RESUMEN
Recombinant SARS-CoV-2 S protein expression was examined in Vero cells by imaging using the human monoclonal antibody panel (PD4, PD5, sc23, and sc29). The PD4 and sc29 antibodies recognised conformational specific epitopes in the S2 protein subunit at the Endoplasmic reticulum and Golgi complex. While PD5 and sc23 detected conformationally specific epitopes in the S1 protein subunit at the Golgi complex, only PD5 recognised the receptor binding domain (RBD). A comparison of the staining patterns of PD5 with non-conformationally specific antibodies that recognises the S1 subunit and RBD suggested the PD5 recognised a conformational structure within the S1 protein subunit. Our data suggests the antibody binding epitopes recognised by the human monoclonal antibodies formed at different locations in the secretory pathway during S protein transport, but a conformational change in the S1 protein subunit at the Golgi complex formed antibody binding epitopes that are recognised by virus neutralising antibodies.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Epítopos , Aparato de Golgi , Conformación Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Aparato de Golgi/metabolismo , Chlorocebus aethiops , Animales , Células Vero , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Humanos , Anticuerpos Neutralizantes/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Epítopos/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Anticuerpos Monoclonales/inmunología , COVID-19/inmunología , COVID-19/virologíaRESUMEN
Evidence for a stable interaction between the respiratory syncytial virus (RSV) F and G proteins on the surface of virus filaments was provided using antibody immunoprecipitation studies on purified RSV particles, and by the in situ analysis on the surface of RSV-infected cells using the proximity ligation assay. Imaging of the F and G protein distribution on virus filaments suggested that this protein complex was localised at the distal ends of the virus filaments, and suggested that this protein complex played a direct role in mediating efficient localised cell-to-cell virus transmission. G protein expression was required for efficient localised cell-to-cell transmission of RSV in cell monolayers which provided evidence that this protein complex mediates efficient multiple cycle infection. Collectively, these data provide evidence that F and G proteins form a complex on the surface of RSV particles, and that a role for this protein complex in promoting virus transmission is suggested.
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Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Proteínas Virales de Fusión/genética , Citoesqueleto , Procesamiento Proteico-Postraduccional , Proteínas de Unión al GTP/metabolismo , Anticuerpos AntiviralesRESUMEN
Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.
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Malaria , Proteómica , Humanos , Malaria/parasitología , Proteoma/metabolismo , Plasmodium berghei/metabolismo , Eritrocitos/parasitologíaRESUMEN
The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum. PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.
Asunto(s)
Diatomeas , Priones , Ribulosa-Bifosfato Carboxilasa/genética , Microscopía por Crioelectrón , Condensados Biomoleculares , Diatomeas/genéticaRESUMEN
Plants accumulate a vast array of secondary metabolites, which constitute a natural resource for pharmaceuticals. Oldenlandia corymbosa belongs to the Rubiaceae family, and has been used in traditional medicine to treat different diseases, including cancer. However, the active metabolites of the plant, their biosynthetic pathway and mode of action in cancer are unknown. To fill these gaps, we exposed this plant to eight different stress conditions and combined different omics data capturing gene expression, metabolic profiles, and anti-cancer activity. Our results show that O. corymbosa extracts are active against breast cancer cell lines and that ursolic acid is responsible for this activity. Moreover, we assembled a high-quality genome and uncovered two genes involved in the biosynthesis of ursolic acid. Finally, we also revealed that ursolic acid causes mitotic catastrophe in cancer cells and identified three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking. Altogether, these results constitute a valuable resource to further characterize the biosynthesis of active metabolites in the Oldenlandia group, while the mode of action of ursolic acid will allow us to further develop this valuable compound.
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Oldenlandia , Oldenlandia/química , Transcriptoma , Metabolómica , Genómica , Ácido UrsólicoRESUMEN
SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that SRRM2 forms biomolecular condensates satisfying most hallmarks of liquid-liquid phase separation, including spherical shape, dynamic rearrangement, coalescence and concentration dependence supported by in vitro experiments. Live-cell imaging shows that SRRM2 organizes nuclear speckles along the cell cycle. As bona-fide splicing factor present in spliceosome structures, SRRM2 deficiency induces skipping of cassette exons with short introns and weak splice sites, tending to change large protein domains. In THP-1 myeloid-like cells, SRRM2 depletion compromises cell viability, upregulates differentiation markers, and sensitizes cells to anti-leukemia drugs. SRRM2 induces a FES splice isoform that attenuates innate inflammatory responses, and MUC1 isoforms that undergo shedding with oncogenic properties. We conclude that SRRM2 acts as a scaffold to organize nuclear speckles, regulating alternative splicing in innate immunity and cell homeostasis.
