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1.
Plant Biotechnol J ; 2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34665915

RESUMO

Seed vigour is an imperative trait for the direct seeding of rice. In this study, we examined the genetic regulation of seedling percentage at the early germination using a genome-wide association study (GWAS) in rice. One major quantitative trait loci qSP3 for seedling percentage was identified, and the candidate gene was validated as qSP3, encoding a cupin domain protein OsCDP3.10 for the synthesis of 52 KDa globulin. Disruption of this gene in Oscdp3.10 mutants reduced the seed vigour, including the germination potential and seedling percentage, at the early germination in rice. The lacking accumulation of 52 KDa globulin was observed in the mature grains of the Oscdp3.10 mutants. The significantly lower amino acid contents were observed in the mature grains and the early germinating seeds of the Oscdp3.10 mutants compared to those of wild-type (WT). Rice OsCDP3.10 regulated seed vigour mainly via modulating the amino acids e.g. Met, Glu, His, and Tyr that contribute to hydrogen peroxide (H2 O2 ) accumulation in the germinating seeds. These results provide important insights into the application of seed priming with the amino acids and the selection of OsCDP3.10 to improve seed vigour in rice.

2.
Chemosphere ; 282: 131110, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34470162

RESUMO

Fibrous activated carbon has attracted emerging research interests due to its remarkable adsorption performance for volatile organic compounds (VOCs). Though this adsorption behavior for VOCs is closely related to the pore structure on the surface of activated carbon fiber (ACF), few researchers paid attentions to the influence of textural properties of this adsorption process. Especially, cotton-based activated carbon fiber (CACF) for adsorbing benzene pollutant is rarely reported. Herein, in order to develop a high-performance adsorbent for the removal of VOCs pollutants, this work studied the influence of textural properties of CACF on the adsorption of benzene. The results showed that the increase of carbonization temperature would lead to the reduction of mesopores but the increase of micropores for CACF; the embedment of phosphoric acid and its derivatives into the carbon layers contributed to the formation of pore structure for CACF; furthermore, specific surface area of CACF can also be enlarged by increasing the concentration of phosphoric acid. More importantly, it was found that the adsorption capacity of CACF for benzene was strongly dependent on the specific surface area and volume of micropores within CACF because micropores can provide more favorable binding sites. This adsorption process preferred to occur on the wall of micropores, then the accumulated benzene would slowly fill the pores. Interestingly, the decrease of pore size of micropores can unexpectedly improve the affinity of CACF to benzene on the contrary. This work provides a new strategy to develop porous structured ACF materials for the high-performance adsorption of VOCs.


Assuntos
Benzeno , Carvão Vegetal , Adsorção , Fibra de Carbono , Porosidade
3.
Arch Toxicol ; 95(11): 3497-3513, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34510229

RESUMO

Cadmium (Cd) has been reported to induce kidney damage by triggering oxidative stress and inflammation. The NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome has been implicated a role in the pathogenesis of inflammation. However, the connection between Cd and NLRP3 inflammasome in the development of renal inflammation remains unknown. In this study, in vitro experiments based on the telomerase-immortalized human renal proximal-tubule epithelial cell line (RPTEC/TERT1) were carried out. Results revealed that CdCl2 (2-8 µM) increased ROS production and activated NLRP3, thereby enhancing secretion of IL-1ß and IL-18 (P < 0.05). Knock-down of NLRP3 rescued the RPTEC/TERT1 cells from Cd-induced inflammatory damage. Cd activated the MAPK/NF-κB signaling pathway in RPTEC/TERT1 cells (P < 0.05). In addition, treatment with N-acetylcysteine (NAC) improved inflammation and blocked the upregulation of the MAPK/NF-κB signaling pathway. Pre-treatment with MAPK and NF-κB inhibitors also suppressed NLRP3 inflammasome activation (P < 0.05). Moreover, CdCl2 (25-00 mg/L) stimulated the MAPK/NF-κB signaling pathway, activated the NLRP3 inflammasome, and increased inflammatory response (P < 0.05) leading to renal injury in rats. Exposure to cadmium elevated serum levels of NLRP3 and IL-1ß in populations (P < 0.05). Further analysis found that serum NLRP3 and IL-1ß levels were positively correlated with urine cadmium (UCd) and urine N-acetyl-ß-D-glucosaminidase (UNAG). Overall, Cd induced renal inflammation through the ROS/MAPK/NF-κB signaling pathway by activating the NLRP3 inflammasome. Our research thus provides new insights into the molecular mechanism that NLRP3 contributes to Cd-induced kidney damage.

