RESUMEN
Cancer immunotherapies are recently gaining attention as viable therapeutic options. There are two types of immunotherapy:passive and active. The passive immunotherapies include several treatments such as monoclonal antibodies,either alone or as antibody-drug conjugates. The active immunotherapies include cancer vaccines which utilize the patient′s own cells as antigen presenting cells and target specific cancer antigens,and chimeric antigen receptor T-cell(CAR-T)therapy which engineers a patient′s T-cells to recognize cancer antigens through chimeric antigen receptors. Recent successes include the US FDA approval of a number of cancer immunotherapies such as treatments utilizing monoclonal antibodies against immune checkpoint inhibitors,the Provenge cancer vaccine that targets prostrate cancer,and a CAR-T against relapsed/refractory acute lymphoblastic leukemia that was designated with breakthrough drug status,all of which has had drug companies investigating cancer immunotherapies with intense enthusiasm. In this review we discuss where the field of immune-oncology stands today,highlight the latest findings and hypothesize future directions.
RESUMEN
Grb2 and ShcA are two phosphotyrosine-binding proteins that link receptor protein-tyrosine kinases to activation of the Ras-Erk pathway. While some receptors bind Grb2 directly, others bind ShcA, which provides a binding site for Grb2. In order to compare signal transduction through a Grb2-binding site with signal transduction through a ShcA-binding site, we replaced the ShcA-binding site in the NGF receptor with a Grb2-binding site. Our results show that the Grb2- and ShcA-binding sites have similar abilities to activate the Ras-Erk and PI 3-kinase-Akt pathways. In contrast, they displayed dramatic differences in their ability to activate DNA synthesis.
Asunto(s)
Proteína Adaptadora GRB2/metabolismo , Ingeniería de Proteínas/métodos , Receptor de Factor de Crecimiento Nervioso/genética , Receptor de Factor de Crecimiento Nervioso/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Animales , Sitios de Unión , ADN/biosíntesis , Humanos , Ratones , Células 3T3 NIH , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Factor de Crecimiento Nervioso/química , Proteínas ras/metabolismoRESUMEN
The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic component of two evolutionarily conserved signaling complexes. mTOR signaling complex 1 (mTORC1) is a key regulator of growth factor and nutrient signaling. S6 kinase is the best-characterized downstream effector of mTORC1. mTOR signaling complex 2 (mTORC2) has a role in regulating the actin cytoskeleton and activating Akt through S473 phosphorylation. Herein, we show that mTOR is phosphorylated differentially when associated with mTORC1 and mTORC2 and that intact complexes are required for these mTORC-specific mTOR phosphorylations. Specifically, we find that mTORC1 contains mTOR phosphorylated predominantly on S2448, whereas mTORC2 contains mTOR phosphorylated predominantly on S2481. Using S2481 phosphorylation as a marker for mTORC2 sensitivity to rapamycin, we find that mTORC2 formation is in fact rapamycin sensitive in several cancer cell lines in which it had been previously reported that mTORC2 assembly and function were rapamycin insensitive. Thus, phospho-S2481 on mTOR serves as a biomarker for intact mTORC2 and its sensitivity to rapamycin.
Asunto(s)
Proteínas Quinasas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Biomarcadores , Células Cultivadas , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Datos de Secuencia Molecular , Complejos Multiproteicos , Fosforilación , Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR , Factores de Transcripción/químicaRESUMEN
Hyperosmotic stress can be encountered by the kidney and the skin, as well as during treatment of acute brain damage. It can lead to cell cycle arrest or apoptosis. Exactly how mammalian cells detect hyperosmolarity and how the cell chooses between cell cycle arrest or death remains to be established. It has been proposed that hyperosmolarity is detected directly by growth factor receptor protein tyrosine kinases. To investigate this, we tested whether growth factors and osmotic stress cooperate in the activation of signaling pathways. Receptors responded normally to the presence of growth factors, and we observed normal levels of GTP-bound Ras under hyperosmotic conditions. In contrast, activation of Raf, Akt, ERK1, ERK2, and c-Jun NH2-terminal kinase was strongly reduced. These observations suggest that hyperosmotic conditions block signaling directly downstream of active Ras. It is thought that apoptotic cell death due to environmental stress is initiated by cytochrome c release from the mitochondria. Visualization of cytochrome c using immunofluorescence showed that hypertonic conditions result in a breakup of the mitochondrial network, which is reestablished within 1 h after hypertonic medium is replaced with isotonic medium. When we carried out live imaging, we observed that the mitochondrial membrane potential disappeared immediately after the onset of hyperosmotic shock. Our observations provide new insights into the hypertonic stress response pathway. In addition, they show that signaling downstream of Ras and mitochondrial dynamics can easily be manipulated by the exposure of cells to hyperosmotic conditions.
