RESUMEN
Mitochondria are highly dynamic organelles that continuously grow, divide, and fuse. The division of mitochondria is crucial for human health. During mitochondrial division, the mechano-guanosine triphosphatase (GTPase) dynamin-related protein (Drp1) severs mitochondria at endoplasmic reticulum (ER)-mitochondria contact sites, where peripheral ER tubules interact with mitochondria. Here, we report that Drp1 directly shapes peripheral ER tubules in human and mouse cells. This ER-shaping activity is independent of GTP hydrolysis and located in a highly conserved peptide of 18 amino acids (termed D-octadecapeptide), which is predicted to form an amphipathic α helix. Synthetic D-octadecapeptide tubulates liposomes in vitro and the ER in cells. ER tubules formed by Drp1 promote mitochondrial division by facilitating ER-mitochondria interactions. Thus, Drp1 functions as a two-in-one protein during mitochondrial division, with ER tubulation and mechano-GTPase activities.
Asunto(s)
Dinaminas/metabolismo , Dinaminas/fisiología , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Mitocondrias/metabolismo , Animales , Dinaminas/genética , Retículo Endoplásmico/efectos de los fármacos , GTP Fosfohidrolasas/genética , Humanos , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Dinámicas Mitocondriales , Oligopéptidos/farmacologíaRESUMEN
Hundreds of effector proteins of the human malaria parasite Plasmodium falciparum constitute a "secretome" carrying a host-targeting (HT) signal, which predicts their export from the intracellular pathogen into the surrounding erythrocyte. Cleavage of the HT signal by a parasite endoplasmic reticulum (ER) protease, plasmepsin V, is the proposed export mechanism. Here, we show that the HT signal facilitates export by recognition of the lipid phosphatidylinositol-3-phosphate (PI(3)P) in the ER, prior to and independent of protease action. Secretome HT signals, including those of major virulence determinants, bind PI(3)P with nanomolar affinity and amino acid specificities displayed by HT-mediated export. PI(3)P-enriched regions are detected within the parasite's ER and colocalize with endogenous HT signal on ER precursors, which also display high-affinity binding to PI(3)P. A related pathogenic oomycete's HT signal export is dependent on PI(3)P binding, without cleavage by plasmepsin V. Thus, PI(3)P in the ER functions in mechanisms of secretion and pathogenesis.
Asunto(s)
Eritrocitos/parasitología , Malaria Falciparum/parasitología , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Eritrocitos/metabolismo , Humanos , Malaria Falciparum/patología , Datos de Secuencia Molecular , Plasmodium falciparum/citología , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteínas Protozoarias/químicaRESUMEN
Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.
Asunto(s)
Membrana Celular , Ebolavirus , Ensamble de Virus , Liberación del Virus , Humanos , Sustitución de Aminoácidos , Membrana Celular/metabolismo , Ebolavirus/metabolismo , Ebolavirus/genética , Células HEK293 , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Mutación , Nucleoproteínas , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Fosfatidilserinas/química , Unión Proteica , Electricidad Estática , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/química , Virión/metabolismo , Virión/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N), and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we used several lipid binding assays and found the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, we show lipid binding occurs in the N protein C-terminal domain, which is supported by extensive in silico analysis. We demonstrate anionic lipid binding occurs for both the free and the N oligomeric forms, suggesting N can associate with membranes in the nucleocapsid form. Based on these results, we present a lipid-dependent model based on in vitro, cellular, and in silico data for the recruitment of N to assembly sites in the lifecycle of SARS-CoV-2.