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Empalme Alternativo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Exones , Células HEK293 , Humanos , Intrones , Isoformas de Proteínas/metabolismoRESUMEN
A human monoclonal antibody panel (PD4, PD5, PD7, SC23, and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2-infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 spike (S) protein, only PD5, PD7, and SC23 were able to bind to the receptor binding domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localized within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD-binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognized the uncleaved S protein, indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining were demonstrated for each isolate, SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection. IMPORTANCE The SARS-CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS-CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection, and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore, an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS- CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID-19 patients to monitor the RBD in cells infected with SARS-CoV-2 clinical isolates. These immunological reagents specifically recognize the correctly folded RBD and were used to monitor the appearance of the RBD in SARS-CoV-2-infected cells and identified the site where the RBD first appears.
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Anticuerpos Monoclonales , Anticuerpos Antivirales , COVID-19 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/metabolismo , Humanos , Unión Proteica , Dominios Proteicos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/síntesis química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Studying the swimming behaviour of bacteria in 3 dimensions (3D) allows us to understand critical biological processes, such as biofilm formation. It is still unclear how near wall swimming behaviour may regulate the initial attachment and biofilm formation. It is challenging to address this as visualizing the movement of bacteria with reasonable spatial and temporal resolution in a high-throughput manner is technically difficult. Here, we compared the near wall (vertical) swimming behaviour of P. aeruginosa (PAO1) and its mutants ΔdipA (reduced in swarming motility and increased in biofilm formation) and ΔfimX (deficient in twitching motility and reduced in biofilm formation) using our new imaging technique based on light sheet microscopy. We found that P. aeruginosa (PAO1) increases its speed and changes its swimming angle drastically when it gets closer to a wall. In contrast, ΔdipA mutant moves toward the wall with steady speed without changing of swimming angle. The near wall behavior of ΔdipA allows it to be more effective to interact with the wall or wall-attached cells, thus leading to more adhesion events and a larger biofilm volume during initial attachment when compared with PAO1. Furthermore, we found that ΔfimX has a similar near wall swimming behavior as PAO1. However, it has a higher dispersal frequency and smaller biofilm formation when compared with PAO1 which can be explained by its poor twitching motility. Together, we propose that near wall swimming behavior of P. aeruginosa plays an important role in the regulation of initial attachment and biofilm formation.
Asunto(s)
Biopelículas , Pseudomonas aeruginosa/fisiología , NataciónRESUMEN
About half the world's population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra-erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin. The replication factor C subunit 1 is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localisation to the nuclear periphery, indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions. Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication, apoptosis and DNA repair pathways leading to cell death. Additionally, mislocalisation of the endogenously tagged protein confirmed its essential role in parasites' replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in P. falciparum.
Asunto(s)
Antimaláricos/farmacología , Daño del ADN , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/fisiología , Proteína de Replicación C/fisiología , Artesunato/farmacología , Muerte Celular , Cloroquina/farmacología , Reparación del ADN , Replicación del ADN , ADN Protozoario , Eritrocitos/parasitología , Regulación de la Expresión Génica , Interacciones Huésped-Parásitos , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Proteínas Protozoarias/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand-receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in-situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.
Asunto(s)
Antígenos de Protozoos/metabolismo , Eritrocitos/parasitología , Interacciones Huésped-Parásitos , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Reticulocitos/metabolismo , Uniones Estrechas/metabolismo , Anticuerpos Monoclonales , Antígenos de Protozoos/genética , Eritrocitos/metabolismo , Proteínas de la Membrana/genética , Merozoítos/metabolismo , Plasmodium falciparum/química , Proteínas Protozoarias/genética , Uniones Estrechas/parasitologíaRESUMEN
In humans, (A549) cells impaired H9N2 virus nuclear export of the ribonucleoprotein (RNP) complex contrasted with the early and efficient nuclear export of the H1N1/WSN and pH1N1 virus RNP complexes. Although nuclear export of the RNP complex occurred via the nuclear pore complex, H9N2 virus infection also induced modifications in the nuclear envelope and induced cell cytotoxicity. Reduced PA protein levels in H9N2 virus-infected A549 cells occurred, and this phenomenon was independent of virus infection. Silencing the H1N1/WSN PA protein expression leads to impaired nuclear export of RNP complexes, suggesting that the impaired nuclear export of the H9N2 virus RNP complex may be one of the consequences of reduced PA protein levels. Early and efficient export of the RNP complex occurred in H9N2 virus-infected avian (CEF) cells, although structural changes in the nuclear envelope also occurred. Collectively our data suggest that a combination of delayed nuclear export and virus-induced cell cytotoxicity restricts H9N2 virus transmission in A549 cells. However, the early and efficient export of the RNP complex mitigated the effects of virus-induced cytotoxicity on H9N2 virus transmission in CEF cells. Our findings highlight the multi-factorial nature of host-adaptation of the polymerase proteins of avian influenza viruses in non-avian cell environments.
