RESUMO
Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of any warm-blooded animal. In a previous screen to identify virulence determinants, disruption of gene TgME49_305140 generated a T. gondii mutant that could not establish a chronic infection in mice. The protein product of TgME49_305140, here named TgPL3, is a 277 kDa protein with a patatin-like phospholipase (PLP) domain and a microtubule binding domain. Antibodies generated against TgPL3 show that it is localized to the apical cap. Using a rapid selection FACS-based CRISPR/Cas-9 method, a TgPL3 deletion strain (ΔTgPL3) was generated. ΔTgPL3 parasites have defects in host cell invasion, which may be caused by reduced rhoptry secretion. We generated complementation clones with either wild type TgPL3 or an active site mutation in the PLP domain by converting the catalytic serine to an alanine, ΔTgPL3::TgPL3S1409A (S1409A). Complementation of ΔTgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. ΔTgPL3 and S1409A parasites are also virtually avirulent in vivo but induce a robust antibody response. Vaccination with ΔTgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type I T. gondii parasites, making ΔTgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival of T. gondii during acute mouse infection.
Assuntos
Proteínas de Protozoários/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/metabolismo , Fatores de Virulência/metabolismo , Animais , Camundongos , Fosfolipases/genética , Fosfolipases/metabolismo , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose/enzimologia , VirulênciaRESUMO
Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.
Assuntos
Antiparasitários/farmacologia , Apicoplastos/efeitos dos fármacos , Clemastina/farmacologia , Parasitos/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Animais , Células Cultivadas , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Trypanosomatids are parasitic eukaryotic organisms that cause human disease. These organisms have complex lifestyles; cycling between vertebrate and insect hosts and alternating between two morphologies; a replicating form and an infective, nonreplicating one. Because trypanosomatids are one of the few organisms that do not synthesize the essential cofactor, heme, these parasites sequester the most common form, heme B, from their hosts. Once acquired, the parasites derivatize heme B to heme A by two sequential enzyme reactions. Although heme C is found in many cytochrome c and c1 proteins, heme A is the cofactor of only one known protein, cytochrome c oxidase (CcO). In a recent issue of the Biochemical Journal, Merli et al. [Biochem. J. (2017) 474, 2315-2332] demonstrate that the final step in the synthesis of heme A by heme A synthase (TcCox15) and the subsequent activity of CcO are essential for infectivity and replication of Trypanosoma cruzi.
Assuntos
Heme/química , Parasitos , Animais , Citocromos c , Complexo IV da Cadeia de Transporte de Elétrons , Humanos , Trypanosoma cruziRESUMO
In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the epsilon-amino group of K40 on alpha-tubulin is a conserved PTM on the luminal side of microtubules that was discovered in the flagella of Chlamydomonas reinhardtii. Studies on the significance of microtubule acetylation have been limited by the undefined status of the alpha-tubulin acetyltransferase. Here we show that MEC-17, a protein related to the Gcn5 histone acetyltransferases and required for the function of touch receptor neurons in Caenorhabditis elegans, acts as a K40-specific acetyltransferase for alpha-tubulin. In vitro, MEC-17 exclusively acetylates K40 of alpha-tubulin. Disruption of the Tetrahymena MEC-17 gene phenocopies the K40R alpha-tubulin mutation and makes microtubules more labile. Depletion of MEC-17 in zebrafish produces phenotypes consistent with neuromuscular defects. In C. elegans, MEC-17 and its paralogue W06B11.1 are redundantly required for acetylation of MEC-12 alpha-tubulin, and contribute to the function of touch receptor neurons partly via MEC-12 acetylation and partly via another function, possibly by acetylating another protein. In summary, we identify MEC-17 as an enzyme that acetylates the K40 residue of alpha-tubulin, the only PTM known to occur on the luminal surface of microtubules.