RESUMEN
Many proteins enhance cancer progression toward life-threatening metastases. These include linking proteins called integrins that mediate cell adhesion to the extracellular matrix (ECM), consequently altering both function and phenotype. Specific neoexpression of the ß6 integrin subunit correlates with the epithelial-to-mesenchymal transition, metastasis, and poor overall patient survival. While ß6 is implicated in these processes, exactly how it affects signaling and/or proteolytic pathways in metastasis remains unclear. A membrane-enriched peptide immobilized pH gradient isoelectric focusing (IPG-IEF) shotgun proteomics study was undertaken in which subclones of the SW480 colorectal cancer cell line transfected with a vector inducing unregulated ß6 integrin overexpression were compared with the "empty" mock vector control cell line. ß6 overexpression induced a significant change in 708 proteins and was found to be localized across most intracellular locations, some involving cellular processes and pathways underpinning cancer progression. Proteomics data have been deposited to the ProteomeXchange with identifier PXD000230. ß6 expression increased cell proliferation 4-fold while decreasing cell adhesion to many integrin ECM substrates. ß6 expression also enhanced cell invasion and promoted the expression/repression of many established cancer-related pathways.
Asunto(s)
Antígenos de Neoplasias/genética , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Integrinas/genética , Proteínas de Neoplasias/aislamiento & purificación , Proteoma/aislamiento & purificación , Secuencia de Aminoácidos , Antígenos de Neoplasias/metabolismo , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Perfilación de la Expresión Génica , Vectores Genéticos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Integrinas/metabolismo , Focalización Isoeléctrica/métodos , Datos de Secuencia Molecular , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Mapeo de Interacción de Proteínas , Proteoma/genética , Proteoma/metabolismo , TransfecciónRESUMEN
A high degree of optimisation is required in native co-immunoprecipitation (co-IP) experiments with added challenges for low-abundant membrane proteins and masking by IgG molecules. Although in vivo tagged-protein purification avoids the IgG masking problem, modifying the terminus of the protein may result in conformational and post-translational modification changes. In this paper, we propose a method which combines four key aspects to improve the solubility and enrichment of low-abundant plasma membrane proteins using the urokinase plasminogen activator receptor (uPAR) as an example. As this GPI-linked receptor predominantly resides in lipid rafts (LR), we used a modified RIPA lysis buffer containing the non-ionic detergent, octyl-glucoside which solubilizes LRs to extract uPAR. This is followed by a modified crosslinking co-IP which covalently crosslinks the antibodies to the beads. Crosslinking allowed for a significant increase in the detection of uPAR with minimal IgG contamination using on-bead digestion or acid elution followed by digestion and analysis on high-throughput one-dimensional (nanoLC) MS/MS instrument (AbSciex 5600). To the best of our knowledge, this method of isolation is the first to be done to increase the yield of a low-abundant membrane protein and may be useful for the purification of other non-raft and raft-residing membrane proteins.
Asunto(s)
Inmunoprecipitación/métodos , Microdominios de Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Glucósidos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
Aneuploidy is a key process in tumorigenesis. Dysfunction of the mitotic spindle checkpoint proteins has been implicated as a cause of aneuploidy in cells. We have previously reported that FAT10, a member of the ubiquitin-like modifier family of proteins, is overexpressed in several gastrointestinal and gynecological cancers. Here we show that FAT10 interacts with MAD2, a spindle checkpoint protein, during mitosis. Notably, we show that localization of MAD2 at the kinetochore during the prometaphase stage of the cell cycle was greatly reduced in FAT10-overexpressing cells. Furthermore, compared with parental HCT116 cells, fewer mitotic cells were observed after double thymidine-synchronized FAT10-overexpressing cells were released into nocodazole for more than 4 h. Nonetheless, when these double thymidine-treated cells were released into media, a similar number of G1 parental and FAT10-overexpressing HCT116 cells was observed throughout the 10-h time course. Additionally, more nocodazole-treated FAT10-overexpressing cells escape mitotic controls and are multinucleate compared with parental cells. Significantly, we observed a higher degree of variability in chromosome number in cells overexpressing FAT10. Hence, our data suggest that high levels of FAT10 protein in cells lead to increased mitotic nondisjunction and chromosome instability, and this effect is mediated by an abbreviated mitotic phase and the reduction in the kinetochore localization of MAD2 during the prometaphase stage of the cell cycle.
Asunto(s)
Cromosomas/ultraestructura , Regulación Neoplásica de la Expresión Génica , Ubiquitinas/fisiología , Aneuploidia , Apoptosis , Western Blotting , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Linaje de la Célula , Núcleo Celular/metabolismo , Inestabilidad Cromosómica , Aberraciones Cromosómicas , Cromosomas/metabolismo , Clonación Molecular , ADN/química , Fase G1 , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Cinetocoros/metabolismo , Proteínas Mad2 , Metafase , Microscopía Fluorescente , Mitosis , Nocodazol/farmacología , No Disyunción Genética , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Represoras/metabolismo , Huso Acromático , Timidina/metabolismo , Factores de TiempoRESUMEN
The ubiquitin-like modifier (UBL) family has recently generated much interest in the scientific community, as it is implicated to play important regulatory roles via novel protein-protein modification. FAT10 (diubiquitin) belongs to this family of proteins, comprising two ubiquitin-like moieties fused in tandem, and has been implicated to be involved in the maintenance of spindle integrity during mitosis. As FAT10 may play a role in the regulation of genomic stability, we examined if there is an association between FAT10 expression and hepatocellular carcinoma (HCC) or other cancers. Northern blot analyses revealed upregulation of FAT10 expression in the tumors of 90% of HCC patients. In situ hybridization as well as immunohistochemistry utilizing anti-FAT10 antibodies localized highest FAT10 expression in the nucleus of HCC hepatocytes rather than the surrounding immune and non-HCC cells. FAT10 expression was also found to be highly upregulated in other cancers of the gastrointestinal tract and female reproductive system. In conclusion, we demonstrated upregulation of FAT10 expression in various gastrointestinal and gynecological cancers. Its overexpression is unrelated to the general increase in protein synthesis or a general immune/inflammatory response to cancer. Rather, FAT10 may modulate tumorigenesis through its reported interaction with the MAD2 spindle-assembly checkpoint protein.