RESUMEN
Ebola virus (EBOV) and Marburg virus (MARV) continue to emerge and cause severe hemorrhagic disease in humans. A comprehensive understanding of the filovirus-host interplay will be crucial for identifying and developing antiviral strategies. The filoviral VP40 matrix protein drives virion assembly and egress, in part by recruiting specific WW domain-containing host interactors via its conserved PPxY late (L) domain motif to positively regulate virus egress and spread. In contrast to these positive regulators of virus budding, a growing list of WW domain-containing interactors that negatively regulate virus egress and spread have been identified, including BAG3, YAP/TAZ, and WWOX. In addition to host WW domain regulators of virus budding, host PPxY-containing proteins also contribute to regulating this late stage of filovirus replication. For example, angiomotin (AMOT) is a multi-PPxY-containing host protein that functionally interacts with many of the same WW domain-containing proteins that regulate virus egress and spread. In this report, we demonstrate that host WWOX, which negatively regulates egress of VP40 virus-like particles (VLPs) and recombinant vesicular stomatitis virus (VSV) M40 virus, interacts with and suppresses the expression of AMOT. We found that WWOX disrupts AMOT's scaffold-like tubular distribution and reduces AMOT localization at the plasma membrane via lysosomal degradation. In sum, our findings reveal an indirect and novel mechanism by which modular PPxY-WW domain interactions between AMOT and WWOX regulate PPxY-mediated egress of filovirus VP40 VLPs. A better understanding of this modular network and competitive nature of protein-protein interactions will help to identify new antiviral targets and therapeutic strategies. IMPORTANCE Filoviruses (Ebola virus [EBOV] and Marburg virus [MARV]) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we reveal a novel mechanism by which host proteins WWOX and AMOTp130 interact with each other and with the filovirus matrix protein VP40 to regulate VP40-mediated egress of virus-like particles (VLPs). Our results highlight the biological impact of competitive interplay of modular virus-host interactions on both the virus life cycle and the host cell.
Asunto(s)
Ebolavirus , Marburgvirus , Oxidorreductasa que Contiene Dominios WW , Angiomotinas/metabolismo , Ebolavirus/fisiología , Humanos , Marburgvirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus/fisiología , Oxidorreductasa que Contiene Dominios WW/metabolismoRESUMEN
Marburg virus (MARV) VP40 protein (mVP40) directs egress and spread of MARV, in part, by recruiting specific host WW domain-containing proteins via its conserved PPxY late (L) domain motif to facilitate efficient virus-cell separation. We reported previously that small-molecule compounds targeting the viral PPxY/host WW domain interaction inhibited VP40-mediated egress and spread. Here, we report on the antiviral potency of novel compound FC-10696, which emerged from extensive structure-activity relationship (SAR) of a previously described series of PPxY inhibitors. We show that FC-10696 inhibits egress of mVP40 virus-like particles (VLPs) and egress of authentic MARV from HeLa cells and primary human macrophages. Moreover, FC-10696 treated-mice displayed delayed onset of weight loss and clinical signs and significantly lower viral loads compared to controls, with 14% of animals surviving 21 days following a lethal MARV challenge. Thus, FC-10696 represents a first-in-class, host-oriented inhibitor effectively targeting late stages of the MARV life cycle.
Asunto(s)
Marburgvirus , Animales , Células HeLa , Humanos , Ratones , Liberación del VirusRESUMEN
Filoviruses Ebola (EBOV) and Marburg (MARV) are devastating high-priority pathogens capable of causing explosive outbreaks with high human mortality rates. The matrix proteins of EBOV and MARV, as well as eVP40 and mVP40, respectively, are the key viral proteins that drive virus assembly and egress and can bud independently from cells in the form of virus-like particles (VLPs). The matrix proteins utilize proline-rich Late (L) domain motifs (e.g., PPxY) to hijack specific host proteins that contain WW domains, such as the HECT family E3 ligases, to facilitate the last step of virus-cell separation. We identified E3 ubiquitin ligase Smad Ubiquitin Regulatory Factor 2 (SMURF2) as a novel interactor with VP40 that positively regulates VP40 VLP release. Our results show that eVP40 and mVP40 interact with the three WW domains of SMURF2 via their PPxY motifs. We provide evidence that the eVP40-SMURF2 interaction is functional as the expression of SMURF2 positively regulates VLP egress, while siRNA knockdown of endogenous SMURF2 decreases VLP budding compared to controls. In sum, our identification of novel interactor SMURF2 adds to the growing list of identified host proteins that can regulate PPxY-mediated egress of VP40 VLPs. A more comprehensive understanding of the modular interplay between filovirus VP40 and host proteins may lead to the development of new therapies to combat these deadly infections.
