RESUMO
The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Most current immunotherapeutic targets are tumor-associated antigens that are also found in healthy tissues and often do not display sufficient cancer selectivity to be used as targets for potent antibody-based immunotherapeutic treatments, such as chimeric antigen receptor (CAR) T cells. Many solid tumors, however, display aberrant glycosylation that results in expression of tumor-associated carbohydrate antigens that are distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes within existing or novel glycoprotein targets may provide the cancer selectivity needed for immunotherapy of solid tumors. However, to date only a few such glycopeptide epitopes have been targeted. Here, we used O-glycoproteomics data from multiple cell lines to identify a glycopeptide epitope in CD44v6, a cancer-associated CD44 isoform, and developed a cancer-specific mAb, 4C8, through a glycopeptide immunization strategy. 4C8 selectively binds to Tn-glycosylated CD44v6 in a site-specific manner with low nanomolar affinity. 4C8 was shown to be highly cancer specific by IHC of sections from multiple healthy and cancerous tissues. 4C8 CAR T cells demonstrated target-specific cytotoxicity in vitro and significant tumor regression and increased survival in vivo. Importantly, 4C8 CAR T cells were able to selectively kill target cells in a mixed organotypic skin cancer model having abundant CD44v6 expression without affecting healthy keratinocytes, indicating tolerability and safety.
Assuntos
Anticorpos Monoclonais , Neoplasias , Humanos , Anticorpos Monoclonais/farmacologia , Neoplasias/patologia , Glicoproteínas , Epitopos , GlicopeptídeosRESUMO
BACKGROUND: Novel immunotherapies targeting cancer-associated truncated O-glycans Tn (GalNAcα-Ser/Thr) and STn (Neu5Acα2-6GalNacα-Ser/Thr) are promising strategies for cancer treatment. However, no comprehensive, antibody-based mapping of truncated O-glycans in tumours exist to guide drug development. METHODS: We used monoclonal antibodies to map the expression of truncated O-glycans in >700 tissue cores representing healthy and tumour tissues originating from breast, colon, lung, pancreas, skin, CNS and mesenchymal tissue. Patient-derived xenografts were used to evaluate Tn expression upon tumour engraftment. RESULTS: The Tn-antigen was highly expressed in breast (57%, n = 64), colorectal (51%, n = 140) and pancreatic (53%, n = 108) tumours, while STn was mainly observed in colorectal (80%, n = 140) and pancreatic (56%, n = 108) tumours. We observed no truncated O-glycans in mesenchymal tumours (n = 32) and low expression of Tn (5%, n = 87) and STn (1%, n = 75) in CNS tumours. No Tn-antigen was found in normal tissue (n = 124) while STn was occasionally observed in healthy gastrointestinal tissue. Surface expression of Tn-antigen was identified across several cancers. Tn and STn expression decreased with tumour grade, but not with cancer stage. Numerous xenografts maintained Tn expression. CONCLUSIONS: Surface expression of truncated O-glycans is limited to cancers of epithelial origin, making Tn and STn attractive immunological targets in the treatment of human carcinomas.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Neoplasias/patologia , Análise Serial de Tecidos/métodos , Animais , Anticorpos Monoclonais/imunologia , Estudos de Casos e Controles , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Gradação de Tumores , Estadiamento de Neoplasias , Transplante de Neoplasias , Neoplasias/classificação , Neoplasias/metabolismo , Regulação para CimaRESUMO
The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of â¼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only â¼17% partition equally. A protein's native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than â¼100 kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1. This resulted in the expected re-localization of proteins toward the nucleus, but only 3% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.