RESUMEN
Ebola virus (EBOV) causes severe hemorrhagic fever in humans and is lethal in a large percentage of those infected. The EBOV matrix protein viral protein 40 kDa (VP40) is a peripheral binding protein that forms a shell beneath the lipid bilayer in virions and virus-like particles (VLPs). VP40 is required for virus assembly and budding from the host cell plasma membrane. VP40 is a dimer that can rearrange into oligomers at the plasma membrane interface, but it is unclear how these structures form and how they are stabilized. We therefore investigated the ability of VP40 to form stable oligomers using in vitro and cellular assays. We characterized two lysine-rich regions in the VP40 C-terminal domain (CTD) that bind phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and play distinct roles in lipid binding and the assembly of the EBOV matrix layer. The extensive analysis of VP40 with and without lipids by hydrogen deuterium exchange mass spectrometry revealed that VP40 oligomers become extremely stable when VP40 binds PI(4,5)P2. The PI(4,5)P2-induced stability of VP40 dimers and oligomers is a critical factor in VP40 oligomerization and release of VLPs from the plasma membrane. The two lysine-rich regions of the VP40 CTD have different roles with respect to interactions with plasma membrane phosphatidylserine (PS) and PI(4,5)P2. CTD region 1 (Lys221, Lys224, and Lys225) interacts with PI(4,5)P2 more favorably than PS and is important for VP40 extent of oligomerization. In contrast, region 2 (Lys270, Lys274, Lys275, and Lys279) mediates VP40 oligomer stability via lipid interactions and has a more prominent role in release of VLPs.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/metabolismo , Lisina/metabolismo , Sitios de Unión , Lípidos , Unión ProteicaRESUMEN
Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Animales , Fiebre Hemorrágica Ebola/metabolismo , Ebolavirus/fisiología , Fosfatidilserinas/metabolismo , Fendilina/metabolismo , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus , Análisis por Conglomerados , Mamíferos/metabolismoRESUMEN
Ebola virus (EBOV) infections continue to pose a global public health threat, with high mortality rates and sporadic outbreaks in Central and Western Africa. A quantitative understanding of the key processes driving EBOV assembly and budding could provide valuable insights to inform drug development. Here, we use a computational model to evaluate EBOV matrix assembly. Our model focuses on the assembly kinetics of VP40, the matrix protein in EBOV, and its interaction with phosphatidylserine (PS) in the host cell membrane. It has been shown that mammalian cells transfected with VP40-expressing plasmids are capable of producing virus-like particles (VLPs) that closely resemble EBOV virions. Previous studies have also shown that PS levels in the host cell membrane affects VP40 association with the plasma membrane inner leaflet and that lower membrane PS levels result in lower VLP production. Our computational findings indicate that PS may also have a direct influence on VP40 VLP assembly and budding, where a higher PS level will result in a higher VLP budding rate and filament dissociation rate. Our results further suggest that the assembly of VP40 filaments follow the nucleation-elongation theory, where initialization and oligomerization of VP40 are two distinct steps in the assembly process. Our findings advance the current understanding of VP40 VLP formation by identifying new possible mechanisms of PS influence on VP40 assembly. We propose that these mechanisms could inform treatment strategies targeting PS alone or in combination with other VP40 assembly steps.
Asunto(s)
Ebolavirus , Fosfatidilserinas , Proteínas de la Matriz Viral , Ensamble de Virus , Animales , Ebolavirus/fisiología , Modelos Moleculares , Fosfatidilserinas/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Liberación del VirusRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic. Research on SARS-CoV-2, as well as the closely related SARS-CoV-1 and MERS coronaviruses, is restricted to BSL-3 facilities. Such BSL-3 classification makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions. These systems should be useful to those looking to circumvent BSL-3 work with SARS-CoV-2 yet study the mechanisms by which SARS-CoV-2 enters and exits human cells.
Asunto(s)
Proteínas de la Envoltura de Coronavirus/genética , Proteínas de la Nucleocápside/genética , SARS-CoV-2/crecimiento & desarrollo , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas de la Matriz Viral/genética , Virión/crecimiento & desarrollo , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Contención de Riesgos Biológicos/clasificación , Proteínas de la Envoltura de Coronavirus/metabolismo , Expresión Génica , Genes Reporteros , Regulación Gubernamental , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica , Proteínas de la Nucleocápside/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestructura , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Virión/genética , Virión/metabolismo , Virión/ultraestructura , Ensamble de Virus/fisiología , Internalización del Virus , Liberación del Virus/fisiología , Proteína Fluorescente RojaRESUMEN
Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD-NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD-CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein-protein or lipid-protein interactions to inhibit virus budding.