5.
J Hazard Mater ; 420: 126550, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34252664

RESUMO

The critical health risks caused by cadmium (Cd) via dietary exposure are commonly assessed by detecting Cd concentrations in foods. Differently, in this study, the bioaccessibility and bioavailability of Cd in major local harvests were introduced to assess the dietary exposure of local residents from a high-level environmental Cd region. The results indicated that certain Cd was released into the digestive juice after in vitro digestion with a bioaccessibility of 20-63% for rice and 3-32% for leafy vegetables, and the released portion was partially absorbed by Caco-2 cells with a bioavailability of 2-21% for rice and 0.2-13% for leafy vegetables. The results obtained from the toxicokinetic model revealed that the predicted urinary Cd values from the estimated daily intake (EDI) of Cd, which accounted for bioaccessibility and bioavailability, were consistent with the actual measured values, and the EDIs were considerably lower than the acceptable daily intake. This suggests that the bioaccessibility and bioavailability adjusted dietary Cd exposure should be more precise. The key issues addressed in our study implores that a potential health risk cannot be neglected in people with high consumption of rice from high-level zone.

6.
Sci Signal ; 14(669)2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563698

RESUMO

Trypanosoma brucei, an important human parasite, has a flagellum that controls cell motility, morphogenesis, proliferation, and cell-cell communication. Inheritance of the newly assembled flagellum during the cell cycle requires the Polo-like kinase homolog TbPLK and the kinetoplastid-specific protein phosphatase KPP1, although whether TbPLK acts on KPP1 or vice versa has been unclear. Here, we showed that dephosphorylation of TbPLK on Thr125 by KPP1 maintained low TbPLK activity in the flagellum-associated hook complex structure, thereby ensuring proper flagellum positioning and attachment. This dephosphorylation event required the recognition of phosphorylated Thr198 in the activation loop of TbPLK by the N-terminal Plus3 domain of KPP1 and the dephosphorylation of phosphorylated Thr125 in TbPLK by the C-terminal catalytic domain of KPP1. Dephosphorylation of TbPLK by KPP1 prevented hyperphosphorylation of the hook complex protein TbCentrin2, thereby allowing timely dephosphorylation of phosphorylated TbCentrin2 for hook complex duplication and flagellum positioning and attachment. Thus, KPP1 attenuates TbPLK activity by dephosphorylating TbPLK to facilitate flagellum inheritance.

7.
BMC Biol ; 19(1): 29, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33568178

RESUMO

BACKGROUND: Faithful DNA replication is essential to maintain genomic stability in all living organisms, and the regulatory pathway for DNA replication initiation is conserved from yeast to humans. The evolutionarily ancient human parasite Trypanosoma brucei, however, lacks many of the conserved DNA replication factors and may employ unusual mechanisms for DNA replication. Neither the S-phase cyclin-dependent kinase (CDK) nor the regulatory pathway governing DNA replication has been previously identified in T. brucei. RESULTS: Here we report that CRK2 (Cdc2-related kinase 2) complexes with CYC13 (Cyclin13) and functions as an S-phase CDK to promote DNA replication in T. brucei. We further show that CRK2 phosphorylates Mcm3, a subunit of the Mcm2-7 sub-complex of the Cdc45-Mcm2-7-GINS complex, and demonstrate that Mcm3 phosphorylation by CRK2 facilitates interaction with Sld5, a subunit of the GINS sub-complex of the Cdc45-Mcm2-7-GINS complex. CONCLUSIONS: These results identify the CRK2-CYC13 complex as an S-phase regulator in T. brucei and reveal its role in regulating DNA replication through promoting the assembly of the Cdc45-Mcm2-7-GINS complex.


