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1.
Circulation ; 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38752370

RESUMO

BACKGROUND: Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. METHODS: We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary sgRNA validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. RESULTS: Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. CONCLUSIONS: These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

2.
Cell Mol Biol Lett ; 28(1): 39, 2023 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-37170209

RESUMO

BACKGROUND: Tissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury. METHODS: We express optogenetic YAP (optoYAP) in three different cell lines. We characterised the behaviour and function of optoYAP using fluorescence imaging and quantitative real-time PCR of downstream YAP target genes. Mutant constructs were generated using site-directed mutagenesis. Nuclear localised optoYAP was functionally tested using wound healing assay. RESULTS: Utilising optoYAP, which enables precise control of pathway activation, we show that YAP induces the expression of downstream genes involved in proliferation and migration. optoYAP can increase the speed of wound healing in H9c2 cardiomyoblasts. Interestingly, this is not driven by an increase in proliferation, but by collective cell migration. We subsequently dissect specific phosphorylation sites in YAP to identify the molecular driver of accelerated healing. CONCLUSIONS: This study shows that optogenetic YAP is functional in H9c2 cardiomyoblasts and its controlled activation can potentially enhance wound healing in a range of conditions.


Assuntos
Infarto do Miocárdio , Transdução de Sinais , Proteínas de Sinalização YAP , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/genética , Proliferação de Células , Optogenética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cicatrização
3.
Proc Natl Acad Sci U S A ; 119(29): e2203257119, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35858299

RESUMO

How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The transcriptional target of the Hippo pathway, the transcriptional coactivator YAP, inhibits the initiation of budding in Hydra and is regulated by Hydra LATS. In addition, we show functions of the Hippo pathway in regulation of actin organization and cell proliferation in Hydra. We hypothesize that the Hippo pathway served as a link between continuous cell division, cell density, and axis formation early in metazoan evolution.


Assuntos
Via de Sinalização Hippo , Hydra , Morfogênese , Animais , Padronização Corporal , Hydra/genética , Hydra/crescimento & desenvolvimento , Hydra/metabolismo , Morfogênese/genética , Transcrição Gênica , Proteínas de Sinalização YAP/metabolismo
4.
EMBO Rep ; 23(9): e54401, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35876586

RESUMO

YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.


Assuntos
Transdução de Sinais , Peixe-Zebra , Animais , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Optogenética , Peixe-Zebra/genética
6.
Cell Death Discov ; 7(1): 369, 2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34845189

RESUMO

Long non-coding RNAs (lncRNAs) regulate numerous biological processes involved in both development and carcinogenesis. Hippo-YAP/TAZ signaling, a critical pathway responsible for organ size control, is often dysregulated in a variety of cancers. However, the nature and function of YAP/TAZ-regulated lncRNAs during tumorigenesis remain largely unexplored. By profiling YAP/TAZ-regulated lncRNAs, we identified SFTA1P as a novel transcriptional target and a positive feedback regulator of YAP/TAZ signaling. Using non-small cell lung cancer (NSCLC) cell lines, we show that SFTA1P is transcriptionally activated by YAP/TAZ in a TEAD-dependent manner. Functionally, knockdown of SFTA1P in NSCLC cell lines inhibited proliferation, induced programmed cell death, and compromised their tumorigenic potential. Mechanistically, SFTA1P knockdown decreased TAZ protein abundance and consequently, the expression of YAP/TAZ transcriptional targets. We provide evidence that this phenomenon could potentially be mediated via its interaction with TAZ mRNA to regulate TAZ translation. Our results reveal SFTA1P as a positive feedback regulator of Hippo-YAP/TAZ signaling, which may serve as the molecular basis for lncRNA-based therapies against YAP/TAZ-driven cancers.

