Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 39
Filtrar
1.
Neuro Oncol ; 2024 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-39248287

RESUMEN

BACKGROUND: Co-amplification of EGFR and EGFRvIII, a tumor-specific truncation mutant of EGFR, represent hallmark genetic lesions in glioblastoma. METHODS: We used phospho-proteomics, RNA-sequencing, TCGA data and glioblastoma cell culture and mouse models to study the signal transduction mediated by EGFR and EGFRvIII. RESULTS: We report that EGFR and EGFRvIII stimulate the innate immune defense receptor Toll-like Receptor 2 (TLR2); and that knockout of TLR2 dramatically improved survival in orthotopic glioblastoma xenografts. EGFR and EGFRvIII activated TLR2 in a ligand-independent manner, promoting tumor growth and immune evasion. We show that EGFR and EGFRvIII cooperate to activate the Rho-associated protein kinase ROCK2, which modulated malignant progression both by activating TLR2 and WNT signaling, and through remodeling the tumor microenvironment. CONCLUSION: Together, our findings show that EGFR and EGFRvIII cooperate to drive tumor progression through ROCK2 and downstream WNT-ß-catenin/TLR2 signaling pathways.

2.
Nat Commun ; 15(1): 7873, 2024 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-39251601

RESUMEN

Meningiomas are associated with inactivation of NF2/Merlin, but approximately one-third of meningiomas with favorable clinical outcomes retain Merlin expression. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that may be used to guide treatment de-escalation or imaging surveillance are lacking. Here, we use single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma xenografts and patients to define biochemical mechanisms and an imaging biomarker that underlie Merlin-intact meningiomas. We find Merlin serine 13 (S13) dephosphorylation drives meningioma Wnt signaling and tumor growth by attenuating inhibitory interactions with ß-catenin and activating the Wnt pathway. MRI analyses show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC). These results define mechanisms underlying a potential imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with Merlin-intact meningiomas.


Asunto(s)
Imagen por Resonancia Magnética , Neoplasias Meníngeas , Meningioma , Neurofibromina 2 , Vía de Señalización Wnt , Meningioma/diagnóstico por imagen , Meningioma/metabolismo , Meningioma/patología , Meningioma/genética , Humanos , Fosforilación , Neurofibromina 2/metabolismo , Neurofibromina 2/genética , Animales , Imagen por Resonancia Magnética/métodos , Neoplasias Meníngeas/diagnóstico por imagen , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patología , Neoplasias Meníngeas/genética , Ratones , Línea Celular Tumoral , beta Catenina/metabolismo , beta Catenina/genética , Femenino , Serina/metabolismo , Masculino , Proteómica/métodos , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética
3.
Cell Host Microbe ; 32(7): 1050-1058.e7, 2024 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-38870941

RESUMEN

Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.


Asunto(s)
ADN Viral , Genoma Viral , Fagos Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/virología , Fagos Pseudomonas/genética , Fagos Pseudomonas/metabolismo , ADN Viral/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Bacteriófagos/genética , Bacteriófagos/fisiología , Virión/metabolismo , Replicación Viral , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Lípidos , Replicación del ADN
4.
ArXiv ; 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38800652

RESUMEN

Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein-coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification-mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein-protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity-tagged "bait" proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity-tagged "bait" gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross-run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al. 20231.

5.
mBio ; 15(4): e0222223, 2024 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-38411080

RESUMEN

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Infecciones por VIH , VIH , Factor 2 Asociado a Receptor de TNF , Ubiquitina-Proteína Ligasas , Latencia del Virus , Humanos , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Linfocitos T CD4-Positivos , Sistemas CRISPR-Cas , Factor 2 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Replicación Viral , VIH/fisiología
6.
Nat Commun ; 15(1): 476, 2024 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-38216587

RESUMEN

Mechanisms specifying cancer cell states and response to therapy are incompletely understood. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas, the most common tumors of the peripheral nervous system. We find schwannomas are comprised of 2 molecular groups that are distinguished by activation of neural crest or nerve injury pathways that specify tumor cell states and the architecture of the tumor immune microenvironment. Moreover, we find radiotherapy is sufficient for interconversion of neural crest schwannomas to immune-enriched schwannomas through epigenetic and metabolic reprogramming. To define mechanisms underlying schwannoma groups, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of tumor evolution and establish a paradigm of epigenetic and metabolic reprograming of cancer cells that shapes the immune microenvironment in response to radiotherapy.