Assuntos
Proteína Quinase CDC2/genética , Ciclinas/genética , Replicação do DNA , Proteínas de Protozoários/genética , Proteínas Quinases Associadas a Fase S/genética , Trypanosoma brucei brucei/genética , Proteína Quinase CDC2/metabolismo , Ciclinas/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Trypanosoma brucei brucei/metabolismo
8.
Phytother Res ; 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33440461

RESUMO

The continued global rise in papillary thyroid carcinoma (PTC) combined with potential adverse effects of regular treatments calls for an alternative therapy. Prunella vulgaris L. (PV) is commonly used as a herbal remedy for thyroid diseases in China, but its influence on PTC is unclear. This study investigated the effect of PV aqueous extract on PTC and its underlying mechanism using a mouse xenograft model and the human PTC cell line K1. PV suppressed tumor growth in PTC-bearing mice at 0.05 and 0.1 g/kg bw, accompanied by improvements in autophagy-related protein expressions in xenografts. In K1 cells, PV inhibited cell growth and induced autophagic flux, manifesting as changes in autophagy-related proteins, the presence of autophagosomes, and a further increase in LC3-II by co-incubation with bafilomycin A1. Autophagy inhibitor 3-methyladenine ameliorated the autophagic cell death caused by PV. The mammalian target of rapamycin (mTOR) activator MHY1485 blocked the antiproliferative activity of PV by regulating mTOR, unc-51-like autophagy activating kinase 1 (ULK1), autophagosomes formation, and autophagy-related proteins. The adenosine monophosphate-activated protein kinase (AMPK) inhibitor compound C attenuated PV-induced inhibition of mTOR. Our results suggest that PV inhibits the growth of PTC in vivo and in vitro via autophagy, which is associated with the AMPK/mTOR/ULK1 pathway. Thus, PV has the potential to function as a therapeutic agent against PTC.

9.
Food Funct ; 11(10): 9157-9167, 2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-33026384

RESUMO

Oenothein B (OEB) has various biological functions, although few studies have focused on its effect on in vivo metabolic phenotypes. In the present study, the systematic antioxidant activity of OEB was evaluated both in vitro and in vivo, and the effect of OEB on metabolic pathways related to antioxidant capacity of Caenorhabditis elegans (C. elegans) was explored. Our findings indicate that OEB exhibits great antioxidant capacity and ability to scavenge free radicals and that OEB treatment can protect RAW 264.7 macrophages from oxidative damage by increasing superoxide dismutase (SOD) activity, catalase (CAT) activity and glutathione (GSH) content and the corresponding gene expression (sod2, cat, gpx1), while decreasing malonic dialdehyde (MDA) content. Moreover, OEB treatment significantly reduced ROS accumulation under oxidative stress conditions and increased glutathione peroxidase (GPx) activity and decreased MDA content in C. elegans. Metabolomics analysis revealed that sixteen out of forty-two significantly altered metabolites were selected as potential biomarkers related to alterations in the antioxidant status of worms, including metabolic pathways involved in amino acid metabolism, taurine and hypotaurine metabolism, lipid metabolism, and purine metabolism. Overall, our results provide new insights into the effects of OEB treatment on antioxidant capacity and metabolism that suggest that OEB could be a potentially good source of natural antioxidants.