Asunto(s)
Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/enzimología , Enfermedad del Virus de Marburg/enzimología , Marburgvirus/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus , Secuencias de Aminoácidos , Animales , Ebolavirus/química , Ebolavirus/genética , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/virología , Humanos , Enfermedad del Virus de Marburg/genética , Enfermedad del Virus de Marburg/virología , Marburgvirus/química , Marburgvirus/genética , Unión Proteica , Ubiquitina-Proteína Ligasas/genética , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Virión/genética , Virión/fisiología , Ensamble de VirusRESUMEN
Multiple copies of WW domains and PPXY motif sequences are often reciprocally presented by regulatory proteins that interact at crucial regulatory steps in the cell life cycle. While biophysical studies of single WW domain-single PPXY motif complexes abound in the literature, the molecular mechanisms of multivalent WW domain-PPXY assemblies are still poorly understood. By way of investigating such assemblies, we characterized the multivalent association of the entire cognate binding domains, two WW sequences and five PPXY motifs respectively, of the Yorkie transcription coactivator and the Warts tumor suppressor. Isothermal titration calorimetry, sedimentation velocity, size-exclusion chromatography coupled to multi-angle light scattering and native-state mass spectrometry of Yorkie WW domains interactions with the full-length Warts PPXY domain, and numerous PPXY motif variants of Warts show that the two proteins assemble via binding of tandem WW domains to adjacent PPXY pairs to produce an ensemble of interconverting complexes of variable stoichiometries, binding energetics and WW domain occupancy. Apparently, the Yorkie tandem WW domains first target the two adjacent PPXY motifs at the C-terminus of the Warts polypeptide and additional WW domains bind unoccupied motifs. Similar ensembles of interconverting conformers may be common in multivalent WW domain-PPXY interactions to promote the adaptability and versatility of WW domain-PPXY mediated cellular processes.
Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Dominios WW , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Unión Proteica , TermodinámicaRESUMEN
Bcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. Both positive and negative modulations of viruses by BAG3 were reported. However, the effects of BAG3 on pseudorabies virus (PRV) remain unknown. To investigate whether BAG3 could modulate the PRV life cycle during a lytic infection, we first identified PRV protein UL56 (pUL56) as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses. The overexpression of pUL56 induced a significant degradation of BAG3 at protein level via the lysosome pathway. The C-terminal mutations of 181L/A, 185L/A, or 181L/A-185L/A in pUL56 resulted in a deficiency in pUL56-induced BAG3 degradation. In addition, the pUL56 C-terminal mutants that lost Golgi retention abrogated pUL56-induced BAG3 degradation, which indicates a Golgi retention-dependent manner. Strikingly, BAG3 was not observed to be degraded in either wild-type or UL56-deleted PRV infected cells as compared to mock infected ones, whereas the additional two adjacent BAG3 cleaved products were found in the infected cells in a species-specific manner. Overexpression of BAG3 significantly suppressed PRV proliferation, while knockdown of BAG3 resulted in increased viral yields in HEK293T cells. Thus, these data indicated a negative regulation role of BAG3 during PRV lytic infection. Collectively, our findings revealed a novel molecular mechanism on host protein degradation induced by PRV pUL56. Moreover, we identified BAG3 as a host restricted protein during PRV lytic infection in cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Herpesvirus Suido 1/fisiología , Interacciones Huésped-Patógeno , Dominios y Motivos de Interacción de Proteínas , Proteínas Estructurales Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Aparato de Golgi/metabolismo , Lisosomas/metabolismo , Modelos Biológicos , Unión Proteica , Transporte de Proteínas , Proteolisis , Seudorrabia/metabolismo , Seudorrabia/virología , Especificidad de la Especie , Proteínas Estructurales Virales/químicaRESUMEN
The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain-bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain-containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.