Asunto(s)
Enfermedad del Virus de Marburg/virología , Marburgvirus/fisiología , Lípidos de la Membrana/metabolismo , Proteínas de la Matriz Viral/metabolismo , Virión/metabolismo , Animales , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Enfermedad del Virus de Marburg/metabolismo , Marburgvirus/química , Lípidos de la Membrana/química , Modelos Moleculares , Multimerización de Proteína , Proteínas de la Matriz Viral/química , Virión/química , Ensamble de VirusRESUMEN
Outbreaks of the Ebola virus (EBOV) continue to occur and while a vaccine and treatment are now available, there remains a dearth of options for those who become sick with EBOV disease. An understanding at the atomic and molecular level of the various steps in the EBOV replication cycle can provide molecular targets for disrupting the virus. An important step in the EBOV replication cycle is the transport of EBOV structural matrix VP40 protein molecules to the plasma membrane inner leaflet, which involves VP40 binding to the host cell's Sec24c protein. Though some VP40 residues involved in the binding are known, the molecular details of VP40-Sec24c binding are not known. We use various molecular computational techniques to investigate the molecular details of how EBOV VP40 binds with the Sec24c complex of the ESCRT-I pathway. We employed different docking programs to identify the VP40-binding site on Sec24c and then performed molecular dynamics simulations to determine the atomic details and binding interactions of the complex. We also investigated how the inter-protein interactions of the complex are affected upon mutations of VP40 amino acids in the Sec24c-binding region. Our results provide a molecular basis for understanding previous coimmunoprecipitation experimental studies. In addition, we found that VP40 can bind to a site on Sec24c that can also bind Sec23 and suggests that VP40 may use the COPII transport mechanism in a manner that may not need the Sec23 protein in order for VP40 to be transported to the plasma membrane.
Asunto(s)
Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/virología , Proteínas de Transporte Vesicular , Proteínas de la Matriz Viral , Humanos , Unión Proteica , Transporte de Proteínas , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismoRESUMEN
Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gßγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.
Asunto(s)
Membrana Celular , Factores de Intercambio de Guanina Nucleótido , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Fosforilación , Dominios ProteicosRESUMEN
Lipid enveloped viruses contain a lipid bilayer coat that protects their genome to help facilitate entry into the new host cell. This lipid bilayer comes from the host cell which they infect. After viral replication, the mature virion hijacks the host cell plasma membrane where it is then released to infect new cells. This process is facilitated by the interaction between phospholipids that make up the plasma membrane and specialized viral matrix proteins. This step in the viral lifecycle may represent a viable therapeutic strategy for small molecules that aim to block enveloped virus spread. In this review, we summarize the current knowledge on the role of plasma membrane lipid-protein interactions on viral assembly and budding.
Asunto(s)
Interacciones Huésped-Patógeno , Lípidos/química , Proteínas/química , Ensamble de Virus , Membrana Celular/metabolismoRESUMEN
Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is an essential step in the development of the disease. Consequently, the molecular players involved in host cell invasion represent important targets for inhibitor design and vaccine development. The process of merozoite invasion is a succession of steps underlined by the sequential secretion of the organelles of the apical complex. However, little is known with regard to how their contents are exocytosed. Here, we identify a phosphoinositide-binding protein conserved in apicomplexan parasites and show that it is important for the attachment and subsequent invasion of the erythrocyte by the merozoite. Critically, removing the protein from its site of action by knock sideways preferentially prevents the secretion of certain types of micronemes. Our results therefore provide evidence for a role of phosphoinositide lipids in the malaria invasion process and provide further insight into the secretion of microneme organelle populations, which is potentially applicable to diverse apicomplexan parasites.
Asunto(s)
Exocitosis , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Eritrocitos/parasitología , Humanos , Estadios del Ciclo de Vida , Fosfatidilinositoles/metabolismo , Dominios Homólogos a Pleckstrina , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genéticaRESUMEN
Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.
Asunto(s)
Antimaláricos/farmacología , Artemisininas/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Resistencia a Medicamentos/genética , Estudio de Asociación del Genoma Completo , Modelos Moleculares , Mutación , Fosfatidilinositol 3-Quinasa/química , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.
Asunto(s)
Membrana Celular/metabolismo , Ceramidas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Necrosis/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Células A549 , Animales , Línea Celular , Membrana Celular/patología , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Humanos , Neoplasias Pulmonares/patología , Simulación del Acoplamiento Molecular , Necrosis/patología , Oogénesis , Ovario/crecimiento & desarrolloRESUMEN
Artemisinin resistance threatens worldwide malaria control and elimination. Elevation of phosphatidylinositol-3-phosphate (PI3P) can induce resistance in blood stages of Plasmodium falciparum The parasite unfolded protein response (UPR) has also been implicated as a proteostatic mechanism that may diminish artemisinin-induced toxic proteopathy. How PI3P acts and its connection to the UPR remain unknown, although both are conferred by mutation in P falciparum Kelch13 (K13), the marker of artemisinin resistance. Here we used cryoimmunoelectron microscopy to show that K13 concentrates at PI3P tubules/vesicles of the parasite's endoplasmic reticulum (ER) in infected red cells. K13 colocalizes and copurifies with the major virulence adhesin PfEMP1. The PfEMP1-K13 proteome is comprehensively enriched in multiple proteostasis systems of protein export, quality control, and folding in the ER and cytoplasm and UPR. Synthetic elevation of PI3P that induces resistance in absence of K13 mutation also yields signatures of proteostasis and clinical resistance. These findings imply a key role for PI3P-vesicle amplification as a mechanism of resistance of infected red cells. As validation, the major resistance mutation K13C580Y quantitatively increased PI3P tubules/vesicles, exporting them throughout the parasite and the red cell. Chemical inhibitors and fluorescence microscopy showed that alterations in PfEMP1 export to the red cell and cytoadherence of infected cells to a host endothelial receptor are features of multiple K13 mutants. Together these data suggest that amplified PI3P vesicles disseminate widespread proteostatic capacity that may neutralize artemisinins toxic proteopathy and implicate a role for the host red cell in artemisinin resistance. The mechanistic insights generated will have an impact on malaria drug development.