Assuntos
Proteínas de Anfíbios/genética , Proteínas Nucleares/genética , Proteoma/genética , Xenopus/genética , Proteínas de Anfíbios/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Proteoma/metabolismo , Xenopus/metabolismoRESUMO
Cell division in prokaryotes and eukaryotes is commonly initiated by the well-controlled binding of proteins to the cytoplasmic side of the cell membrane. However, a precise characterization of the spatiotemporal dynamics of membrane-bound proteins is often difficult to achieve in vivo. Here, we present protocols for the use of supported lipid bilayers to rebuild the cytokinetic machineries of cells with greatly different dimensions: the bacterium Escherichia coli and eggs of the vertebrate Xenopus laevis. Combined with total internal reflection fluorescence microscopy, these experimental setups allow for precise quantitative analyses of membrane-bound proteins. The protocols described to obtain glass-supported membranes from bacterial and vertebrate lipids can be used as starting points for other reconstitution experiments. We believe that similar biochemical assays will be instrumental to study the biochemistry and biophysics underlying a variety of complex cellular tasks, such as signaling, vesicle trafficking, and cell motility.
Assuntos
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Xenopus laevis/metabolismo , Animais , Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia de Fluorescência/métodos , Óvulo/metabolismo , Ligação Proteica/fisiologia , Extratos de Tecidos/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Previous study of self-organization of Taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle. We revisit this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. We added probes for NUMA and Aurora B to reveal microtubule polarity. Taxol and dimethyl sulfoxide promote rapid polymerization of microtubules that slowly self-organize into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends align in NUMA-containing foci on the outside, and plus ends in Aurora B-containing foci on the inside. Assemblies have a well-defined width that depends on initial assembly conditions, but microtubules within them have a broad length distribution. Electron microscopy shows that plus-end foci are coated with electron-dense material and resemble similar foci in monopolar midzones in cells. Functional tests show that two key spindle assembly factors, dynein and kinesin-5, act during assembly as they do in spindles, whereas two key midzone assembly factors, Aurora B and Kif4, act as they do in midzones. These data reveal the richness of self-organizing mechanisms that operate on microtubules after they polymerize in meiotic cytoplasm and provide a biochemically tractable system for investigating plus-end organization in midzones.
Assuntos
Citosol/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Aurora Quinase B/metabolismo , Sistema Livre de Células , Citocinese , Dineínas/metabolismo , Cinesinas/metabolismo , Meiose , Microtúbulos/ultraestrutura , Oócitos/metabolismo , Estabilidade Proteica , Fuso Acromático/ultraestrutura , Imagem com Lapso de Tempo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
BACKGROUND: Bipolar spindle assembly is critical for achieving accurate segregation of chromosomes. In the absence of centrosomes, meiotic spindles achieve bipolarity by a combination of chromosome-initiated microtubule nucleation and stabilization and motor-driven organization of microtubules. Once assembled, the spindle structure is maintained on a relatively long time scale despite the high turnover of the microtubules that comprise it. To study the underlying mechanisms responsible for spindle assembly and steady-state maintenance, we used microneedle manipulation of preassembled spindles in Xenopus egg extracts. RESULTS: When two meiotic spindles were brought close enough together, they interacted, creating an interconnected microtubule structure with supernumerary poles. Without exception, the perturbed system eventually re-established bipolarity, forming a single spindle of normal shape and size. Bipolar spindle fusion was blocked when cytoplasmic dynein function was perturbed, suggesting a critical role for the motor in this process. The fusion of Eg5-inhibited monopoles also required dynein function but only occurred if the initial interpolar separation was less than twice the microtubule radius of a typical monopole. CONCLUSIONS: Our experiments uniquely illustrate the architectural plasticity of the spindle and reveal a robust ability of the system to attain a bipolar morphology. We hypothesize that a major mechanism driving spindle fusion is dynein-mediated sliding of oppositely oriented microtubules, a novel function for the motor, and posit that this same mechanism might also be involved in normal spindle assembly and homeostasis.