Asunto(s)
Ebolavirus/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Secuencias de Aminoácidos , Angiomotinas , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/transmisión , Fiebre Hemorrágica Ebola/virología , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Microscopía Confocal , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Virión/fisiología , Liberación del VirusRESUMEN
The WW domain is a modular protein structure that recognizes the proline-rich Pro-Pro-x-Tyr (PPxY) motif contained in specific target proteins. The compact modular nature of the WW domain makes it ideal for mediating interactions between proteins in complex networks and signaling pathways of the cell (e.g. the Hippo pathway). As a result, WW domains play key roles in a plethora of both normal and disease processes. Intriguingly, RNA and DNA viruses have evolved strategies to hijack cellular WW domain-containing proteins and thereby exploit the modular functions of these host proteins for various steps of the virus life cycle, including entry, replication, and egress. In this review, we summarize key findings in this rapidly expanding field, in which new virus-host interactions continue to be identified. Further unraveling of the molecular aspects of these crucial virus-host interactions will continue to enhance our fundamental understanding of the biology and pathogenesis of these viruses. We anticipate that additional insights into these interactions will help support strategies to develop a new class of small-molecule inhibitors of viral PPxY-host WW-domain interactions that could be used as antiviral therapeutics.
Asunto(s)
Ubiquitina-Proteína Ligasas/química , Proteínas de la Matriz Viral/química , Virus/metabolismo , Secuencias de Aminoácidos , Virus ADN/fisiología , Interacciones Huésped-Patógeno , Humanos , Virus ARN/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de la Matriz Viral/metabolismo , Internalización del Virus , Dominios WW/fisiologíaRESUMEN
The second WW domain (WW2) of the kidney and brain scaffolding protein, KIBRA, has an isoleucine (Ile-81) rather than a second conserved tryptophan and is primarily unstructured. However, it adopts the canonical triple-stranded antiparallel ß-sheet structure of WW domains when bound to a two-PPXY motif peptide of the synaptic protein Dendrin. Here, using a series of biophysical experiments, we demonstrate that the WW2 domain remains largely disordered when bound to a 69-residue two-PPXY motif polypeptide of the synaptic and podocyte protein synaptopodin (SYNPO). Isothermal titration calorimetry and CD experiments revealed that the interactions of the disordered WW2 domain with SYNPO are significantly weaker than SYNPO's interactions with the well-folded WW1 domain and that an I81W substitution in the WW2 domain neither enhances binding affinity nor induces substantial WW2 domain folding. In the tandem polypeptide, the two WW domains synergized, enhancing the overall binding affinity with the I81W variant tandem polypeptide 2-fold compared with the WT polypeptide. Solution NMR results showed that SYNPO binding induces small but definite chemical shift perturbations in the WW2 domain, confirming the disordered state of the WW2 domain in this complex. These analyses also disclosed that SYNPO binds the tandem WW domain polypeptide in an antiparallel manner, that is, the WW1 domain binds the second PPXY motif of SYNPO. We propose a binding model consisting of a bipartite interaction mode in which the largely disordered WW2 forms a "fuzzy" complex with SYNPO. This binding mode may be important for specific cellular functions.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de Microfilamentos/química , Unión Proteica/genética , Dominios WW/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Calorimetría , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Isoleucina/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/ultraestructura , Péptidos/química , Péptidos/genética , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
Recent findings indicate that the WW domain-containing oxidoreductase (WWOX) is a tumor suppressor protein that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. WWOX protein mediates multiple signaling networks that suppress carcinogenesis through binding of its first WW domain to various cancer-associated proteins, i.e., p73, AP-2γ, and others. Although the tumor suppressor property of WWOX is inarguable, WWOX is not inactivated in the manner characteristic of the canonical Knudson hypothesis. Impairment of both alleles of WWOX is thought to be a rare event, only occurring in a few cancer cell lines. How is the tumor suppressor function of WWOX impaired in cancer cells? Recent advances highlight that a small transmembrane protein possessing a PPxY motif, called TMEM207, and its relatives are aberrantly expressed in various cancer cells and hinder the tumor suppressor function of WWOX through inhibiting its WW domain. Here, we review the recent findings related to the pathobiological properties of TMEM207 and its relatives based on clinicopathological and experimental pathological studies.
