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1.
PLoS Biol ; 17(10): e3000475, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584943

RESUMO

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite's plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein-protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein-protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.


Assuntos
Azidas/química , Reagentes de Ligações Cruzadas/química , Proteínas do Citoesqueleto/química , Citoesqueleto/metabolismo , Membranas Intracelulares/metabolismo , Fenilalanina/análogos & derivados , Proteínas de Protozoários/química , Toxoplasma/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Expressão Gênica , Membranas Intracelulares/ultraestrutura , Fenilalanina/química , Processos Fotoquímicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/ultraestrutura , Raios Ultravioleta
2.
PLoS One ; 15(5): e0232552, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374791

RESUMO

Toxoplasma gondii is an obligate intracellular parasite which is capable of establishing life-long chronic infection in any mammalian host. During the intracellular life cycle, the parasite secretes an array of proteins into the parasitophorous vacuole (PV) where it resides. Specialized organelles called the dense granules secrete GRA proteins that are known to participate in nutrient acquisition, immune evasion, and host cell-cycle manipulation. Although many GRAs have been discovered which are expressed during the acute infection mediated by tachyzoites, little is known about those that participate in the chronic infection mediated by the bradyzoite form of the parasite. In this study, we sought to uncover novel bradyzoite-upregulated GRA proteins using proximity biotinylation, which we previously used to examine the secreted proteome of the tachyzoites. Using a fusion of the bradyzoite upregulated protein MAG1 to BirA* as bait and a strain with improved switch efficiency, we identified a number of novel GRA proteins which are expressed in bradyzoites. After using the CRISPR/Cas9 system to characterize these proteins by gene knockout, we focused on one of these GRAs (GRA55) and found it was important for the establishment or maintenance of cysts in the mouse brain. These findings highlight new components of the GRA proteome of the tissue-cyst life stage of T. gondii and identify potential targets that are important for maintenance of parasite persistence in vivo.


Assuntos
Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Animais , Biotinilação , Encéfalo/metabolismo , Encéfalo/parasitologia , Sistemas CRISPR-Cas , Feminino , Técnicas de Inativação de Genes , Genes de Protozoários , Humanos , Estágios do Ciclo de Vida , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/metabolismo , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose Animal/metabolismo , Toxoplasmose Animal/parasitologia , Toxoplasmose Cerebral/metabolismo , Toxoplasmose Cerebral/parasitologia , Vacúolos/metabolismo , Virulência
3.
Mol Cell Biol ; 37(11)2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28289076

RESUMO

Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.


Assuntos
Carcinoma de Células Renais/patologia , Ferritinas/genética , Fumarato Hidratase/metabolismo , Fumaratos/farmacologia , Neoplasias Renais/patologia , Leiomiomatose/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Sequência de Aminoácidos , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Forkhead Box M1/metabolismo , Humanos , Espaço Intracelular/metabolismo , Proteína 2 Reguladora do Ferro/química , Proteína 2 Reguladora do Ferro/metabolismo , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Leiomiomatose/enzimologia , Leiomiomatose/genética , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido Succínico/metabolismo
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