Asunto(s)
Neurilemoma , Humanos , Neurilemoma/genética , Neurilemoma/patología , Epigénesis Genética , Reprogramación Celular/genética , Microambiente Tumoral/genética
7.
Mol Biol Cell ; 35(3): ar27, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38117589

RESUMEN

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.


Asunto(s)
Legionella pneumophila , Proteínas de Unión al GTP Monoméricas , Animales , Legionella pneumophila/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Bacterianas/metabolismo , Ubiquitinación , Ubiquitina/metabolismo , Vacuolas/metabolismo , Ligasas/metabolismo , Mamíferos/metabolismo
8.
Sci Adv ; 9(49): eadj4884, 2023 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-38064566

RESUMEN

Oxygen deprivation and excess are both toxic. Thus, the body's ability to adapt to varying oxygen tensions is critical for survival. While the hypoxia transcriptional response has been well studied, the post-translational effects of oxygen have been underexplored. In this study, we systematically investigate protein turnover rates in mouse heart, lung, and brain under different inhaled oxygen tensions. We find that the lung proteome is the most responsive to varying oxygen tensions. In particular, several extracellular matrix (ECM) proteins are stabilized in the lung under both hypoxia and hyperoxia. Furthermore, we show that complex 1 of the electron transport chain is destabilized in hyperoxia, in accordance with the exacerbation of associated disease models by hyperoxia and rescue by hypoxia. Moreover, we nominate MYBBP1A as a hyperoxia transcriptional regulator, particularly in the context of rRNA homeostasis. Overall, our study highlights the importance of varying oxygen tensions on protein turnover rates and identifies tissue-specific mediators of oxygen-dependent responses.


Asunto(s)
Hiperoxia , Oxígeno , Animales , Ratones , Encéfalo/metabolismo , Hiperoxia/genética , Hiperoxia/metabolismo , Hipoxia/metabolismo , Pulmón/metabolismo , Oxígeno/metabolismo
9.
bioRxiv ; 2023 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-37873080

RESUMEN

ApoE4 is the primary risk factor for Alzheimer's Disease. While apoE is primarily expressed by astrocytes, AD pathology including endosomal abnormalities and mitochondrial dysfunction first occurs in neurons. Lysosomes are poised at the convergence point between these features. We find that apoE4-expressing cells exhibit lysosomal alkalinization, reduced lysosomal proteolysis, and impaired mitophagy. To identify driving factors for this lysosomal dysfunction, we performed quantitative lysosomal proteome profiling. This revealed that apoE4 expression results in lysosomal depletion of Lgals3bp and accumulation of Tmed5 in both Neuro-2a cells and postmitotic human neurons. Modulating the expression of both proteins affected lysosomal function, with Tmed5 knockdown rescuing lysosomal alkalinization in apoE4 cells, and Lgals3bp knockdown causing lysosomal alkalinization and reduced lysosomal density in apoE3 cells. Taken together, our work reveals that apoE4 exerts gain-of-toxicity by alkalinizing the lysosomal lumen, pinpointing lysosomal Tmed5 accumulation and Lgals3bp depletion as apoE4-associated drivers for this phenotype.

10.
Cell Rep Med ; 4(11): 101244, 2023 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-37858338

RESUMEN

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.


