RESUMEN
Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6,000 mouse self-peptides presented on MHC Class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.
RESUMEN
With current trends in proteomics, especially regarding clinical and low input (to single cell) samples, it is increasingly important to both maximize the throughput of the analysis and maintain as much sensitivity as possible. The new generation of mass spectrometers (MS) are taking a huge leap in sensitivity, allowing analysis of samples with shorter liquid chromatography (LC) methods while digging as deep in the proteome. However, the throughput can be doubled by implementing a dual column nano-LC-MS configuration. For this purpose, we used a dual-column setup with a two-outlet electrospray source and compared it to a classic dual-column setup with a single-outlet source.
Asunto(s)
Nanotecnología , Proteómica , Espectrometría de Masa por Ionización de Electrospray , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Humanos , Cromatografía Liquida/métodos , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and (V600E)BRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like (V600E)BRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.
Asunto(s)
Astrocitoma/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Niño , Dimerización , Inhibidores Enzimáticos/farmacología , Vectores Genéticos , Células HEK293 , Humanos , Indoles/farmacología , Ratones , Ratones Endogámicos BALB C , Mutación , Células 3T3 NIH , Trasplante de Neoplasias , Fenotipo , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Transducción de Señal , Sulfonamidas/farmacología , VemurafenibRESUMEN
The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.
Asunto(s)
Fibroblastos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Androstadienos/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Mamíferos/citología , Activación Enzimática/efectos de los fármacos , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células HEK293 , Humanos , Immunoblotting , Fosfatos de Inositol/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/genética , Fosfatos de Fosfatidilinositol/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , WortmaninaRESUMEN
Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.
Asunto(s)
Vacunas contra el Cáncer , Galactosilceramidas , Ratones Endogámicos C57BL , Nanopartículas , Ovalbúmina , Animales , Galactosilceramidas/administración & dosificación , Galactosilceramidas/química , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Femenino , Nanopartículas/química , Nanopartículas/administración & dosificación , Ovalbúmina/inmunología , Ovalbúmina/administración & dosificación , Vacunas de ARNm , Adyuvantes Inmunológicos/administración & dosificación , Linfocitos T CD8-positivos/inmunología , ARN Mensajero/administración & dosificación , Ratones , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Lípidos/química , LiposomasRESUMEN
Pediatric brain tumors are the leading cause of cancer-related death in children in the United States and contribute a disproportionate number of potential years of life lost compared to adult cancers. Moreover, survivors frequently suffer long-term side effects, including secondary cancers. The Children's Brain Tumor Network (CBTN) is a multi-institutional international clinical research consortium created to advance therapeutic development through the collection and rapid distribution of biospecimens and data via open-science research platforms for real-time access and use by the global research community. The CBTN's 32 member institutions utilize a shared regulatory governance architecture at the Children's Hospital of Philadelphia to accelerate and maximize the use of biospecimens and data. As of August 2022, CBTN has enrolled over 4700 subjects, over 1500 parents, and collected over 65,000 biospecimen aliquots for research. Additionally, over 80 preclinical models have been developed from collected tumors. Multi-omic data for over 1000 tumors and germline material are currently available with data generation for > 5000 samples underway. To our knowledge, CBTN provides the largest open-access pediatric brain tumor multi-omic dataset annotated with longitudinal clinical and outcome data, imaging, associated biospecimens, child-parent genomic pedigrees, and in vivo and in vitro preclinical models. Empowered by NIH-supported platforms such as the Kids First Data Resource and the Childhood Cancer Data Initiative, the CBTN continues to expand the resources needed for scientists to accelerate translational impact for improved outcomes and quality of life for children with brain and spinal cord tumors.
Asunto(s)
Neoplasias Encefálicas , Calidad de Vida , Adulto , Humanos , Niño , Neoplasias Encefálicas/terapiaRESUMEN
Listeria monocytogenes is a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine against Listeria is available. Attenuated Listeria strains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screen for Listeria peptides presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detect 68 Listeria immunopeptides from 42 different bacterial proteins, including several known antigens. Peptides presented on different cell lines are often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations results in specific CD8+ T-cell responses and induces protection in vaccination challenge experiments in mice. Our results can serve as a starting point for the development of a clinical mRNA vaccine against Listeria and aid to improve attenuated Listeria vaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.
Asunto(s)
Listeria monocytogenes , Listeria , Listeriosis , Animales , Proteínas Bacterianas/genética , Vacunas Bacterianas/genética , Linfocitos T CD8-positivos , Humanos , Epítopos Inmunodominantes , Liposomas , Listeria/genética , Listeria monocytogenes/genética , Listeriosis/prevención & control , Proteínas de la Membrana , Ratones , Nanopartículas , Vacunas Atenuadas , Vacunas Sintéticas/genética , Vacunas de ARNmRESUMEN
RNF213 is a large, poorly characterized interferon-induced protein. Mutations in RNF213 are associated with predisposition for Moyamoya disease (MMD), a rare cerebrovascular disorder. Recently, RNF213 was found to have broad antimicrobial activity in vitro and in vivo, yet the molecular mechanisms behind this function remain unclear. Using mass spectrometry-based proteomics and validation by real-time PCR we report here that knockdown of RNF213 leads to transcriptional upregulation of MVP and downregulation of CYR61, in line with reported pro- and anti-bacterial activities of these proteins. Knockdown of RNF213 also results in downregulation of DDAH1, which we discover to exert antimicrobial activity against Listeria monocytogenes infection. DDAH1 regulates production of nitric oxide (NO), a molecule with both vascular and antimicrobial effects. We show that NO production is reduced in macrophages from RNF213 KO mice, suggesting that RNF213 controls Listeria infection through regulation of DDAH1 transcription and production of NO. Our findings propose a potential mechanism for the antilisterial activity of RNF213 and highlight NO as a potential link between RNF213-mediated immune responses and the development of MMD.
Asunto(s)
Enfermedad de Moyamoya , Óxido Nítrico , Adenosina Trifosfatasas/genética , Animales , Predisposición Genética a la Enfermedad , Ratones , Proteoma , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antiinfecciosos/metabolismo , Citocinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Células A549 , Animales , Enterovirus/fisiología , Células HEK293 , Células HeLa , Herpesvirus Humano 1/fisiología , Humanos , Interferón Tipo I/metabolismo , Gotas Lipídicas/metabolismo , Listeria monocytogenes/fisiología , Masculino , Ratones Endogámicos C57BL , Unión Proteica , Multimerización de Proteína , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Células THP-1 , Ubiquitina/metabolismoRESUMEN
Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along with the sample preparation protocol, in particular, after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in the MS-loading solvent, consuming only 2 µL of sample. We compared the performance of the microfluidic spectrophotometer with a standard device and determined the optimal sample amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. A careful evaluation of selected LC and MS parameters allowed us to define 3 µg as an optimal peptide amount to be injected into this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells and showing that injecting equal peptide amounts, rather than approximate ones, result in less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.
RESUMEN
Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.
RESUMEN
Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.