7.
PLoS One ; 16(6): e0251955, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34106957

RESUMO

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , Proteínas do Envelope de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , COVID-19/patologia , Interações Hospedeiro-Patógeno , Humanos , Domínios PDZ , Ligação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SARS-CoV-2/isolamento & purificação , Junções Íntimas/metabolismo
8.
Cancers (Basel) ; 13(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670427

RESUMO

The Hippo-YAP/TAZ signaling pathway is an evolutionarily conserved signaling pathway involved in a broad spectrum of biological processes, including tumorigenesis. Whilst aberrant Hippo-YAP/TAZ signaling is frequently reported in various cancers, the genetic alterations of this pathway are relatively rare, suggesting regulation at the post-transcriptional level. MicroRNAs play key role in tumorigenesis by regulating gene expression post-transcriptionally. Amongst the cancer-relevant microRNAs, miR-582-5p suppresses cell growth and tumorigenesis by inhibiting the expression of oncogenes, including AKT3, MAP3K2 and NOTCH1. Given the oncogenic role of YAP/TAZ in solid tumors, we scrutinized the possible interplay between miR-582-5p and Hippo-YAP/TAZ signaling. Correlation analysis in NSCLC cells revealed a positive relationship between the expression of mature miR-582-5p and the proportion of phosphorylated YAP/TAZ. Intriguingly, YAP/TAZ knockdown reduced the expression of mature miR-582-5p but increased that of primary miR-582. Overexpression of miR-582-5p resulted in increased phosphorylation of YAP/TAZ with a concomitant reduction in cell proliferation and enhanced apoptosis. Mechanistically, we find that miR-582-5p targets actin regulators NCKAP1 and PIP5K1C, which may be responsible for the observed alteration in F-actin, known to modulate YAP/TAZ. We postulate that regulation of the actin cytoskeleton by miR-582-5p may attenuate YAP/TAZ activity. Altogether, this study reveals a novel mechanism of YAP/TAZ regulation by miR-582-5p in a cytoskeleton-dependent manner and suggests a negative feedback loop, highlighting the therapeutic potential of restoring miR-582-5p expression in treating NSCLC.

9.
Genomics ; 113(3): 1349-1365, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33713822

RESUMO

Yes-associated protein 1 (YAP1) is a transcriptional co-activator downstream of Hippo pathway. The pathway exerts crucial roles in organogenesis and its dysregulation is associated with the spreading of different cancer types. YAP1 gene encodes for multiple protein isoforms, whose specific functions are not well defined. We demonstrate the splicing of isoform-specific mRNAs is controlled in a stage- and tissue-specific fashion. We designed expression vectors encoding for the most-represented isoforms of YAP1 with either one or two WW domains and studied their specific signaling activities in YAP1 knock-out cell lines. YAP1 isoforms display both common and unique functions and activate distinct transcriptional programs, as the result of their unique protein interactomes. By generating TEAD-based transcriptional reporter cell lines, we demonstrate individual YAP1 isoforms display unique effects on cell proliferation and differentiation. Finally, we illustrate the complexity of the regulation of Hippo-YAP1 effector in physiological and in pathological conditions of the heart.


Assuntos
Proteínas de Ciclo Celular , Isoformas de RNA , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Sinalização YAP
10.
J Virol ; 95(8)2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33536174

RESUMO

Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection.Author Summary Filoviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) 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 identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

11.
Cancer Lett ; 500: 51-63, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33296708

RESUMO

Lung cancer is the leading cause of cancer-related deaths, worldwide. Non-small cell lung cancer (NSCLC) is the most prevalent lung cancer subtype. YAP and TAZ have been implicated in lung cancer by acting as transcriptional co-activators of oncogenes or as transcriptional co-repressors of tumor suppressor genes. Previously we reported that YAP and TAZ regulate microRNAs expression in NSCLC. Among the set of regulated miRNAs, the oncogenic miR-25, 93, and 106b, clustering within the MCM7 gene were selected for further studies. We firstly identified Transforming Growth Factor-ß (TGF-ß) Receptor 2 (TGFBR2), a member of the TGF-ß signaling, as a target of the miRNA cluster, which exhibited prognostic value because of its tumor suppressor activity. We found that YAP/TAZ-mediated repression of TGFBR2 occurs both: post-transcriptionally through the miR-106b-25 cluster and transcriptionally by engaging the EZH2 epigenetic repressor that we reported here as a novel target gene of YAP/TAZ. Furthermore, we document that YAP/TAZ and EZH2 cooperate in lung tumorigenesis by transcriptionally repressing a specific subset of tumor suppressor genes, including TGFBR2. Our findings point to YAP/TAZ and EZH2 as potential therapeutic targets for NSCLC treatment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Fatores de Transcrição/genética , Células A549 , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MicroRNAs/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Proteínas Nucleares/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Fatores de Transcrição de Domínio TEA , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Fator de Crescimento Transformador beta1/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAP
12.
Cancers (Basel) ; 12(12)2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-33297432