Asunto(s)
Melanoma , Neoplasias Cutáneas , Neoplasias de la Úvea , Animales , Ratones , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/uso terapéutico , Evaluación Preclínica de Medicamentos , Neoplasias de la Úvea/tratamiento farmacológico , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Inhibidores de Proteínas Quinasas/farmacología
11.
Nat Cell Biol ; 25(11): 1600-1615, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37857833

RESUMEN

A widespread strategy employed by pathogens to establish infection is to inhibit host-cell protein synthesis. Legionella pneumophila, an intracellular bacterial pathogen and the causative organism of Legionnaires' disease, secretes a subset of protein effectors into host cells that inhibit translation elongation. Mechanistic insights into how the bacterium targets translation elongation remain poorly defined. We report here that the Legionella effector SidI functions in an unprecedented way as a transfer-RNA mimic that directly binds to and glycosylates the ribosome. The 3.1 Å cryo-electron microscopy structure of SidI reveals an N-terminal domain with an 'inverted L' shape and surface-charge distribution characteristic of tRNA mimicry, and a C-terminal domain that adopts a glycosyl transferase fold that licenses SidI to utilize GDP-mannose as a sugar precursor. This coupling of tRNA mimicry and enzymatic action endows SidI with the ability to block protein synthesis with a potency comparable to ricin, one of the most powerful toxins known. In Legionella-infected cells, the translational pausing activated by SidI elicits a stress response signature mimicking the ribotoxic stress response, which is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAKα and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus and orchestrates the transcription of stress-inducible genes that promote cell death, revealing a major role for ATF3 in the response to collided ribosome stress. Together, our findings elucidate a novel mechanism by which a pathogenic bacterium employs tRNA mimicry to hijack a ribosome-to-nuclear signalling pathway that regulates cell fate.


Asunto(s)
Legionella pneumophila , Legionella , Enfermedad de los Legionarios , Humanos , Legionella/metabolismo , Microscopía por Crioelectrón , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/genética , Enfermedad de los Legionarios/microbiología , Transferasas/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología
12.
Cell ; 186(21): 4597-4614.e26, 2023 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-37738970

RESUMEN

SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/virología , Inmunidad Innata/genética , Pandemias , SARS-CoV-2/genética
13.
Nat Commun ; 14(1): 6030, 2023 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-37758692

RESUMEN

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.


Asunto(s)
COVID-19 , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Proteómica , Replicación Viral/genética , SARS-CoV-2 , Antivirales/metabolismo , Interacciones Huésped-Patógeno/genética
14.
bioRxiv ; 2023 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-37577546

RESUMEN

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s arsenal of ~330 secreted effector proteins have been biochemically characterized as ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p hijacks ubiquitin signaling within the host cell, we undertook a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection results in increased ubiquitination of host proteins regulating subcellular trafficking and membrane dynamics, most notably 63 of ~160 mammalian Ras superfamily small GTPases. We determine that these small GTPases predominantly undergo non-degradative monoubiquitination, and link ubiquitination to recruitment to the Legionella-containing vacuole membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central, but likely indirect, role in cross-family small GTPase ubiquitination. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. This work positions L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

15.
Res Sq ; 2023 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-37645943

RESUMEN

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.

16.
Res Sq ; 2023 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-36993679

RESUMEN

Meningiomas are the most common primary intracranial tumors and are associated with inactivation of the tumor suppressor NF2/Merlin, but one-third of meningiomas retain Merlin expression and typically have favorable clinical outcomes. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that predict meningioma outcomes and could be used to guide treatment de-escalation or imaging surveillance of Merlin-intact meningiomas are lacking. Here we integrate single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma cells, xenografts, and human patients to define biochemical mechanisms and an imaging biomarker that distinguish Merlin-intact meningiomas with favorable clinical outcomes from meningiomas with unfavorable clinical outcomes. We find Merlin drives meningioma Wnt signaling and tumor growth through a feed-forward mechanism that requires Merlin dephosphorylation on serine 13 (S13) to attenuate inhibitory interactions with ß-catenin and activate the Wnt pathway. Meningioma MRI analyses of xenografts and human patients show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC) on diffusion-weighted imaging. In sum, our results shed light on Merlin posttranslational modifications that regulate meningioma Wnt signaling and tumor growth in tumors without NF2/Merlin inactivation. To translate these findings to clinical practice, we establish a non-invasive imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with favorable meningiomas.