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Tubulina (Proteína)/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Acetilação , Animais , Caenorhabditis elegans/metabolismo , Linhagem Celular , Dipodomys , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Tetrahymena/metabolismo , Tato , Tubulina (Proteína)/química , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Toxoplasma gondii is an obligate intracellular parasite that causes serious opportunistic infections, birth defects, and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout the Toxoplasma life cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive "zoites," and motility follows a spiral trajectory that reflects the path of these microtubules. Toxoplasma zoites also construct an intricate, tubulin-based apical structure, termed the conoid, which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Last, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins in Toxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Toxoplasma/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Esquizontes , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade , Tubulina (Proteína)/genéticaRESUMO
Apicomplexa are intracellular parasites that cause important human diseases including malaria and toxoplasmosis. During host cell infection new parasites are formed through a budding process that parcels out nuclei and organelles into multiple daughters. Budding is remarkably flexible in output and can produce two to thousands of progeny cells. How genomes and daughters are counted and coordinated is unknown. Apicomplexa evolved from single celled flagellated algae, but with the exception of the gametes, lack flagella. Here we demonstrate that a structure that in the algal ancestor served as the rootlet of the flagellar basal bodies is required for parasite cell division. Parasite striated fiber assemblins (SFA) polymerize into a dynamic fiber that emerges from the centrosomes immediately after their duplication. The fiber grows in a polarized fashion and daughter cells form at its distal tip. As the daughter cell is further elaborated it remains physically tethered at its apical end, the conoid and polar ring. Genetic experiments in Toxoplasma gondii demonstrate two essential components of the fiber, TgSFA2 and 3. In the absence of either of these proteins cytokinesis is blocked at its earliest point, the initiation of the daughter microtubule organizing center (MTOC). Mitosis remains unimpeded and mutant cells accumulate numerous nuclei but fail to form daughter cells. The SFA fiber provides a robust spatial and temporal organizer of parasite cell division, a process that appears hard-wired to the centrosome by multiple tethers. Our findings have broader evolutionary implications. We propose that Apicomplexa abandoned flagella for most stages yet retained the organizing principle of the flagellar MTOC. Instead of ensuring appropriate numbers of flagella, the system now positions the apical invasion complexes. This suggests that elements of the invasion apparatus may be derived from flagella or flagellum associated structures.
Assuntos
Divisão Celular , Eucariotos/metabolismo , Flagelos/metabolismo , Parasitos/citologia , Toxoplasma/citologia , Animais , Polaridade Celular , Centrossomo/metabolismo , Flagelos/ultraestrutura , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Modelos Biológicos , Parasitos/ultraestrutura , Proteínas de Protozoários/metabolismo , Toxoplasma/ultraestruturaRESUMO
A mild protocol for the synthesis of diaryl and heteroaryl sulfides is described. In a one-pot procedure, thiols are converted to sulfenyl chlorides and reacted with arylzinc reagents. This method tolerates functional groups including aryl fluorides and chlorides, ketones, as well as N-heterocycles including pyrimidines, imidazoles, tetrazoles, and oxadiazoles. Two compounds synthesized by this method exhibited selective activity against the MCF-7 breast cancer cell line in the micromolar range.
Assuntos
Antineoplásicos/síntese química , Cloretos/química , Imidazóis/química , Cetonas/química , Células MCF-7/química , Células MCF-7/efeitos dos fármacos , Ácidos Sulfênicos/síntese química , Compostos de Sulfidrila/química , Sulfetos/síntese química , Antineoplásicos/química , Catálise , Humanos , Estrutura Molecular , Sulfetos/químicaRESUMO
SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.
Assuntos
Evolução Biológica , Flagelos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Citoesqueleto de Actina/metabolismo , Axonema/metabolismo , Axonema/ultraestrutura , Cílios/metabolismo , Flagelos/ultraestrutura , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/ultraestruturaRESUMO
Alkyl Grignard reagents that contain ß-hydrogen atoms were used in a stereospecific nickel-catalyzed cross-coupling reaction to form C(sp(3))-C(sp(3)) bonds. Aryl Grignard reagents were also utilized to synthesize 1,1-diarylalkanes. Several compounds synthesized by this method exhibited selective inhibition of proliferation of MCF-7 breast cancer cells.