Asunto(s)
Artemisininas/farmacología , Resistencia a Medicamentos , Retículo Endoplásmico , Eritrocitos/parasitología , Lactonas/farmacología , Plasmodium falciparum , Proteínas Protozoarias , Respuesta de Proteína Desplegada , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Eritrocitos/metabolismo , Humanos , Mutación , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteostasis/efectos de los fármacos , Proteostasis/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genéticaRESUMEN
During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Citocinesis/genética , Citocinesis/fisiología , Dinaminas/genética , Dinaminas/metabolismo , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Red trans-Golgi/metabolismo , Red trans-Golgi/fisiologíaRESUMEN
The two-photon absorption properties of a pyrene-pyridinium dye (1) were studied for potential application in two-photon spectroscopy. When probe 1 was used in cellular two-photon fluorescence microscopy imaging, it allowed the visualization of nuclei in live cells with a relatively low probe concentration (such as 1 µM). Spectroscopic evidence further revealed that probe 1 interacted with DNA as an intercalator. The proposed DNA intercalation properties of probe 1 were consistent with the experimental findings that suggested that the observed nucleus staining ability is dependent on the substituents on the pyridinium fragment of the probe.
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Núcleo Celular/química , Colorantes Fluorescentes/química , Fotones , Pirenos/química , Animales , Células COS , Bovinos , Supervivencia Celular , Células Cultivadas , Chlorocebus aethiops , ADN/química , Microscopía Fluorescente , Estructura Molecular , Compuestos de Piridinio/químicaRESUMEN
HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A's cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A's PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A's lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A's secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A's ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A's refolding activities. Furthermore, using a Lysine-71-Alanine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the wild-type (WT), the mutated protein associates with lipids three times faster and dissociates 300 times faster than the WT HspA1A. These observations suggest a two-step binding model including an initial interaction of HspA1A with lipids followed by a conformational change of the HspA1A-lipid complex, which accelerates the binding reaction. Together these findings strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap and provide the basis for future interventions to inhibit HspA1A's PM-translocation in tumor cells, making them sensitive to radiation therapy.
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Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Liposomas/metabolismo , Fosfatidilserinas/metabolismo , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Animales , Dicroismo Circular , Proteínas HSP70 de Choque Térmico/genética , Liposomas/química , Lisina/genética , Ratones , Chaperonas Moleculares/metabolismo , Mutación , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Unión Proteica , Replegamiento Proteico , Estructura Secundaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Manganese porphyrins (MnPs), MnTE-2-PyP5+, MnTnHex-2-PyP5+ and MnTnBuOE-2-PyP5+, are superoxide dismutase (SOD) mimetics and form a redox cycle between O2 and reductants, including ascorbic acid, ultimately producing hydrogen peroxide (H2O2). We previously found that MnPs oxidize hydrogen sulfide (H2S) to polysulfides (PS; H2Sn, n = 2-6) in buffer. Here, we examine the effects of MnPs for 24 h on H2S metabolism and PS production in HEK293, A549, HT29 and bone marrow derived stem cells (BMDSC) using H2S (AzMC, MeRho-AZ) and PS (SSP4) fluorophores. All MnPs decreased intracellular H2S production and increased intracellular PS. H2S metabolism and PS production were unaffected by cellular O2 (5% versus 21% O2), H2O2 or ascorbic acid. We observed with confocal microscopy that mitochondria are a major site of H2S production in HEK293 cells and that MnPs decrease mitochondrial H2S production and increase PS in what appeared to be nucleoli and cytosolic fibrillary elements. This supports a role for MnPs in the metabolism of H2S to PS, the latter serving as both short- and long-term antioxidants, and suggests that some of the biological effects of MnPs may be attributable to sulfur metabolism.