, we report the upregulation of PfCAF1 after treatment with genotoxic agents like MMS and HU. Together, these findings establish role of PfCAF1 in heterochromatin maintenance and as histone chaperone in nucleosome assembly and DNA damage repair.
Assuntos
Fator 1 de Modelagem da Cromatina/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA de Protozoário/genética , Nucleossomos/genética , Plasmodium falciparum/genéticaRESUMO
DNA replication in Helicobacter pylori is initiated from a unique site (oriC) on its chromosome where several proteins assemble to form a functional replisome. The assembly of H. pylori replication machinery is similar to that of the model gram negative bacterium Escherichia coli except for the absence of DnaC needed to recruit the hexameric DnaB helicase at the replisome assembly site. In the absence of an obvious DnaC homologue inH. pylori, the question arises as to whether HpDnaB helicase is loaded at theHp-replication origin by itself or is assisted by other unidentified protein(s). A high-throughput yeast two-hybrid study has revealed two proteins of unknown functions (Hp0897 and Hp0340) that interact with HpDnaB. Here we demonstrate that Hp0897 interacts with HpDnaB helicase in vitro as well as in vivo Furthermore, the interaction stimulates the DNA binding activity of HpDnaB and modulates its adenosine triphosphate hydrolysis and helicase activities significantly. Prior complex formation of Hp0897 and HpDnaB enhances the binding/loading of DnaB onto DNA. Hp0897, along with HpDnaB, colocalizes with replication complex at initiation but does not move with the replisome during elongation. Together, these results suggest a possible role of Hp0897 in loading of HpDnaB at oriC.
Assuntos
Proteínas de Bactérias/metabolismo , DnaB Helicases/metabolismo , Helicobacter pylori/enzimologia , Proteínas de Bactérias/química , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DnaB Helicases/química , Helicobacter pylori/metabolismo , Complexos Multienzimáticos/metabolismo , Ligação Proteica , Multimerização ProteicaRESUMO
In molecular biology, understanding the functional and structural aspects of DNA requires sequence-specific DNA binding probes. Especially, sequence-specific fluorescence probes offer the advantage of real-time monitoring of the conformational and structural reorganization of DNA in living cells. Herein, we designed a new class of D2A (one-donor-two-acceptor) near-infrared (NIR) fluorescence switch-on probe named quinone cyanine-dithiazole ( QCY-DT: ) based on the distinctive internal charge transfer (ICT) process for minor groove recognition of AT-rich DNA. Interestingly, QCY-DT: exhibited strong NIR-fluorescence enhancement in the presence of AT-rich DNA compared to GC-rich and single-stranded DNAs. We show sequence-specific minor groove recognition of QCY-DT: for DNA containing 5'-AATT-3' sequence over other variable (A/T)4 sequences and local nucleobase variation study around the 5'-X(AATT)Y-3' recognition sequence revealed that X = A and Y = T are the most preferable nucleobases. The live cell imaging studies confirmed mammalian cell permeability, low-toxicity and selective staining capacity of nuclear DNA without requiring RNase treatment. Further, Plasmodium falciparum with an AT-rich genome showed specific uptake with a reasonably low IC50 value (<4 µM). The ease of synthesis, large Stokes shift, sequence-specific DNA minor groove recognition with switch-on NIR-fluorescence, photostability and parasite staining with low IC50 make QCY-DT: a potential and commercially viable DNA probe.
Assuntos
Benzotiazóis/química , DNA/química , Corantes Fluorescentes/química , Sequência Rica em At , Pareamento de Bases , Benzotiazóis/metabolismo , Benzotiazóis/toxicidade , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Células MCF-7 , Microscopia de Fluorescência , Modelos Moleculares , Conformação de Ácido Nucleico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Plasmodium falciparumâ Origin Recognition Complex subunit 1 (PfORC1) has been implicated in DNA replication and var gene regulation. While the C-terminus is involved in DNA replication, the specific role of N-terminus has been suggested in var gene regulation in a Sir2-dependent manner. PfORC1 is localized at the nuclear periphery, where the clustering of chromosomal ends at the early stage of parasite development may be crucial for the regulation of subtelomeric var gene expression. Upon disassembly of telomeric clusters at later stages of parasite development, ORC1 is distributed in the nucleus and parasite cytoplasm where it may be required for its other cellular functions including DNA replication. The level of ORC1 decreases dramatically at the late schizont stage. The mechanisms that mediate regulation of PfORC1 function are largely unknown. Here we show, by the use of recombinant proteins and of transgenic parasites expressing wild type or mutant forms of ORC1, that phosphorylation of the PfORC1-N terminal domain by the cyclin-dependent kinase (CDK) PfPK5 abolishes DNA-binding activity and leads to changes in subcellular localization and proteasome-mediated degradation of the protein in schizonts. These results reveal that PfORC1 phosphorylation by a CDK is central to the regulation of important biological functions like DNA replication and var gene silencing.
Assuntos
Complexo de Reconhecimento de Origem/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Replicação do DNA , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Complexo de Reconhecimento de Origem/genética , Fosforilação , Fosfotransferases , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Esquizontes/fisiologiaRESUMO
Regulatory roles of CDKs in fundamental processes including cell cycle progression and transcription are well conserved in metazoans. This family of proteins has undergone significant evolutionary divergence and specialization. Several CDK-like kinases have been identified and characterized in parasitic protozoans. However, clear functional role and physiological relevance of these proteins in protozoans still remain elusive. In continuation with the recent finding that CDK-like protein PfPK5 regulates important DNA replication protein like origin recognition complex subunit 1 in Plasmodium falciparum, here we have discussed the emerging significance of CDK1/2 homologs in DNA replication of parasitic protozoans. In fact, involvement of these proteins in crucial cellular processes projects them as potential drug targets. The possibilities that CDKs offer as potential therapeutic targets in controlling parasite progression have also been explored.
Assuntos
Replicação do DNA , Regulação da Expressão Gênica , Parasitos/genética , Parasitos/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Antiparasitários/farmacologia , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Descoberta de Drogas , Regulação da Expressão Gênica/efeitos dos fármacos , Família Multigênica , Parasitos/efeitos dos fármacos , Proteínas de Protozoários/genéticaRESUMO
Eukaryotic PCNAs (proliferating-cell nuclear antigens) play diverse roles in nucleic acid metabolism in addition to DNA replication. Plasmodium falciparum, which causes human malaria, harbours two PCNA homologues: PfPCNA1 and PfPCNA2. The functional role of two distinct PCNAs in the parasite still eludes us. In the present study, we show that, whereas both PfPCNAs share structural and biochemical properties, only PfPCNA1 functionally complements the ScPCNA mutant and forms distinct replication foci in the parasite, which PfPCNA2 fails to do. Although PfPCNA1 appears to be the primary replicative PCNA, both PfPCNA1 and PfPCNA2 participate in an active DDR (DNA-damage-response) pathway with significant accumulation in the parasite upon DNA damage induction. Interestingly, PfPCNA genes were found to be regulated not at the transcription level, but presumably at the protein stability level upon DNA damage. Such regulation of PCNA has not been shown in eukaryotes before. Moreover, overexpression of PfPCNA1 and PfPCNA2 in the parasite confers a survival edge on the parasite in a genotoxic environment. This is the first evidence of a PfPCNA-mediated DDR in the parasite and gives new insights and rationale for the presence of two PCNAs as a parasite survival strategy and its probable success.
Assuntos
Dano ao DNA/fisiologia , Replicação do DNA/fisiologia , Plasmodium falciparum/fisiologia , Antígeno Nuclear de Célula em Proliferação/fisiologia , Células Cultivadas , Humanos , Antígeno Nuclear de Célula em Proliferação/química , Estrutura Secundária de ProteínaRESUMO
We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.
Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA/fisiologia , DNA Bacteriano/fisiologia , Proteínas de Ligação a DNA/metabolismo , Helicobacter pylori/metabolismo , Transporte Proteico/fisiologia , Proteínas de Bactérias/genética , Divisão Celular/fisiologia , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Helicobacter pylori/citologia , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimentoRESUMO
Plasmodium falciparum origin recognition complex 1 (ORC1) protein has been implicated in DNA replication and silencing var gene family. However, the mechanism and the domain structure of ORC1 related to the regulation of var gene family are unknown. Here we show that the unique N-terminus of PfORC1 (PfORC1N(1-238)) is targeted to the nuclear periphery in vivo and this region binds to the telomeric DNA in vitro due to the presence of a leucine heptad repeats. Like PfORC1N(1-238), endogenous full length ORC1, was found to be associated with sub telomeric repeat regions and promoters of various var genes. Additionally, binding and propagation of ORC1 to telomeric and subtelomeric regions was severely compromised in PfSir2 deficient parasites suggesting the dependence of endogenous ORC1 on Sir2 for var gene regulation. This feature is not previously described for Plasmodium ORC1 and contrary to yeast Saccharomyces cerevisiae where ORC function as a landing pad for Sir proteins. Interestingly, the overexpression of ORC1N(1-238) compromises the binding of Sir2 at the subtelomeric loci and var gene promoters consistent with de-repression of some var genes. These results establish role of the N-terminus of PfORC1 in heterochromatin formation and regulation of var gene expression in co-ordination with Sir2 in P. falciparum.