Asunto(s)
Núcleo Celular/metabolismo , Patos/virología , Subtipo H9N2 del Virus de la Influenza A/fisiología , Pulmón/patología , Pulmón/virología , Ribonucleoproteínas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Muerte Celular , Línea Celular , Pollos , Humanos , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
High level of the multifunctional AAA-ATPase p97/VCP is often correlated to the development of cancer; however, the underlying mechanism is not understood completely. Here, we report a novel function of p97/VCP in actin regulation and cell motility. We found that loss of p97/VCP promotes stabilization of F-actin, which cannot be reversed by actin-destabilizing agent, Cytochalasin D. Live-cell imaging demonstrated reduced actin dynamics in p97/VCP-knockdown cells, leading to compromised cell motility. We further examined the underlying mechanism and found elevated RhoA protein levels along with increased phosphorylation of its downstream effectors, ROCK, LIMK, and MLC upon the knockdown of p97/VCP. Since p97/VCP is indispensable in the ubiquitination-dependent protein degradation pathway, we investigated if the loss of p97/VCP hinders the protein degradation of RhoA. Knockdown of p97/VCP resulted in a higher amount of ubiquitinated RhoA, suggesting p97/VCP involvement in the proteasome-dependent protein degradation pathway. Finally, we found that p97/VCP interacts with FBXL19, a molecular chaperone known to guide ubiquitinated RhoA for proteasomal degradation. Reduction of p97/VCP may result in the accumulation of RhoA which, in turn, enhances cytoplasmic F-actin formation. In summary, our study uncovered a novel function of p97/VCP in actin regulation and cell motility via the Rho-ROCK dependent pathway which provides fundamental insights into how p97/VCP is involved in cancer development.
RESUMEN
The slow and promiscuous properties of the CO2-fixing enzyme Rubisco constrain photosynthetic efficiency and have prompted the evolution of powerful CO2 concentrating mechanisms (CCMs). In eukaryotic microalgae a key strategy involves sequestration of the enzyme in the pyrenoid, a liquid non-membranous compartment of the chloroplast stroma. Here we show using pure components that two proteins, Rubisco and the linker protein Essential Pyrenoid Component 1 (EPYC1), are both necessary and sufficient to phase separate and form liquid droplets. The phase-separated Rubisco is functional. Droplet composition is dynamic and components rapidly exchange with the bulk solution. Heterologous and chimeric Rubiscos exhibit variability in their tendency to demix with EPYC1. The ability to dissect aspects of pyrenoid biochemistry in vitro will permit us to inform and guide synthetic biology ambitions aiming to engineer microalgal CCMs into crop plants.
Asunto(s)
Microalgas/enzimología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/enzimología , Cloroplastos/metabolismo , Fotosíntesis/fisiologíaRESUMEN
We double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist) to generate "ΔA mutant" and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES) cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen) in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading.
RESUMEN
The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.
Asunto(s)
Basigina/metabolismo , Señalización del Calcio/fisiología , Proteínas Portadoras/metabolismo , Eritrocitos/fisiología , Merozoítos/patogenicidad , Plasmodium falciparum/patogenicidad , Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/biosíntesis , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/inmunología , Línea Celular , Citoesqueleto/parasitología , Citoesqueleto/patología , Eritrocitos/parasitología , Interacciones Huésped-Parásitos/fisiología , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/biosíntesisRESUMEN
The Runt-related transcription factors (RUNX) are master regulators of development and major players in tumorigenesis. Interestingly, unlike most transcription factors, RUNX proteins are detected on the mitotic chromatin and apparatus, suggesting that they are functionally active in mitosis. Here, we identify key sites of RUNX phosphorylation in mitosis. We show that the phosphorylation of threonine 173 (T173) residue within the Runt domain of RUNX3 disrupts RUNX DNA binding activity during mitotic entry to facilitate the recruitment of RUNX proteins to mitotic structures. Moreover, knockdown of RUNX3 delays mitotic entry. RUNX3 phosphorylation is therefore a regulatory mechanism for mitotic entry. Cancer-associated mutations of RUNX3 T173 and its equivalent in RUNX1 further corroborate the role of RUNX phosphorylation in regulating proper mitotic progression and genomic integrity.