Assuntos
Dineínas/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Polaridade Celular , Segregação de Cromossomos , Cinesinas/genética , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
Assuntos
Cinesinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Células HeLa , Humanos , Cinesinas/genética , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas de Xenopus/genéticaRESUMO
The tetrameric plus-end-directed motor, kinesin-5, is essential for bipolar spindle assembly. Small-molecule inhibitors of kinesin-5 have been important tools for investigating its function, and some are currently under evaluation as anti-cancer drugs. Most inhibitors reported to date are ;non-competitive' and bind to a specific site on the motor head, trapping the motor in an ADP-bound state in which it has a weak but non-zero affinity for microtubules. Here, we used a novel ATP-competitive inhibitor, FCPT, developed at Merck (USA). We found that it induced tight binding of kinesin-5 onto microtubules in vitro. Using Xenopus egg-extract spindles, we found that FCPT not only blocked poleward microtubule sliding but also selectively induced loss of microtubules at the poles of bipolar spindles (and not asters or monoasters). We also found that the spindle-pole proteins TPX2 and gamma-tubulin became redistributed to the spindle equator, suggesting that proper kinesin-5 function is required for pole assembly.
Assuntos
Ciclopropanos/farmacologia , Cinesinas/antagonistas & inibidores , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Tiazóis/farmacologia , Proteínas de Xenopus/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Ciclopropanos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Piridinas/química , Bibliotecas de Moléculas Pequenas/química , Tiazóis/química , Tubulina (Proteína)/metabolismo , Xenopus , Proteínas de Xenopus/metabolismoRESUMO
The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM--a known TPX2 partner--and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.
Assuntos
Proteína BRCA1/metabolismo , Fuso Acromático/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Proteína BRCA1/genética , Proteínas de Ciclo Celular/metabolismo , Extratos Celulares/química , Dimerização , Feminino , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/química , Fosfoproteínas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Xenopus , Proteínas de Xenopus/metabolismoRESUMO
In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of gamma-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of gamma-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of gamma-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.
Assuntos
Proteína BRCA1/metabolismo , Centrossomo/ultraestrutura , Ubiquitina/metabolismo , Aneuploidia , Proteína BRCA1/fisiologia , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Centrossomo/metabolismo , Relação Dose-Resposta a Droga , Humanos , Lisina/química , Microscopia de Fluorescência , Microtúbulos/ultraestrutura , Mutação , Peptídeos/química , Fenótipo , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Fase S , Fatores de Tempo , Transfecção , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The Nercc1 protein kinase autoactivates in vitro and is activated in vivo during mitosis. Autoactivation in vitro requires phosphorylation of the activation loop at threonine 210. Mitotic activation of Nercc1 in mammalian cells is accompanied by Thr210 phosphorylation and involves a small fraction of total Nercc1. Mammalian Nercc1 coimmunoprecipitates gamma-tubulin and the activated Nercc1 polypeptides localize to the centrosomes and spindle poles during early mitosis, suggesting that active Nercc has important functions at the microtubular organizing center during cell division. To test this hypothesis, we characterized the Xenopus Nercc1 orthologue (XNercc). XNercc endogenous to meiotic egg extracts coprecipitates a multiprotein complex that contains gamma-tubulin and several components of the gamma-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the gamma-tubulin ring complex polypeptide Xgrip109 contain XNercc. Immunodepletion of XNercc from egg extracts results in delayed spindle assembly, fewer bipolar spindles, and the appearance of aberrant microtubule structures, aberrations corrected by addition of purified recombinant XNercc. XNercc immunodepletion also slows aster assembly induced by Ran-GTP, producing Ran-asters of abnormal size and morphology. Thus, Nercc1 contributes to both the centrosomal and the chromatin/Ran pathways that collaborate in the organization of a bipolar spindle.
Assuntos
Centrossomo/enzimologia , Mitose/fisiologia , Proteínas Quinases/metabolismo , Fuso Acromático/fisiologia , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Oócitos/enzimologia , Ligação Proteica , Proteínas Quinases/genética , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , Proteína ran de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.