Assuntos
Inativação Gênica , Complexo de Reconhecimento de Origem/química , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Telômero/metabolismo , Núcleo Celular/metabolismo , DNA/metabolismo , Dimerização , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/fisiologia , Plasmodium falciparum/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Sequências Repetitivas de Aminoácidos , Sequências Repetitivas de Ácido Nucleico , Sirtuína 2/metabolismoRESUMO
Plasmodium falciparum, one of the deadliest parasites on earth causes human malaria resulting one million deaths annually. Central to the parasite pathogenicity and morbidity is the switching of parasite virulence (var) gene expression causing host immune evasion. The regulation of Plasmodium var gene expression is poorly understood. The complex life cycle of Plasmodium and mutually exclusive expression pattern of var genes make this disease difficult to control. Recent studies have demonstrated the pivotal role of epigenetic mechanism for control of coordinated expression of var genes, important for various clinical manifestations of malaria. In this review, we discuss about different Plasmodium histones and their various modifications important for gene expression and gene repression.Contribution of epigenetic mechanism to understand the var gene expression is also highlighted. We also describe in details P. falciparum nuclear architecture including heterochromatin, euchromatin and telomeric regions and their importance in subtelomeric and centrally located var gene expression. Finally, we explore the possibility of using Histone Acetyl Transferase (HAT) and Histone Deacetylase (HDAC)inhibitors against multi-drug resistance malaria parasites to provide another line of treatment for malaria.
Assuntos
Antimaláricos/uso terapêutico , Epigênese Genética , Regulação da Expressão Gênica , Malária Falciparum/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Acetilação , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Resistência a Medicamentos/genética , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Humanos , Malária Falciparum/diagnóstico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/virologia , Fenótipo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , Processamento de Proteína Pós-Traducional , Virulência/genéticaRESUMO
To better understand the poor conservation of the helicase binding domain of primases (DnaGs) among the eubacteria, we determined the crystal structure of the Helicobacter pylori DnaG C-terminal domain (HpDnaG-CTD) at 1.78 Å. The structure has a globular subdomain connected to a helical hairpin. Structural comparison has revealed that globular subdomains, despite the variation in number of helices, have broadly similar arrangements across the species, whereas helical hairpins show different orientations. Further, to study the helicase-primase interaction in H. pylori, a complex was modeled using the HpDnaG-CTD and HpDnaB-NTD (helicase) crystal structures using the Bacillus stearothermophilus BstDnaB-BstDnaG-CTD (helicase-primase) complex structure as a template. By using this model, a nonconserved critical residue Phe534 on helicase binding interface of DnaG-CTD was identified. Mutation guided by molecular dynamics, biophysical, and biochemical studies validated our model. We further concluded that species-specific helicase-primase interactions are influenced by electrostatic surface potentials apart from the critical hydrophobic surface residues.
Assuntos
DNA Primase/química , DNA Primase/metabolismo , Helicobacter pylori/enzimologia , Cristalografia por Raios X , DNA Primase/genética , Geobacillus stearothermophilus/química , Helicobacter pylori/química , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Actively treadmilling FtsZ acts as the pivotal scaffold for bacterial cell divisome components providing them with a circumferential ride along the site of future division. FtsZ from slow growing Helicobacter pylori (HpFtsZ), a class I carcinogen which thrives abundantly in the acidic environment is poorly understood. We studied HpFtsZ as a function of pH, cations and time and compared it with well-studied E. coli FtsZ (EcFtsZ). HpFtsZ shows pH dependent GTPase activity which is inhibited under acidic conditions. Mg+2 ions play an indispensable role in its GTPase activity, however, higher Mg+2 levels negatively affect its activity. As compared to EcFtsZ, HpFtsZ exhibits lower and slower nucleotide hydrolyzing activity. Molecular Dynamics Simulation studies of FtsZ reveal that GTP binding induces a rewiring of the hydrogen bond network which results in reduction of the binding cleft volume leading to the spontaneous release of GTP. The GTPase activity is linked to the extent of reduction in the binding cleft volume, which is also supported by the binding free energy analysis. Evidently, HpFtsZ is a pH sensitive GTPase with low efficiency that may reflect on the overall slow growth rate of H. pylori.