RESUMO

YAP and its paralog TAZ are the nuclear effectors of the Hippo tumour-suppressor pathway, and function as transcriptional co-activators to control gene expression in response to mechanical cues. To identify both common and unique transcriptional targets of YAP and TAZ in gastric cancer cells, we carried out RNA-sequencing analysis of overexpressed YAP or TAZ in the corresponding paralogous gene-knockouts (KOs), TAZ KO or YAP KO, respectively. Gene Ontology (GO) analysis of the YAP/TAZ-transcriptional targets revealed activation of genes involved in platelet biology and lipoprotein particle formation as targets that are common for both YAP and TAZ. However, the GO terms for cell-substrate junction were a unique function of YAP. Further, we found that YAP was indispensable for the gastric cancer cells to re-establish cell-substrate junctions on a rigid surface following prolonged culture on a soft substrate. Collectively, our study not only identifies common and unique transcriptional signatures of YAP and TAZ in gastric cancer cells but also reveals a dominant role for YAP over TAZ in the control of cell-substrate adhesion.

13.
Oncogene ; 39(24): 4621-4635, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393834

RESUMO

WW domain-binding protein 2 (WBP2) is an emerging oncoprotein. Over the past decade, WBP2 surfaced as a key node connecting key signaling pathways associated with ER/PR, EGFR, PI3K, Hippo, and Wnt in cancer. In addition to the oncogenic functions of WBP2, this review discusses the latest research regarding the multilevel regulation and modes of action of WBP2 and how they can be exploited for molecular medicine. In translational research, evidence supports the role of WBP2 as a biomarker for early detection, prognosis, and companion diagnostics in breast cancer. Finally, we envision new trends in WBP2 research in the space of molecular etiology of cancer, targeted therapeutics, and precision medicine.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas Oncogênicas/metabolismo , Transativadores/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Proteínas Oncogênicas/genética , Transativadores/genética
14.
J Biol Chem ; 295(14): 4604-4616, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32111739

RESUMO

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.


Assuntos
Ubiquitina-Proteína Ligases/química , Proteínas da Matriz Viral/química , Vírus/metabolismo , Motivos de Aminoácidos , Vírus de DNA/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Vírus de RNA/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas da Matriz Viral/metabolismo , Internalização do Vírus , Domínios WW/fisiologia
15.
PLoS Pathog ; 16(1): e1008231, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31905227

RESUMO

Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.


Assuntos
Filoviridae/fisiologia , Marburgvirus/fisiologia , Mimetismo Molecular , Proteínas Proto-Oncogênicas c-yes/metabolismo , Proteínas da Matriz Viral/fisiologia , Liberação de Vírus , Angiomotinas , Sítios de Ligação , Membrana Celular/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Domínios PDZ , Domínios Proteicos , Proteínas Recombinantes de Fusão/metabolismo
16.
Nat Commun ; 11(1): 507, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980612

RESUMO

The timing and characteristics of neuronal death in Alzheimer's disease (AD) remain largely unknown. Here we examine AD mouse models with an original marker, myristoylated alanine-rich C-kinase substrate phosphorylated at serine 46 (pSer46-MARCKS), and reveal an increase of neuronal necrosis during pre-symptomatic phase and a subsequent decrease during symptomatic phase. Postmortem brains of mild cognitive impairment (MCI) rather than symptomatic AD patients reveal a remarkable increase of necrosis. In vivo imaging reveals instability of endoplasmic reticulum (ER) in mouse AD models and genome-edited human AD iPS cell-derived neurons. The level of nuclear Yes-associated protein (YAP) is remarkably decreased in such neurons under AD pathology due to the sequestration into cytoplasmic amyloid beta (Aß) aggregates, supporting the feature of YAP-dependent necrosis. Suppression of early-stage neuronal death by AAV-YAPdeltaC reduces the later-stage extracellular Aß burden and cognitive impairment, suggesting that preclinical/prodromal YAP-dependent neuronal necrosis represents a target for AD therapeutics.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Animais , Núcleo Celular/metabolismo , Disfunção Cognitiva/líquido cefalorraquidiano , Disfunção Cognitiva/patologia , Simulação por Computador , Modelos Animais de Doenças , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Feminino , Proteína HMGB1/líquido cefalorraquidiano , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisofosfolipídeos/metabolismo , Masculino , Camundongos Transgênicos , Necrose , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Imagem com Lapso de Tempo , Proteínas de Sinalização YAP
17.
bioRxiv ; 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33398268

RESUMO

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2 induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe respiratory dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway barrier damage and mitigate virus spread.