RESUMEN
The Hippo tumor suppressor pathway is an important regulator of cell proliferation and apoptosis, and signal transduction occurs through phosphorylation of the effector protein TAZ by the serine/threonine kinase LATS1/2. Here, we report the biophysical and computational studies to characterize the interaction between TAZ and LATS1/2 through WW domain-PPxY motif binding. We show that the TAZ WW domain exhibits a binding preference for the second of the two PPxY motifs of LATS1 in vitro. We modelled the structure of the domain in complex with LATS1 PPxY2 peptide and, through molecular dynamics simulations, show that WW domain-PPxY2 complex is stable with some flexibility in the peptide region. Next, we predict and verify that L143 and T150 of the WW domain are important for TAZ binding with the PPxY2 peptide using mutational and isothermal titration calorimetric studies. Furthermore, we suggest that the electrostatic potential of charged residues within the binding pocket may influence the ligand affinity among otherwise highly similar WW domains.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Fenómenos Biofísicos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Dominios WW/genéticaRESUMEN
YES-associated protein (YAP) is a major effector protein of the Hippo tumor suppressor pathway, and is phosphorylated by the serine/threonine kinase LATS. Their binding is mediated by the interaction between WW domains of YAP and PPxY motifs of LATS. Their isoforms, YAP2 and LATS1 contain two WW domains and two PPxY motifs respectively. Here, we report the study of the interaction of these domains both in vitro and in human cell lines, to better understand the mechanism of their binding. We show that there is a reciprocal binding preference of YAP2-WW1 with LATS1-PPxY2, and YAP2-WW2 with LATS1-PPxY1. We solved the NMR structures of these complexes and identified several conserved residues that play a critical role in binding. We further created a YAP2 mutant by swapping the WW domains, and found that YAP2 phosphorylation at S127 by LATS1 is not affected by the spatial configuration of its WW domains. This is likely because the region between the PPxY motifs of LATS1 is unstructured, even upon binding with its partner. Based on our observations, we propose possible models for the interaction between YAP2 and LATS1.
RESUMEN
Thioredoxin-interacting protein (TXNIP) is a multifunctional protein involved in diverse cellular processes such as cell proliferation and apoptosis. TXNIP stability is controlled by the ubiquitin-proteasome pathway, and the E3 ubiquitin ligase Itch directly interacts with TXNIP via PPxY motifs of TXNIP. In a previously published study, we have shown that phosphorylation of the PPxY tyrosyl residue switches TXNIP selectivity between different binding partners. Here, we describe that tyrosine-phosphorylated PPxY motifs also bind to SH2 domains of Vav2 and Src with dissociation constants around 10 µm and that phosphorylation is indispensable for these interactions as well. The crystal structure of the complex between a phosphorylated PPxY motif, and the SH2 domain of Vav2 reveals a conserved recognition mechanism.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Proto-Oncogénicas c-vav/química , Proteínas Proto-Oncogénicas c-vav/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Tirosina/química , Ubiquitina-Proteína Ligasas/metabolismo , Dominios Homologos srcRESUMEN
ASPP2 is an important tumor suppressor protein promoting p53-dependent and-independent apoptosis. However, it has been unclear how ASPP2 protein is regulated. Here, we identified Itch as the E3 ubiquitin ligase for ASPP2. Itch interacts with ASPP2 and mediates its degradation and ubiquitination in vivo. The PPXY motif of ASPP2 interacts with the WW domains of Itch. Yap1 competes with Itch for binding to ASPP2, and prevents Itch-mediated degradation and ubiquitination of ASPP2. Together, these observations reveal that Itch and Yap1 have antagonistic roles in the regulation of ASPP2 protein stability through competing post-translational regulatory mechanism of ASPP2.