Asunto(s)
Aurora Quinasas/metabolismo , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Mitosis/fisiología , Animales , Aurora Quinasas/genética , Células COS , Chlorocebus aethiops , Cromatina/metabolismo , Subunidad alfa 3 del Factor de Unión al Sitio Principal/química , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Subunidades alfa del Factor de Unión al Sitio Principal/genética , ADN/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mutación , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/químicaRESUMEN
The RUNX genes encode transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here, we show that Runx1;Runx3 double-knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes such as Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independently of CBFß, suggesting a nontranscriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes the development of leukemias and other cancers.
Asunto(s)
Médula Ósea/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Reparación del ADN , Anemia de Fanconi/genética , Leucemia/genética , Animales , Médula Ósea/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Eliminación de Gen , Predisposición Genética a la Enfermedad , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Leucemia/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
The kinesin motors are important in the regulation of cellular functions such as protein trafficking, spindle organization and centrosome separation. In this study, we have identified POPX2, a serine-threonine phosphatase, as an interacting partner of the KAP3 subunit of the kinesin-2 motor. The kinesin-2 motor is a heterotrimeric complex composed of KIF3A, KIF3B motor subunits and KAP3, the non-motor subunit, which binds the cargo. Here we report that the phosphatase POPX2 is a negative regulator of the trafficking of N-cadherin and other cargoes; consequently, it markedly influences cell-cell adhesion. POPX2 affects trafficking by determining the phosphorylation status of KIF3A at serine 690. This is consistent with the observation that the KIF3A-S690A mutant is defective in cargo trafficking. Our studies also implicate CaMKII as the kinase that phosphorylates KIF3A at serine 690. These results strongly suggest that POPX2 and CaMKII are a phosphatase-kinase pair that regulates kinesin-mediated transport and cell-cell adhesion.
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Cinesinas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Células COS , Cadherinas/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Adhesión Celular , Chlorocebus aethiops , Secuencia Conservada , Células HeLa , Humanos , Cinesinas/química , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , beta Catenina/metabolismoRESUMEN
Under the fluctuating circumstances provided by the innate dynamics of microtubules and opposing tensions resulted from microtubule-associated motors, it is vital to ensure stable kinetochore-microtubule attachments for accurate segregation. However, a comprehensive understanding of how this regulation is mechanistically achieved remains elusive. Using our newly designed live cell FRET time-lapse imaging, we found that post-metaphase RanGTP is crucial in the maintenance of stable kinetochore-microtubule attachments by regulating Aurora B kinase via the NES-bearing Mst1. More importantly, our study demonstrates that by ensuring stable alignment of metaphase chromosomes prior to segregation, RanGTP is indispensible in governing the genomic integrity and the fidelity of cell cycle progression. Our findings suggest an additional role of RanGTP beyond its known function in mitotic spindle assembly during the prometaphase-metaphase transition.
Asunto(s)
Cinetocoros/enzimología , Microtúbulos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP ran/fisiología , Animales , Aurora Quinasa B , Aurora Quinasas , Proteínas de Ciclo Celular/metabolismo , Cromosomas de los Mamíferos/metabolismo , Cricetinae , Transferencia Resonante de Energía de Fluorescencia , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Carioferinas/metabolismo , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Metafase , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteolisis , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Imagen de Lapso de Tiempo , Proteína de Unión al GTP ran/metabolismo , Proteína Exportina 1RESUMEN
RUNX family proteins are critical regulators of lineage differentiation during development. The high prevalence of RUNX mutation/epigenetic inactivation in human cancer indicates a causative role for dysfunctional RUNX in carcinogenesis. This is supported by well-documented evidence of functional interaction of RUNX with components of major oncogenic or tumor suppressive signaling pathways such as TGFß and Wnt. Here, we explore the binding partners of RUNX3 proteins to further define the scope of RUNX3 function. Using a mass spectrometry-based approach, we found that RUNX3 binds to centrosomal protein rootletin. This led us to uncover the presence of RUNX proteins at the centrosome. Our findings suggest a potential function for RUNX3 during mitosis.