RESUMO
Acetyl-CoA participates in post-translational modification of proteins and in central carbon and lipid metabolism in several cell compartments. In mammals, acetyl-CoA transporter 1 (AT1, also known as SLC33A1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with the activity of dedicated acetyltransferases such as NAT8. However, the involvement of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. Here, we identified homologs of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei parasites. Proteome-wide analyses revealed widespread N-terminal acetylation of secreted proteins in both species. Such extensive acetylation of N-terminally processed proteins has not been observed previously in any other organism. Deletion of AT1 homologs in both T. gondii and P. berghei resulted in considerable reductions in parasite fitness. In P. berghei, AT1 was found to be important for growth of asexual blood stages, production of female gametocytes and male gametocytogenesis, implying its requirement for parasite transmission. In the absence of AT1, lysine acetylation and N-terminal acetylation in T. gondii remained globally unaltered, suggesting an uncoupling between the role of AT1 in development and active acetylation occurring along the secretory pathway.
Assuntos
Parasitos , Toxoplasma , Acetilcoenzima A/metabolismo , Acetilação , Animais , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Mamíferos/metabolismo , Parasitos/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
BACKGROUND: BMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension. METHODS: To get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry. RESULTS: Large-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45ß. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1. CONCLUSIONS: Overall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.
Assuntos
Proteínas Morfogenéticas Ósseas , Células Endoteliais , Pontos de Checagem do Ciclo Celular , Fosforilação , Pontos de Checagem da Fase G1 do Ciclo CelularRESUMO
Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.
Assuntos
Carcinoma Hepatocelular/virologia , Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Neoplasias Hepáticas/virologia , Proteínas Repressoras/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas de Ciclo Celular/genética , Vírus da Hepatite B/genética , Hepatócitos/virologia , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteômica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Fatores de Processamento de Serina-Arginina/genética , Proteínas do Core Viral/genética , Replicação ViralRESUMO
The general perception of viruses is that they are small in terms of size and genome, and that they hijack the host machinery to glycosylate their capsid. Giant viruses subvert all these concepts: their particles are not small, and their genome is more complex than that of some bacteria. Regarding glycosylation, this concept has been already challenged by the finding that Chloroviruses have an autonomous glycosylation machinery that produces oligosaccharides similar in size to those of small viruses (6-12 units), albeit different in structure compared to the viral counterparts. We report herein that Mimivirus possesses a glycocalyx made of two different polysaccharides, now challenging the concept that all viruses coat their capsids with oligosaccharides of discrete size. This discovery contradicts the paradigm that such macromolecules are absent in viruses, blurring the boundaries between giant viruses and the cellular world and opening new avenues in the field of viral glycobiology.
Assuntos
Mimiviridae/metabolismo , Polissacarídeos/biossíntese , Glicosilação , Mimiviridae/química , Polissacarídeos/químicaRESUMO
Type 2 diabetic cardiomyopathy features Ca2+ signaling abnormalities, notably an altered mitochondrial Ca2+ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca2+ homeostasis, the reticulum-mitochondrial Ca2+ coupling, and/or the mitochondrial Ca2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum-mitochondria interface revealed tighter interactions not compatible with Ca2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca2+ sensors were performed to measure Ca2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R-VDAC interaction and a reduced IP3-stimulated Ca2+ transfer to mitochondria, with no changes in reticular Ca2+ level, cytosolic Ca2+ transients, and mitochondrial Ca2+ uniporter function. Disruption of organelle Ca2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum-mitochondria Ca2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Canais de Cálcio/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/dietoterapia , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Dieta Hiperlipídica , Sacarose Alimentar , Retículo Endoplasmático/patologia , Metabolismo Energético , Acoplamento Excitação-Contração , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Resistência à Insulina , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/patologia , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/patologia , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
Bone morphogenetic protein 9 (BMP9) is a circulating factor produced by hepatic stellate cells that plays a critical role in vascular quiescence through its endothelial receptor activin receptor-like kinase 1 (ALK1). Mutations in the gene encoding ALK1 cause hereditary hemorrhagic telangiectasia type 2, a rare genetic disease presenting hepatic vessel malformations. Variations of both the circulating levels and the hepatic mRNA levels of BMP9 have been recently associated with various forms of hepatic fibrosis. However, the molecular mechanism that links BMP9 with liver diseases is still unknown. Here, we report that Bmp9 gene deletion in 129/Ola mice triggers hepatic perisinusoidal fibrosis that was detectable from 15 weeks of age. An inflammatory response appeared within the same time frame as fibrosis, whereas sinusoidal vessel dilation developed later on. Proteomic and mRNA analyses of primary liver sinusoidal endothelial cells (LSECs) both revealed that the expression of the LSEC-specifying transcription factor GATA-binding protein 4 was strongly reduced in Bmp9 gene knockout (Bmp9-KO) mice as compared with wild-type mice. LSECs from Bmp9-KO mice also lost the expression of several terminal differentiation markers (Lyve1, Stab1, Stab2, Ehd3, Cd209b, eNos, Maf, Plvap). They gained CD34 expression and deposited a basal lamina, indicating that they were capillarized. Another main characteristic of differentiated LSECs is the presence of permeable fenestrae. LSECs from Bmp9-KO mice had a significantly reduced number of fenestrae. This was already observable in 2-week-old pups. Moreover, we could show that addition of BMP9 to primary cultures of LSECs prevented the loss of their fenestrae and maintained the expression levels of Gata4 and Plvap. Conclusion: Taken together, our observations show that BMP9 is a key paracrine regulator of liver homeostasis, controlling LSEC fenestration and protecting against perivascular hepatic fibrosis.
Assuntos
Receptores de Activinas Tipo II/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Fator 2 de Diferenciação de Crescimento/genética , Cirrose Hepática/genética , Cirrose Hepática/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/citologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteômica , RNA Mensageiro/genética , Distribuição Aleatória , Estatísticas não Paramétricas , Técnicas de Cultura de Tecidos/métodosRESUMO
Generation of dimers, trimers and dendrimers of bioactive compounds is an approach that has recently been developed for the discovery of new potent drug candidates. Herein, we present the synthesis of new artemisinin-derived dimers and dendrimers and investigate their action against malaria parasite Plasmodium falciparum 3D7 strain and human cytomegalovirus (HCMV). Dimer 7 was the most active compound (EC50 1.4â nm) in terms of antimalarial efficacy and was even more effective than the standard drugs dihydroartemisinin (EC50 2.4â nm), artesunic acid (EC50 8.9â nm) and chloroquine (EC50 9.8â nm). Trimer 4 stood out as the most active agent against HCMV in vitro replication and exerted an EC50 value of 0.026â µm, representing an even higher activity than the two reference drugs ganciclovir (EC50 2.60â µm) and artesunic acid (EC50 5.41â µm). In addition, artemisinin-derived dimer 13 and trimer 15 were for the first time both immobilized on TOYOPEARL AF-Amino-650M beads and used for mass spectrometry-based target identification experiments using total lysates of HCMV-infected primary human fibroblasts. Two major groups of novel target candidates, namely cytoskeletal and mitochondrial proteins were obtained. Two putatively compound-binding viral proteins, namely major capsid protein (MCP) and envelope glycoprotein pUL132, which are both essential for HCMV replication, were identified.
Assuntos
Antimaláricos/farmacologia , Antivirais/farmacologia , Artemisininas/síntese química , Citomegalovirus/efeitos dos fármacos , Dendrímeros/farmacologia , Succinatos/farmacologia , Antimaláricos/química , Antivirais/química , Artemisininas/química , Artemisininas/farmacologia , Citomegalovirus/química , Dendrímeros/química , Humanos , Succinatos/químicaRESUMO
The phospholipid (PL) composition of bacterial membranes varies as a function of growth rate and in response to changes in the environment. While growth adaptation can be explained by biochemical feedback in the PL synthesis pathway, recent transcriptome studies have revealed that the expression of PL synthesis genes can also be tuned in response to various stresses. We previously showed that the BasRS two-component pathway controls the expression of the diacylglycerol kinase gene, dgkA, in Escherichia coli (A. Wahl, L. My, R. Dumoulin, J. N. Sturgis, and E. Bouveret, Mol Microbiol, 80:1260-1275, 2011, https://doi.org/10.1111/j.1365-2958.2011.07641.x). In this study, we set up a strategy to identify the mutation responsible for the upregulation of pssA observed in the historical pssR1 mutant and supposedly corresponding to a transcriptional repressor (C. P. Sparrow and J. Raetz, J Biol Chem, 258:9963-9967, 1983). pssA encodes phosphatidylserine synthase, the first step of phosphatidylethanolamine synthesis. We showed that this mutation corresponded to a single nucleotide change in the anti-Shine-Dalgarno sequence of the 16S rRNA encoded by the rrnC operon. We further demonstrated that this mutation enhanced the translation of pssA Though this effect appeared to be restricted to PssA among phospholipid synthesis enzymes, it was not specific, as evidenced by a global effect on the production of unrelated proteins.IMPORTANCE Bacteria adjust the phospholipid composition of their membranes to the changing environment. In addition to enzymatic regulation, stress response regulators control specific steps of the phospholipid synthesis pathway. We wanted to identify a potential regulator controlling the expression of the phosphatidylserine synthase gene. We showed that it was not the previously suggested hdfR gene and instead that a mutation in the anti-Shine-Dalgarno sequence of 16S RNA was responsible for an increase in pssA translation. This example underlines the fact that gene expression can be modulated by means other than specific regulatory processes.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfolipídeos/biossíntese , Ribossomos/metabolismo , Fatores de Transcrição/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Ribossomos/genética , Fatores de Transcrição/genéticaRESUMO
Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.
RESUMO
The mimivirus 1.2 Mb genome was shown to be organized into a nucleocapsid-like genomic fiber encased in the nucleoid compartment inside the icosahedral capsid. The genomic fiber protein shell is composed of a mixture of two GMC-oxidoreductase paralogs, one of them being the main component of the glycosylated layer of fibrils at the surface of the virion. In this study, we determined the effect of the deletion of each of the corresponding genes on the genomic fiber and the layer of surface fibrils. First, we deleted the GMC-oxidoreductase, the most abundant in the genomic fiber, and determined its structure and composition in the mutant. As expected, it was composed of the second GMC-oxidoreductase and contained 5- and 6-start helices similar to the wild-type fiber. This result led us to propose a model explaining their coexistence. Then we deleted the GMC-oxidoreductase, the most abundant in the layer of fibrils, to analyze its protein composition in the mutant. Second, we showed that the fitness of single mutants and the double mutant were not decreased compared with the wild-type viruses under laboratory conditions. Third, we determined that deleting the GMC-oxidoreductase genes did not impact the glycosylation or the glycan composition of the layer of surface fibrils, despite modifying their protein composition. Because the glycosylation machinery and glycan composition of members of different clades are different, we expanded the analysis of the protein composition of the layer of fibrils to members of the B and C clades and showed that it was different among the three clades and even among isolates within the same clade. Taken together, the results obtained on two distinct central processes (genome packaging and virion coating) illustrate an unexpected functional redundancy in members of the family Mimiviridae, suggesting this may be the major evolutionary force behind their giant genomes.
RESUMO
Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cell dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cell ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulate lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation lose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo. Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.
RESUMO
IFITMs are a family of highly related interferon-induced transmembrane proteins that interfere with the processes of fusion between viral and cellular membranes and are thus endowed with broad antiviral properties. A number of studies have shown how the antiviral potency of IFITMs is highly dependent on their steady-state levels, their intracellular distribution and a complex pattern of post-translational modifications, parameters that are overall tributary of a number of cellular partners. In an effort to identify additional protein partners involved in the biology of IFITMs, we devised a proteomics-based approach based on the piggyback incorporation of IFITM3 partners into extracellular vesicles. MS analysis of the proteome of vesicles bearing or not bearing IFITM3 identified the NDFIP2 protein adaptor protein as an important regulator of IFITM3 levels. NDFIP2 is a membrane-anchored adaptor protein of the E3 ubiquitin ligases of the NEDD4 family that have already been found to be involved in IFITM3 regulation. We show here that NDFIP2 acts as a recruitment factor for both IFITM3 and NEDD4 and mediates their distribution in lysosomal vesicles. The genetic inactivation and overexpression of NDFIP2 drive, respectively, lower and higher levels of IFITM3 accumulation in the cell, overall suggesting that NDFIP2 locally competes with IFITM3 for NEDD4 binding. Given that NDFIP2 is itself tightly regulated and highly responsive to external cues, our study sheds light on a novel and likely dynamic layer of regulation of IFITM3.
Assuntos
Proteômica , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Processamento de Proteína Pós-Traducional , Lisossomos/metabolismo , Antivirais/metabolismoRESUMO
Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Lignina , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Processamento de Proteína Pós-Traducional , Parede Celular/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cells dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cells ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulates lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation loose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo . Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.
RESUMO
Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.
Assuntos
Vírus Gigantes , Mimiviridae , Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Genoma Viral , Vírus Gigantes/genética , Mimiviridae/genética , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Oxirredutases/metabolismoRESUMO
Upon pathogen detection, macrophages normally stay sessile in tissues while dendritic cells (DCs) migrate to secondary lymphoid tissues. The obligate intracellular protozoan Toxoplasma gondii exploits the trafficking of mononuclear phagocytes for dissemination via unclear mechanisms. We report that, upon T. gondii infection, macrophages initiate the expression of transcription factors normally attributed to DCs, upregulate CCR7 expression with a chemotactic response, and perform systemic migration when adoptively transferred into mice. We show that parasite effector GRA28, released by the MYR1 secretory pathway, cooperates with host chromatin remodelers in the host cell nucleus to drive the chemotactic migration of parasitized macrophages. During in vivo challenge studies, bone marrow-derived macrophages infected with wild-type T. gondii outcompeted those challenged with MYR1- or GRA28-deficient strains in migrating and reaching secondary organs. This work reveals how an intracellular parasite hijacks chemotaxis in phagocytes and highlights a remarkable migratory plasticity in differentiated cells of the mononuclear phagocyte system.