10.
Open Biol ; 10(10): 200189, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33050792

RESUMO

Polo-like kinases (Plks) are evolutionarily conserved serine/threonine protein kinases playing crucial roles during multiple stages of mitosis and cytokinesis in yeast and animals. Plks are characterized by a unique Polo-box domain, which plays regulatory roles in controlling Plk activation, interacting with substrates and targeting Plk to specific subcellular locations. Plk activity and protein abundance are subject to temporal and spatial control through transcription, phosphorylation and proteolysis. In the early branching protists, Plk orthologues are present in some taxa, such as kinetoplastids and Giardia, but are lost in apicomplexans, such as Plasmodium. Works from characterizing a Plk orthologue in Trypanosoma brucei, a kinetoplastid protozoan, discover its essential roles in regulating the inheritance of flagellum-associated cytoskeleton and the initiation of cytokinesis, but not any stage of mitosis. These studies reveal evolutionarily conserved and species-specific features in the control of Plk activation, substrate recognition and protein abundance, and suggest the divergence of Plk function and regulation for specialized needs in this flagellated unicellular eukaryote.

11.
Food Res Int ; 136: 109302, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32846514

RESUMO

While eucalyptus leaf polyphenols extract (EPE) has been evaluated for its various bioactivities, few studies thus far have focused on its systemic antioxidant activity or its effects in chickens in relation to meat quality or the intestinal microbiome. Therefore, the goal of this study was to investigate the antioxidant activity of EPE in vitro and in vivo, and to evaluate its effect on chicken meat quality and cecum microbiota. In this study, EPE scavenged DPPH free radical, ABTS free radical, and superoxide radical, and showed strong reducing power in chemical-based assay. EPE protected RAW264.7 cells from H2O2-induced oxidative damage by improving total superoxide dismutase (T-SOD) activity, catalase (CAT) activity and glutathione (GSH) content, decreasing malondialdehyde (MDA) content. Additionally, EPE dietary supplementation was found to increase chicken meat antioxidant levels and quality. Furthermore, chickens fed a diet supplemented with EPE had differentially changed cecal microbial compositions when compared to controls. EPE supplementation notably improved the α-diversity of the cecum. The Firmicutes/Bacteroidetes ratio and the relative abundance of Verrucomicrobia at the phylum level were clearly enhanced in the cecum with EPE supplementation (p < 0.05), with the relative abundance of Subdivision 5 genera incertae sedis and Aminivibrio enriched at genus level (p < 0.05). Therefore, these findings indicate that EPE is a good source of natural antioxidants and could be used as antioxidant supplements in animal feed and other foods, contributing to gut health improvement.


Assuntos
Ceco/microbiologia , Galinhas/fisiologia , Eucalyptus/química , Carne , Extratos Vegetais/administração & dosagem , Polifenóis/administração & dosagem , Animais , Antioxidantes/administração & dosagem , Galinhas/microbiologia , Dieta/veterinária , Suplementos Nutricionais , Qualidade dos Alimentos , Microbioma Gastrointestinal/fisiologia , Camundongos , Folhas de Planta/química , Células RAW 264.7
12.
J Biol Chem ; 295(37): 12962-12974, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32675283

RESUMO

Inheritance of the newly assembled flagellum in the human parasite Trypanosoma brucei depends on the faithful duplication and segregation of multiple flagellum-associated cytoskeletal structures, including the hook complex and its associated centrin arm. The biological functions of this unique hook complex-centrin arm assembly remain poorly understood. Here, we report a hook complex-associated protein named BOH2 that plays an essential role in promoting flagellum inheritance. BOH2 localizes to the hooked part of the hook complex by bridging the hook complex, the centrin arm, and the flagellum attachment zone filament. Depletion of BOH2 caused the loss of the shank part of the hook complex and its associated protein TbSmee1, disrupted the assembly of the centrin arm and the recruitment of centrin arm-associated protein CAAP1, inhibited the assembly of the flagellum attachment zone, and caused flagellum mispositioning and detachment. These results demonstrate crucial roles of BOH2 in maintaining hook complex integrity and promoting centrin arm formation and suggest that proper assembly of the hook complex-centrin arm structure facilitates flagellum inheritance.