17.
Children (Basel) ; 10(3)2023 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-36980055

RESUMEN

Physical literacy (PL) is gaining more attention from educational policy-makers, practitioners, and researchers as a way to improve health and wellness outcomes for children and youth. While the development of PL is important for early years children, there is limited attention in the literature that explores the political, cultural, and social discourses imbued in colonialism that implicate how PL is actualized in Indigenous early childhood education (ECE) contexts. This case assemblage explores how the culturally rooted, interdisciplinary, and community-based PL initiative, Nature's Way-Our Way (NWOW), negotiated movement with three early childhood educators in the pilot project with an early childhood education centre (ECEC) in Saskatchewan, Canada. Through postqualitative approaches to research, this case assemblage adopts new materialist methodologies to show how the natural order of knowing in movement was disrupted through moments of rupture generating stories of PL to encompass radical relationality with land. As land becomes a vital and lively part of PL storying, it can function as an important protective factor for Indigenous preschool-aged children's wholistic wellness.

18.
Cancer Res ; 82(21): 3950-3961, 2022 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-36273492

RESUMEN

Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription-replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs. SIGNIFICANCE: Loss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs.


Asunto(s)
Neoplasias Gástricas , Humanos , Proteómica , Inhibidores de Proteínas Quinasas , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas de Unión al ARN , Proteínas de la Ataxia Telangiectasia Mutada
19.
J Proteome Res ; 21(4): 1124-1136, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35234472

RESUMEN

The use of multiple proteases has been shown to increase protein sequence coverage in proteomics experiments; however, due to the additional analysis time required, it has not been widely adopted in routine data-dependent acquisition (DDA) proteomic workflows. Alternatively, data-independent acquisition (DIA) has the potential to analyze multiplexed samples from different protease digests, but has been primarily optimized for fragmenting tryptic peptides. Here we evaluate a DIA multiplexing approach that combines three proteolytic digests (Trypsin, AspN, and GluC) into a single sample. We first optimize data acquisition conditions for each protease individually with both the canonical DIA fragmentation mode (beam type CID), as well as resonance excitation CID, to determine optimal consensus conditions across proteases. Next, we demonstrate that application of these conditions to a protease-multiplexed sample of human peptides results in similar protein identifications and quantitative performance as compared to trypsin alone, but enables up to a 63% increase in peptide detections, and a 45% increase in nonredundant amino acid detections. Nontryptic peptides enabled noncanonical protein isoform determination and resulted in 100% sequence coverage for numerous proteins, suggesting the utility of this approach in applications where sequence coverage is critical, such as protein isoform analysis.


Asunto(s)
Proteoma , Proteómica , Secuencia de Aminoácidos , Humanos , Péptido Hidrolasas/genética , Péptidos/química , Proteoma/genética , Proteómica/métodos
20.
Cell ; 185(4): 712-728.e14, 2022 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-35063084

RESUMEN

Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.


Asunto(s)
Mitocondrias/metabolismo , Degeneración Nerviosa/metabolismo , Mapas de Interacción de Proteínas , Sinapsis/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Aminoácidos/metabolismo , Biotinilación , Encéfalo/metabolismo , Encéfalo/patología , Núcleo Celular/metabolismo , Progresión de la Enfermedad , Metabolismo Energético , Demencia Frontotemporal/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Degeneración Nerviosa/patología , Neuronas/metabolismo , Unión Proteica , Dominios Proteicos , Proteómica , Índice de Severidad de la Enfermedad , Fracciones Subcelulares/metabolismo , Tauopatías/genética , Proteínas tau/química
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA