RESUMO
Identifying major histocompatibility complex (MHC) class I immunopeptide antigens represents a key step in the development of immune-based targeted therapeutics and vaccines. However, the complete characterization of these antigens by tandem mass spectrometry remains challenging due to their short sequence length, high degree of hydrophobicity, and/or lack of sufficiently basic amino acids. This study seeks to address the potential for 193 nm ultraviolet photodissociation (UVPD) to improve the analysis of MHC class I immunopeptides by offering enhanced characterization of these sequences in lower charge states and differentiation of prominent isomeric leucine and isoleucine residues in the HLA-A*02:01 motif. Although electron transfer dissociation-higher energy collisional dissociation (EThcD) offered some success in the differentiation of leucine and isoleucine, 193 nm UVPD was able to confirm the identity of nearly 60% of leucine and isoleucine residues in a synthetic peptide mixture. Furthermore, 193 nm UVPD led to significantly more peptide identifications and higher scoring metrics than EThcD for peptides obtained from immunoprecipitation of MHC class I immunopeptides from in vitro cell culture. Additionally, 193 nm UVPD represents a promising complementary technique to higher-energy collisional dissociation (HCD), in which 424 of the 2593 peptides identified by 193 nm UVPD were not identified by HCD in HLA-A*02:01-specific immunoprecipitation and 804 of the 3300 peptides identified by 193 nm UVPD were not identified by HCD for pan HLA-A, -B, and -C immunoprecipitation. These results highlight that 193 nm UVPD offers an option for the characterization of immunopeptides, including differentiation of leucine and isoleucine residues.
Assuntos
Antígenos HLA-A , Humanos , Espectrometria de MassasRESUMO
We previously showed that the cell-cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here we show that PG deficiency results in extracellular matrix (ECM)-dependent disruption of mature focal adhesions and cortical actin organization. Plating PGâ»/â» cells onto ECM deposited by PG+/â» cells partially restored normal cell morphology and inhibited PGâ»/â» cell motility. In over 70 adhesion molecules whose expression we previously showed to be altered in PGâ»/â» cells, a substantial decrease in fibronectin (FN) in PGâ»/â» cells stood out. Re-introduction of PG into PGâ»/â» cells restored FN expression, and keratinocyte motility was reversed by plating PGâ»/â» cells onto FN. Somewhat surprisingly, based on previously reported roles for PG in regulating gene transcription, PG-null cells exhibited an increase, not a decrease, in FN promoter activity. Instead, PG was required for maintenance of FN mRNA stability. PGâ»/â» cells exhibited an increase in activated Src, one of the kinases controlled by FN, a phenotype reversed by plating PGâ»/â» cells on ECM deposited by PG+/â» keratinocytes. PGâ»/â» cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PGâ»/â» keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM-Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability.
Assuntos
Movimento Celular/fisiologia , Fibronectinas/metabolismo , Transdução de Sinais/fisiologia , gama Catenina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Western Blotting , Movimento Celular/genética , Células Cultivadas , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Técnica Indireta de Fluorescência para Anticorpo , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , gama Catenina/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mM ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin beta(4), and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Queratinócitos/metabolismo , Espectrometria de Massas/métodos , Proteínas/genética , Proteínas/metabolismo , Hidróxido de Amônia , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Hidróxidos/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Peptídeos/química , Peptídeos/metabolismo , gama Catenina/deficiência , gama Catenina/metabolismoRESUMO
Aberrant gene activation driven by the histone acetyltransferases p300 and CREB binding protein (CBP) has been linked to several diseases, including cancers. Because of this, many efforts have been aimed toward the targeting of the closely related paralogues, p300 and CBP, but these endeavors have been exclusively directed toward noncovalent inhibitors. X-ray crystallography of A-485 revealed that both p300 and CBP possess a cysteine (C1450) near the active site, thus rendering covalent inhibition an attractive chemical approach. Herein we report the development of compound 2, an acrylamide-based inhibitor of p300/CBP that forms a covalent adduct with C1450. We demonstrated using mass spectrometry that compound 2 selectively targets C1450, and we also validated covalent binding using kinetics experiments and cellular washout studies. The discovery of covalent inhibitor 2 gives us a unique tool for the study of p300/CBP biology.
RESUMO
Peptides presented by the class-I major histocompatibility complex (MHC-I) are important targets for immunotherapy. The identification of these peptide targets greatly facilitates the generation of T-cell-based therapeutics. Herein, we report the capability of proteolysis targeting chimera (PROTAC) compounds to induce the presentation of specific MHC class-I peptides derived from endogenous cellular proteins. Using LC-MS/MS, we identified several BET-derived MHC-I peptides induced by treatment with three BET-directed PROTAC compounds. To understand our ability to tune this process, we measured the relative rate of presentation of these peptides under varying treatment conditions using label-free mass spectrometry quantification. We found that the rate of peptide presentation reflected the rate of protein degradation, indicating a direct relationship between PROTAC treatment and peptide presentation. We additionally analyzed the effect of PROTAC treatment on the entire immunopeptidome and found many new peptides that were displayed in a PROTAC-specific fashion: we determined that these identifications map to the BET pathway, as well as, potential off-target or unique-to-PROTAC pathways. This work represents the first evidence of the use of PROTAC compounds to induce the presentation of MHC-I peptides from endogenous cellular proteins, highlighting the capability of PROTAC compounds for the discovery and generation of new targets for immunotherapy.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/química , Peptídeos/imunologia , Proteólise , Linhagem Celular , HumanosRESUMO
PURPOSE: PARP inhibitors are being developed as therapeutic agents for cancer. More than six compounds have entered clinical trials. The majority of these compounds are ß-nicotinamide adenine dinucleotide (NAD(+))-competitive inhibitors. One exception is iniparib, which has been proposed to be a noncompetitive PARP inhibitor. In this study, we compare the biologic activities of two different structural classes of NAD(+)-competitive compounds with iniparib and its C-nitroso metabolite. EXPERIMENTAL DESIGN: Two chemical series of NAD(+)-competitive PARP inhibitors, iniparib and its C-nitroso metabolite, were analyzed in enzymatic and cellular assays. Viability assays were carried out in MDA-MB-436 (BRCA1-deficient) and DLD1(-/-) (BRCA2-deficient) cells together with BRCA-proficient MDA-MB-231 and DLD1(+/+) cells. Capan-1 and B16F10 xenograft models were used to compare iniparib and veliparib in vivo. Mass spectrometry and the (3)H-labeling method were used to monitor the covalent modification of proteins. RESULTS: All NAD(+)-competitive inhibitors show robust activity in a PARP cellular assay, strongly potentiate the activity of temozolomide, and elicit robust cell killing in BRCA-deficient tumor cells in vitro and in vivo. Cell killing was associated with an induction of DNA damage. In contrast, neither iniparib nor its C-nitroso metabolite inhibited PARP enzymatic or cellular activity, potentiated temozolomide, or showed activity in a BRCA-deficient setting. We find that the nitroso metabolite of iniparib forms adducts with many cysteine-containing proteins. Furthermore, both iniparib and its nitroso metabolite form protein adducts nonspecifically in tumor cells. CONCLUSIONS: Iniparib nonselectively modifies cysteine-containing proteins in tumor cells, and the primary mechanism of action for iniparib is likely not via inhibition of PARP activity.