Assuntos
Flagelos , Proteínas de Protozoários , Trypanosoma brucei brucei , Flagelos/genética , Flagelos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo
13.
Toxicol Lett ; 331: 159-166, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522577

RESUMO

Cadmium, which is extensively distributed in the environment, accumulates in organisms through the trophic chain. Although cadmium can cause bone injury, its role in osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) remains unclear. The present study investigated the effect of cadmium chloride (CdCl2) on osteogenesis of hBMSCs and the underlying mechanism. CdCl2 dose-dependently reduced the viability of hBMSCs. Concentrations of CdCl2 (2.5 and 5.0 µM) increased miR-143-3p levels; decreased levels of adenosine diphosphate-ribosylation factor-like protein 6 (ARL6); inhibited Wnt family member 3A (Wnt3a), ß-catenin, lymphoid enhancer factor (LEF1), and T-cell factor 1 (TCF1); and suppressed osteogenesis of hBMSCs. Inhibition of miR-143-3p or overexpression of ARL6 with lentivirus blocked these CdCl2-induced changes. Luciferase reporter assays confirmed that miR-143-3p binds to the 3'-UTR regions of ARL6 mRNA. Reduced-expression of miR-143-3p enhanced the CdCl2-induced suppression of the osteogenesis of hBMSCs and inhibition of the Wnt/ß-catenin pathway, effects that were reversed by down-regulated expression of ARL6. Thus, miR-143-3p targets ARL6 to down-regulate the Wnt/ß-catenin pathway, which is involved in the suppression of osteogenic differentiation of hBMSCs. The results provide new directions for clinical treatment of bone diseases resulting from cadmium toxicity.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Cádmio/toxicidade , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/genética , Osteogênese/efeitos dos fármacos , Regiões 3' não Traduzidas , Fatores de Ribosilação do ADP/genética , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Via de Sinalização Wnt/efeitos dos fármacos
14.
J Cell Sci ; 133(11)2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393602

RESUMO

The human parasite Trypanosoma brucei transitions from the trypomastigote form to the epimastigote form in the insect vector by repositioning its mitochondrial genome and flagellum-associated cytoskeleton. The molecular mechanisms underlying such changes in cell morphology remain elusive, but recent works demonstrated the involvement of three flagellar proteins, FLAM3, ClpGM6 and KIN-E, in this process by controlling the elongation of the flagellum attachment zone (FAZ). In this report, we identified a FAZ flagellum domain-localizing protein named FAZ27 and characterized its role in cell morphogenesis. Depletion of FAZ27 in the trypomastigote form caused major morphological changes and repositioning of the mitochondrial genome and flagellum-associated cytoskeleton, generating epimastigote-like cells. Furthermore, proximity biotinylation and co-immunoprecipitation identified FLAM3 and ClpGM6 as FAZ27-interacting proteins, and analyses of their functional interplay revealed an interdependency for assembly into the FAZ flagellum domain. Finally, we showed that assembly of FAZ27 occurred proximally, identical to the assembly pattern of other FAZ sub-domain proteins. Taken together, these results demonstrate a crucial role for the FAZ flagellum domain in controlling cell morphogenesis and suggest a coordinated assembly of all the FAZ sub-domains at the proximal end of the FAZ.


Assuntos
Trypanosoma brucei brucei , Citoesqueleto , Flagelos , Humanos , Microtúbulos , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética
15.
J Biol Chem ; 295(3): 729-742, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31819011

RESUMO

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.


Assuntos
Corpos Basais/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Animais , Axonema/química , Axonema/genética , Axonema/metabolismo , Corpos Basais/química , Corpos Basais/parasitologia , Centríolos/química , Centríolos/genética , Centríolos/parasitologia , Flagelos/química , Flagelos/genética , Flagelos/parasitologia , Humanos , Microtúbulos/química , Microtúbulos/parasitologia , Estabilidade Proteica , Proteínas de Protozoários/química , Interferência de RNA , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/patogenicidade
16.
Sensors (Basel) ; 20(1)2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878254

RESUMO

It very important to be able to efficiently detect hydrazine hydrate in an aqueous medium due to its high toxicity. Here, we have proposed a new idea: to construct a sensor for the rapid determination of hydrazine hydrate based on the nano-CuO derived by controlled pyrolysis of HKUST-1 [Cu3(BTC)2(H2O)3]. The as-prepared CuO at 400 °C possesses a uniform appearance with nano-structure via SEM images, and the nano-CuO-400 has exhibited excellent electrocatalytic activity towards hydrazine oxidation. Amperometric i-t curves shows the peak current as linearly proportional to the hydrazine concentration within 1.98-169.3 µmol L-1 and 232-2096 µmol L-1 with the detection limit of 2.55 × 10-8 mol L-1 and 7.01 × 10-8 mol L-1, respectively. Moreover, the sensor constructed in the experiment shows good selectivities, and it is feasible to determining actual water samples.

17.
Mol Microbiol ; 112(6): 1718-1730, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515877

RESUMO

The flagellated eukaryote Trypanosoma brucei alternates between the insect vector and the mammalian host and proliferates through an unusual mode of cell division. Cell division requires flagellum motility-generated forces, but flagellum motility exerts distinct effects between different life cycle forms. Motility is required for the final cell abscission of the procyclic form in the insect vector, but is necessary for the initiation of cell division of the bloodstream form in the mammalian host. The underlying mechanisms remain elusive. Here we carried out functional analyses of a flagellar axonemal inner-arm dynein complex in the bloodstream form and investigated its mechanistic role in cytokinesis initiation. We showed that the axonemal inner-arm dynein heavy chain TbIAD5-1 and TbCentrin3 form a complex, localize to the flagellum, and are required for viability in the bloodstream form. We further demonstrated the interdependence between TbIAD5-1 and TbCentrin3 for maintenance of protein stability. Finally, we showed that depletion of TbIAD5-1 and TbCentrin3 arrested cytokinesis initiation and disrupted the localization of multiple cytokinesis initiation regulators. These findings identified the essential role of an axonemal inner-arm dynein complex in cell division, and provided molecular insights into the flagellum motility-mediated cytokinesis initiation in the bloodstream form of T. brucei.


Assuntos
Dineínas do Axonema/metabolismo , Proteínas Contráteis/metabolismo , Citocinese/fisiologia , Proteínas de Protozoários/metabolismo , Dineínas do Axonema/fisiologia , Axonema/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Movimento Celular , Proteínas Contráteis/genética , Proteínas Contráteis/fisiologia , Dineínas/metabolismo , Dineínas/fisiologia , Flagelos/metabolismo , Flagelos/fisiologia , Estágios do Ciclo de Vida , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Interferência de RNA , Trypanosoma brucei brucei/metabolismo
18.
J Biol Chem ; 294(45): 16672-16683, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31540971

RESUMO

The evolutionarily early divergent human parasite Trypanosoma brucei proliferates through binary cell fission in both its tsetse fly vector and mammalian host. The parasite divides unidirectionally along the longitudinal cell axis from the anterior cell tip toward the posterior cell tip through a mechanism distinct from that in the cells of its human host. Initiation of cytokinesis in T. brucei is regulated by two evolutionarily conserved protein kinases, the Polo-like kinase TbPLK and the Aurora B kinase TbAUK1, and a cohort of trypanosome-specific proteins, including the three cytokinesis initiation factors CIF1, CIF2, and CIF3. Here, using RNAi, in situ epitope tagging of proteins, GST pulldown, and coimmunoprecipitation assays, and immunofluorescence and scanning electron microscopy analyses, we report the identification and functional characterization of two trypanosome-specific proteins, flagellum attachment zone tip-localizing protein required for cytokinesis (FPRC) and CIF4. We found that the two proteins colocalize to the distal tips of the new and the old flagellum attachment zones and are required for cytokinesis initiation. Knockdown of FPRC or CIF4 disrupted the localization of CIF1, suggesting that they function upstream of CIF1. Moreover, depletion of CIF4 abolished FPRC localization, indicating that CIF4 acts upstream of FPRC. Together, these results identify two new cytokinesis regulators in T. brucei and integrate them into the CIF1-mediated cytokinesis regulatory pathway. These findings highlight the existence of a cytokinesis pathway in T. brucei that is different from that of its mammalian host and therefore suggest that cytokinesis in T. brucei could potentially be exploited as a new drug target.


Assuntos
Citocinese/fisiologia , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Ciclo Celular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Interferência de RNA
19.
J Cell Sci ; 132(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31217284

RESUMO

Trypanosoma brucei possesses a motile flagellum that determines cell morphology and the cell division plane. Inheritance of the newly assembled flagellum during the cell cycle is controlled by the Polo-like kinase homolog TbPLK, which also regulates cytokinesis initiation. How TbPLK is targeted to its subcellular locations remains poorly understood. Here we report the trypanosome-specific protein BOH1 that cooperates with TbPLK to regulate flagellum inheritance and cytokinesis initiation in the procyclic form of T. brucei BOH1 localizes to an unusual sub-domain in the flagellum-associated hook complex, bridging the hook complex, the centrin arm and the flagellum attachment zone. Depletion of BOH1 disrupts hook-complex morphology, inhibits centrin-arm elongation and abolishes flagellum attachment zone assembly, leading to flagellum mis-positioning and detachment. Further, BOH1 deficiency impairs the localization of TbPLK and the cytokinesis regulator CIF1 to the cytokinesis initiation site, providing a molecular mechanism for its role in cytokinesis initiation. These findings reveal the roles of BOH1 in maintaining hook-complex morphology and regulating flagellum inheritance, and establish BOH1 as an upstream regulator of the TbPLK-mediated cytokinesis regulatory pathway.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese , Flagelos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Flagelos/ultraestrutura , Técnicas de Silenciamento de Genes , Ligação Proteica , Domínios Proteicos , Proteínas de Protozoários/química , Trypanosoma brucei brucei/ultraestrutura
20.
Nucleic Acids Res ; 47(15): 7973-7988, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31147720

RESUMO

DNA damage-induced cell cycle checkpoints serve as surveillance mechanisms to maintain genomic stability, and are regulated by ATM/ATR-mediated signaling pathways that are conserved from yeast to humans. Trypanosoma brucei, an early divergent microbial eukaryote, lacks key components of the conventional DNA damage-induced G2/M cell cycle checkpoint and the spindle assembly checkpoint, and nothing is known about how T. brucei controls its cell cycle checkpoints. Here we discover a kinetochore-based, DNA damage-induced metaphase checkpoint in T. brucei. MMS-induced DNA damage triggers a metaphase arrest by modulating the abundance of the outer kinetochore protein KKIP5 in an Aurora B kinase- and kinetochore-dependent, but ATM/ATR-independent manner. Overexpression of KKIP5 arrests cells at metaphase through stabilizing the mitotic cyclin CYC6 and the cohesin subunit SCC1, mimicking DNA damage-induced metaphase arrest, whereas depletion of KKIP5 alleviates the DNA damage-induced metaphase arrest and causes chromosome mis-segregation and aneuploidy. These findings suggest that trypanosomes employ a novel DNA damage-induced metaphase checkpoint to maintain genomic integrity.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Dano ao DNA , Genoma de Protozoário/genética , Instabilidade Genômica , Cinetocoros/metabolismo , Trypanosoma/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Humanos , Metáfase , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo
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