Asunto(s)
Caspasas/metabolismo , Activación Enzimática/fisiología , Mitocondrias/patología , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 3 , Caspasas/efectos de los fármacos , Chlorocebus aethiops , Citocromos c/efectos de los fármacos , Citocromos c/metabolismo , Activación Enzimática/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Immunoblotting , Inmunoprecipitación , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Presión Osmótica/efectos de los fármacos , Solución Salina Hipertónica , Transducción de Señal/efectos de los fármacos , Células Vero , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
Protein-tyrosine kinases are known regulators of cell division that have been implicated in the onset of a variety of malignancies. They act through cellular signaling proteins that bind to specific autophosphorylation sites. To find out whether these autophosphorylation sites can be used to identify downstream signaling proteins, synthetic peptides based on an autophosphorylation site in the colony-stimulating factor-1 (CSF-1) receptor were linked to agarose beads and incubated with lysates from macrophages. Bound proteins were analyzed by MS, leading to the identification of both known and novel CSF-1 receptor-interacting proteins. The approach presented here can be applied to phosphorylation sites in a wide variety of proteins. It will lead to the identification of novel protein-protein interactions and provide new insights into the mechanics of signal transduction. Novel protein-protein interactions may provide useful targets for the development of drugs that interfere with the activation of signaling cascades used by protein-tyrosine kinases to turn on cell division.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Proteínas Tirosina Quinasas/metabolismo , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Secuencia Conservada , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Ratones , Datos de Secuencia Molecular , Fosfopéptidos , Señales de Clasificación de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Homología de Secuencia de Aminoácido , Tirosina/químicaRESUMEN
The Listeria monocytogenes protein InlB promotes invasion of mammalian cells through activation of the receptor tyrosine kinase Met. The InlB N-cap, a approximately 40 residue part of the domain that binds Met, was previously observed to bind two calcium ions in a novel and unusually exposed manner. Because subsequent work raised questions about the existence of these calcium-binding sites, we assayed calcium binding in solution to the InlB N-cap. We show that calcium ions are bound with dissociation constants in the low micromolar range at the two identified sites, and that the sites interact with one another. We demonstrate that the calcium ions are not required for structure, and also find that they have no appreciable effect on Met activation or intracellular invasion. Therefore, our results indicate that the sites are fortuitous in InlB, but also suggest that the simple architecture of the sites may be adaptable for protein engineering purposes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al Calcio/química , Calcio/metabolismo , Listeria monocytogenes , Proteínas de la Membrana/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación , Secuencias Repetitivas de AminoácidoRESUMEN
The Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met. Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond. In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N-terminal LRR region and C-terminal GW domains. The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans. Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor-mediated mode, as opposed to the ligand-mediated mode observed for the LRR fragment. Surprisingly, we find that gC1q-R, a host protein implicated in InlB-mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q-R is unnecessary for bacterial invasion. Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone-like mimicry of HGF.
Asunto(s)
Listeria monocytogenes/patogenicidad , Glicoproteínas de Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Estructura Terciaria de Proteína , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Células CHO , Línea Celular , Chlorocebus aethiops , Cricetinae , Dimerización , Glicosaminoglicanos/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Proteínas de la Membrana/genética , Modelos Moleculares , Mutación , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Complemento/fisiología , Eliminación de Secuencia/genética , Eliminación de Secuencia/fisiología , Células VeroRESUMEN
Internalin B (InlB) is a protein present on the surface of Listeria monocytogenes that mediates bacterial entry into mammalian cells. It is thought that InlB acts by binding directly to the hepatocyte growth factor (HGF) receptor, present on the surface of host cells. Binding of InlB to the HGF receptor results in mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase activation, followed by changes in the organization of the actin cytoskeleton. Here we have compared signaling by HGF and InlB. Whereas stimulation with equivalent concentrations of HGF and InlB elicits similar activation of the HGF receptor, we observed striking differences in downstream activation of MAP kinase. InlB leads to a greater activation of the Ras-MAP kinase pathway than does HGF. The leucine-rich repeat region, which was previously shown to be sufficient for binding and activation of the HGF receptor, lacks the ability to super-activate the Ras-MAP kinase pathway. Analysis of a series of deletion mutants suggests that it is the B repeat region between the leucine-rich repeat and GW domains that endows InlB with an increased ability to turn on the Ras-MAP kinase pathway. These unexpected observations suggest that HGF and InlB use alternative mechanisms to turn on cellular signaling pathways.