Assuntos
Alcanos/síntese química , Alcanos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Níquel/química , Alcanos/química , Antineoplásicos/química , Catálise , Linhagem Celular Tumoral , Feminino , Humanos , Hidrocarbonetos Aromáticos/síntese química , Hidrocarbonetos Aromáticos/química , Hidrocarbonetos Aromáticos/farmacologia , Indicadores e Reagentes , EstereoisomerismoRESUMO
Protozoan parasites cause life-threatening infections in both humans and animals, including agriculturally significant livestock. Available treatments are typically narrow spectrum and are complicated by drug toxicity and the development of resistant parasites. Protozoan tubulin is an attractive target for the development of broad-spectrum antimitotic agents. The Medicines for Malaria Pathogen Box compound MMV676477 was previously shown to inhibit replication of kinetoplastid parasites, such as Leishmania amazonensis and Trypanosoma brucei, and the apicomplexan parasite Plasmodium falciparum by selectively stabilizing protozoan microtubules. In this report, we show that MMV676477 inhibits intracellular growth of the human apicomplexan pathogen Toxoplasma gondii with an EC50 value of ~50 nM. MMV676477 does not stabilize vertebrate microtubules or cause other toxic effects in human fibroblasts. The availability of tools for genetic studies makes Toxoplasma a useful model for studies of the cytoskeleton. We conducted a forward genetics screen for MMV676477 resistance, anticipating that missense mutations would delineate the binding site on protozoan tubulin. Unfortunately, we were unable to use genetics to dissect target interactions because no resistant parasites emerged. This outcome suggests that future drugs based on the MMV676477 scaffold would be less likely to be undermined by the emergence of drug resistance.
RESUMO
We have identified two novel proteins that colocalize with the subpellicular microtubules in the protozoan parasite Toxoplasma gondii and named these proteins SPM1 and SPM2. These proteins have basic isoelectric points and both have homologs in other apicomplexan parasites. SPM1 contains six tandem copies of a 32-amino-acid repeat, whereas SPM2 lacks defined protein signatures. Alignment of Toxoplasma SPM2 with apparent Plasmodium SPM2 homologs indicates that the greatest degree of conservation lies in the carboxy-terminal half of the protein. Analysis of Plasmodium homologs of SPM1 indicates that while the central 32-amino-acid repeats have expanded to different degrees (7, 8, 9, 12, or 13 repeats), the amino- and carboxy-terminal regions remain conserved. In contrast, although the Cryptosporidium SPM1 homolog has a conserved carboxy tail, the five repeats are considerably diverged, and it has a smaller amino-terminal domain. SPM1 is localized along the full length of the subpellicular microtubules but does not associate with the conoid or spindle microtubules. SPM2 has a restricted localization along the middle region of the subpellicular microtubules. Domain deletion analysis indicates that four or more copies of the SPM1 repeat are required for localization to microtubules, and the amino-terminal 63 residues of SPM2 are required for localization to the subpellicular microtubules. Gene deletion studies indicate that neither SPM1 nor SPM2 is essential for tachyzoite viability. However, loss of SPM1 decreases overall parasite fitness and eliminates the stability of subpellicular microtubules to detergent extraction.
Assuntos
Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Humanos , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Alinhamento de Sequência , Sequências de Repetição em Tandem , Tubulina (Proteína)/metabolismoRESUMO
Microtubules and specialized microtubule-containing structures are assembled from tubulins, an ancient superfamily of essential eukaryotic proteins. Here, we use bioinformatic approaches to analyze features of tubulins in organisms from the phylum Apicomplexa. Apicomplexans are protozoan parasites that cause a variety of human and animal infectious diseases. Individual species harbor one to four genes each for α- and ß-tubulin isotypes. These may specify highly similar proteins, suggesting functional redundancy, or exhibit key differences, consistent with specialized roles. Some, but not all apicomplexans harbor genes for δ- and ε-tubulins, which are found in organisms that construct appendage-containing basal bodies. Critical roles for apicomplexan δ- and ε-tubulin are likely to be limited to microgametes, consistent with a restricted requirement for flagella in a single developmental stage. Sequence divergence or the loss of δ- and ε-tubulin genes in other apicomplexans appears to be associated with diminished requirements for centrioles, basal bodies, and axonemes. Finally, because spindle microtubules and flagellar structures have been proposed as targets for anti-parasitic therapies and transmission-blocking strategies, we discuss these ideas in the context of tubulin-based structures and tubulin superfamily properties.
RESUMO
Microtubules are polymeric filaments, constructed of α-ß tubulin heterodimers that underlie critical subcellular structures in eukaryotic organisms. Four homologous proteins (γ-, δ-, ε- and ζ-tubulin) additionally contribute to specialized microtubule functions. Although there is an immense volume of publicly available data pertaining to tubulins, it is difficult to assimilate all potentially relevant information across diverse organisms, isotypes, and categories of data. We previously assembled an extensive web-based catalogue of published missense mutations to tubulins with >1,500 entries that each document a specific substitution to a discrete tubulin, the species where the mutation was described and the associated phenotype with hyperlinks to the amino acid sequence and citation(s) for research. This report describes a significant update and expansion of our online resource (TubulinDB.bio.uci.edu) to nearly 18,000 entries. It now encompasses a cross-referenced catalog of post-translational modifications (PTMs) to tubulin drawn from public datasets, primary literature, and predictive algorithms. In addition, tubulin protein structures were used to define local interactions with bound ligands (GTP, GDP and diverse microtubule-targeting agents) and amino acids at the intradimer interface, within the microtubule lattice and with associated proteins. To effectively cross-reference these datasets, we established a universal tubulin numbering system to map entries into a common framework that accommodates specific insertions and deletions to tubulins. Indexing and cross-referencing permitted us to discern previously unappreciated patterns. We describe previously unlinked observations of loss of PTM sites in the context of cancer cells and tubulinopathies. Similarly, we expanded the set of clinical substitutions that may compromise MAP or microtubule-motor interactions by collecting tubulin missense mutations that alter amino acids at the interface with dynein and doublecortin. By expanding the database as a curated resource, we hope to relate model organism data to clinical findings of pathogenic tubulin variants. Ultimately, we aim to aid researchers in hypothesis generation and design of studies to dissect tubulin function.
Assuntos
Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Citoesqueleto/metabolismo , Mutação , Ligantes , Aminoácidos/metabolismoRESUMO
Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.
Assuntos
Divisão Celular , Membrana Celular/metabolismo , Fibroblastos/parasitologia , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células Cultivadas , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Prepúcio do Pênis/parasitologia , Humanos , Imunização , Imunoglobulina G/imunologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Toxoplasmose/genética , Toxoplasmose/parasitologiaRESUMO
Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogen-derived material from the host cell's periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Microtúbulos/parasitologia , Toxoplasma/patogenicidade , Sequência de Aminoácidos , Animais , Antígenos CD59/genética , Antígenos CD59/fisiologia , Linhagem Celular , Citoesqueleto/parasitologia , Citoesqueleto/fisiologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Dados de Sequência Molecular , Nocodazol/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tromboplastina/genética , Tromboplastina/fisiologia , Virulência/fisiologiaRESUMO
Plant and protozoan microtubules are selectively sensitive to dinitroanilines, which do not disrupt vertebrate or fungal microtubules. Tetrahymena thermophila is an abundant source of dinitroaniline-sensitive tubulin, and we have modified the single T. thermophila α-tubulin gene to create strains that solely express mutant α-tubulin in functional dimers. Previous research identified multiple α-tubulin mutations that confer dinitroaniline resistance in the human parasite Toxoplasma gondii, and when two of these mutations (L136F and I252L) were introduced into T. thermophila, they conferred resistance in these free-living ciliates. Purified tubulin heterodimers composed of L136F or I252L α-tubulin display decreased affinity for the dinitroaniline oryzalin relative to wild-type T. thermophila tubulin. Moreover, the L136F substitution dramatically reduces the critical concentration for microtubule assembly relative to the properties of wild-type T. thermophila tubulin. Our data provide additional support for the proposed dinitroaniline binding site on α-tubulin and validate the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.
Assuntos
Coccidiostáticos/farmacologia , Dinitrobenzenos/farmacologia , Microtúbulos/metabolismo , Mutação , Proteínas de Protozoários/genética , Sulfanilamidas/farmacologia , Tetrahymena thermophila/efeitos dos fármacos , Tetrahymena thermophila/metabolismo , Tubulina (Proteína)/genética , Sítios de Ligação , Microtúbulos/genética , Ligação Proteica , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/genética , Tubulina (Proteína)/metabolismoRESUMO
Fas-associated death domain protein (FADD) and caspase-8 (casp8) are vital intermediaries in apoptotic signaling induced by tumor necrosis factor family ligands. Paradoxically, lymphocytes lacking FADD or casp8 fail to undergo normal clonal expansion following antigen receptor cross-linking and succumb to caspase-independent cell death upon activation. Here we show that T cells lacking FADD or casp8 activity are subject to hyperactive autophagic signaling and subvert a cellular survival mechanism into a potent death process. T cell autophagy, enhanced by mitogenic signaling, recruits casp8 through interaction with FADD:Atg5-Atg12 complexes. Inhibition of autophagic signaling with 3-methyladenine, dominant-negative Vps34, or Atg7 shRNA rescued T cells expressing a dominant-negative FADD protein. The necroptosis inhibitor Nec-1, which blocks receptor interacting protein kinase 1 (RIP kinase 1), also completely rescued T cells lacking FADD or casp8 activity. Thus, while autophagy is necessary for rapid T cell proliferation, our findings suggest that FADD and casp8 form a feedback loop to limit autophagy and prevent this salvage pathway from inducing RIPK1-dependent necroptotic cell death. Thus, linkage of FADD and casp8 to autophagic signaling intermediates is essential for rapid T cell clonal expansion and may normally serve to promote caspase-dependent apoptosis under hyperautophagic conditions, thereby averting necrosis and inflammation in vivo.
Assuntos
Autofagia , Caspase 8/fisiologia , Proliferação de Células , Proteína de Domínio de Morte Associada a Fas/fisiologia , Linfócitos T/citologia , Animais , Apoptose , Caspase 8/genética , Proteína de Domínio de Morte Associada a Fas/genética , Retroalimentação Fisiológica , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Transdução de Sinais/imunologiaRESUMO
Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated auranofin resistant T. gondii lines through chemical mutagenesis to identify the molecular target of this drug. Resistant clones were confirmed with a competition assay using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal a consensus resistance locus, although many have point mutations in genes encoding redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient to confer resistance when introduced into wild-type parasites. Resistant clones accumulated less ROS than their wild type counterparts. Our results demonstrate that resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin anti-parasitic activity as disruption of redox homeostasis.
Assuntos
Parasitos , Toxoplasma , Animais , Auranofina/farmacologia , Espécies Reativas de Oxigênio , Tiorredoxina Dissulfeto Redutase/genética , Toxoplasma/genéticaRESUMO
Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand-binding sites in tubulin makes this protein an attractive target for anti-parasite drug discovery. However, despite remarkable successes as anti-cancer agents, the rational development of protozoan parasite-specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug-binding sites. Based on this information, we rationally designed the parasite-specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species-specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much-needed, novel parasite inhibitors.
Assuntos
Antiparasitários , Parasitos , Animais , Antiparasitários/farmacologia , Sítios de Ligação , Proliferação de Células , Humanos , Microtúbulos/metabolismo , Parasitos/metabolismo , Tubulina (Proteína) , Moduladores de Tubulina/farmacologiaRESUMO
The human parasite Toxoplasma gondii is sensitive to dinitroaniline compounds which selectively disrupt microtubules in diverse protozoa but which have no detectable effect on vertebrate host cell microtubules or other functions. Replication of wild-type T. gondii is inhibited by 0.5 to 2.5 microM oryzalin, but mutant parasites harboring amino acid substitutions in the predicted dinitroaniline binding site confer resistance up to 40 microM oryzalin. However, the precise interaction between dinitroanilines and the binding site in alpha-tubulin remains unclear. We have investigated the activity of 12 dinitroanilines and the related compound amiprophos methyl on wild-type and dinitroaniline-resistant parasite lines that contain proposed binding site mutations. These data indicate that dinitramine is the most effective dinitroaniline to inhibit Toxoplasma growth in wild-type parasites and most resistant lines. Dinitramine has an amine group at the meta position not present in any of the other dinitroanilines tested here that is predicted to form hydrogen bonds with residues Arg2 and Gln133 according to docking data. Remarkably, although the binding site mutation Ile235Val confers increased resistance to most dinitroanilines, it confers increased sensitivity to GB-II-5, a compound optimized for activity against kinetoplastid tubulin. Kinetoplastid parasites have a valine at position 235 of alpha-tubulin, whereas apicomplexan parasites have an isoleucine at this site. We suggest that this heterogeneity in binding site environment influences relative dinitroaniline sensitivity in distinct protozoan lineages and hypothesize that a mutation that makes the apicomplexan dinitroaniline binding site more like the kinetoplastid site increases sensitivity to a dinitroaniline optimized for activity in the latter parasites.