Assuntos
Parasitos , Toxoplasma , Camundongos , Animais , Toxoplasma/fisiologia , Células Dendríticas/fisiologia , Movimento Celular , MacrófagosRESUMO
The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include Toxoplasma gondii, Plasmodium, and Cryptosporidium spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against T. gondii. We have identified TgPRP4K of T. gondii as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for TgPRP4K and for its Plasmodium falciparum counterpart, PfCLK3. Our data identified structural features critical for binding of the other PfCLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum. Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.
Assuntos
Criptosporidiose , Cryptosporidium , Malária Falciparum , Toxoplasma , Inibidores da Angiogênese/uso terapêutico , Animais , Humanos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum , Inibidores de Proteínas Quinases/farmacologia , Spliceossomos , Toxoplasma/genéticaRESUMO
Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.
Assuntos
Genes Controladores do Desenvolvimento , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Poliadenilação , Toxoplasma/metabolismo , Transcriptoma , Arabidopsis/genética , Sítios de Ligação , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Regulação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/química , Metiltransferases/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Fatores de Processamento de RNA/química , RNA Mensageiro/metabolismo , Leitura , Análise de Sequência de RNA , Dedos de ZincoRESUMO
Toxoplasma gondii has a complex life cycle that is typified by asexual development that takes place in vertebrates, and sexual reproduction, which occurs exclusively in felids and is therefore less studied. The developmental transitions rely on changes in the patterns of gene expression, and recent studies have assigned roles for chromatin shapers, including histone modifications, in establishing specific epigenetic programs for each given stage. Here, we identified the T. gondii microrchidia (MORC) protein as an upstream transcriptional repressor of sexual commitment. MORC, in a complex with Apetala 2 (AP2) transcription factors, was shown to recruit the histone deacetylase HDAC3, thereby impeding the accessibility of chromatin at the genes that are exclusively expressed during sexual stages. We found that MORC-depleted cells underwent marked transcriptional changes, resulting in the expression of a specific repertoire of genes, and revealing a shift from asexual proliferation to sexual differentiation. MORC acts as a master regulator that directs the hierarchical expression of secondary AP2 transcription factors, and these transcription factors potentially contribute to the unidirectionality of the life cycle. Thus, MORC plays a cardinal role in the T. gondii life cycle, and its conditional depletion offers a method to study the sexual development of the parasite in vitro, and is proposed as an alternative to the requirement of T. gondii infections in cats.
Assuntos
Adenosina Trifosfatases/genética , Histona Desacetilases/genética , Histonas/metabolismo , Proteínas de Protozoários/genética , Toxoplasma/genética , Fatores de Transcrição/genética , Transcrição Gênica , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Animais , Gatos , Cromatina , Fibroblastos/parasitologia , Código das Histonas , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Histonas/genética , Humanos , Estágios do Ciclo de Vida/genética , Modelos Moleculares , Cultura Primária de Células , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Wilson's disease (WD), a rare genetic disease caused by mutations in the ATP7B gene, is associated with altered expression and/or function of the copper-transporting ATP7B protein, leading to massive toxic accumulation of copper in the liver and brain. The Atp7b-/- mouse, a genetic and phenotypic model of WD, was developed to provide new insights into the pathogenic mechanisms of WD. Many plasma proteins are secreted by the liver, and impairment of liver function can trigger changes to the plasma proteome. High standard proteomics workflows can identify such changes. Here, we explored the plasma proteome of the Atp7b-/- mouse using a mass spectrometry (MS)-based proteomics workflow combining unbiased discovery analysis followed by targeted quantification. Among the 367 unique plasma proteins identified, 7 proteins were confirmed as differentially abundant between Atp7b-/- mice and wild-type littermates, and were directly linked to WD pathophysiology (regeneration of liver parenchyma, plasma iron depletion, etc.). We then adapted our targeted proteomics assay to quantify human orthologues of these proteins in plasma from copper-chelator-treated WD patients. The plasma proteome changes observed in the Atp7b-/- mouse were not confirmed in these samples, except for alpha-1 antichymotrypsin, levels of which were decreased in WD patients compared to healthy individuals. Plasma ceruloplasmin was investigated in both the Atp7b-/- mouse model and human patients; it was significantly decreased in the human form of WD only. In conclusion, MS-based proteomics is a method of choice to identify proteome changes in murine models of disrupted metal homeostasis, and allows their validation in human cohorts.