18.
Mol Syst Biol ; 16(12): e9310, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33438817

RESUMO

Many proteins involved in signal transduction contain peptide recognition modules (PRMs) that recognize short linear motifs (SLiMs) within their interaction partners. Here, we used large-scale peptide-phage display methods to derive optimal ligands for 163 unique PRMs representing 79 distinct structural families. We combined the new data with previous data that we collected for the large SH3, PDZ, and WW domain families to assemble a database containing 7,984 unique peptide ligands for 500 PRMs representing 82 structural families. For 74 PRMs, we acquired enough new data to map the specificity profiles in detail and derived position weight matrices and binding specificity logos based on multiple peptide ligands. These analyses showed that optimal peptide ligands resembled peptides observed in existing structures of PRM-ligand complexes, indicating that a large majority of the phage-derived peptides are likely to target natural peptide-binding sites and could thus act as inhibitors of natural protein-protein interactions. The complete dataset has been assembled in an online database (http://www.prm-db.org) that will enable many structural, functional, and biological studies of PRMs and SLiMs.


Assuntos
Bases de Dados de Proteínas , Peptídeos/metabolismo , Inquéritos e Questionários , Sequência de Aminoácidos , Bacteriófagos/metabolismo , Humanos , Ligantes , Peptídeos/química
19.
J Exp Clin Cancer Res ; 38(1): 349, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399037

RESUMO

BACKGROUND: Over the past decade, newly designed cancer therapies have not significantly improved the survival of patients diagnosed with Malignant Pleural Mesothelioma (MPM). Among a limited number of genes that are frequently mutated in MPM several of them encode proteins that belong to the HIPPO tumor suppressor pathway. METHODS: The anticancer effects of the top flower standardized extract of Filipendula vulgaris (Dropwort) were characterized in "in vitro" and "in vivo" models of MPM. At the molecular level, two "omic" approaches were used to investigate Dropwort anticancer mechanism of action: a metabolomic profiling and a phosphoarray analysis. RESULTS: We found that Dropwort significantly reduced cell proliferation, viability, migration and in vivo tumor growth of MPM cell lines. Notably, Dropwort affected viability of tumor-initiating MPM cells and synergized with Cisplatin and Pemetrexed in vitro. Metabolomic profiling revealed that Dropwort treatment affected both glycolysis/tricarboxylic acid cycle as for the decreased consumption of glucose, pyruvate, succinate and acetate, and the lipid metabolism. We also document that Dropwort exerted its anticancer effects, at least partially, promoting YAP and TAZ protein ubiquitination. CONCLUSIONS: Our findings reveal that Dropwort is a promising source of natural compound(s) for targeting the HIPPO pathway with chemo-preventive and anticancer implications for MPM management.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Metabolismo Energético/efeitos dos fármacos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Mesotelioma/etiologia , Mesotelioma/metabolismo , Extratos Vegetais/farmacologia , Fatores de Transcrição/metabolismo , Aciltransferases , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Filipendula/química , Humanos , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , Extratos Vegetais/química , Ligação Proteica
20.
Diseases ; 6(3)2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30011814

RESUMO

Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like particles (VLPs) that mimic egress of infectious virus. LFV Z contains both PTAP and PPPY L-domain motifs that are known to recruit host proteins that are important for mediating efficient virus egress and spread. The viral PPPY motif is known to interact with specific host WW-domain bearing proteins. Here we identified host WW-domain bearing protein BCL2 Associated Athanogene 3 (BAG3) as a LFV Z PPPY interactor using our proline-rich reading array of WW-domain containing mammalian proteins. BAG3 is a stress-induced molecular co-chaperone that functions to regulate cellular protein homeostasis and cell survival via Chaperone-Assisted Selective Autophagy (CASA). Similar to our previously published findings for the VP40 proteins of Ebola and Marburg viruses, our results using VLP budding assays, BAG3 knockout cells, and confocal microscopy indicate that BAG3 is a WW-domain interactor that negatively regulates egress of LFV Z VLPs, rather than promoting VLP release. Our results suggest that CASA and specifically BAG3 may represent a novel host defense mechanism, whereby BAG3 may dampen egress of several hemorrhagic fever viruses by interacting and interfering with the budding function of viral PPxY-containing matrix proteins.

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