RESUMEN
Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.
Asunto(s)
Cationes , Ciclización , Indicadores y Reactivos , Proteínas , Triptófano , Cationes/química , Indicadores y Reactivos/química , Oxidación-Reducción , Proteoma/química , Triptófano/química , Péptidos/química , Química Clic , Proteínas/químicaRESUMEN
Sexual dimorphisms in the brain underlie behavioral sex differences, but the function of individual sexually dimorphic neuronal populations is poorly understood. Neuronal sexual dimorphisms typically represent quantitative differences in cell number, gene expression, or other features, and it is unknown whether these dimorphisms control sex-typical behavior exclusively in one sex or in both sexes. The progesterone receptor (PR) controls female sexual behavior, and we find many sex differences in number, distribution, or projections of PR-expressing neurons in the adult mouse brain. Using a genetic strategy we developed, we have ablated one such dimorphic PR-expressing neuronal population located in the ventromedial hypothalamus (VMH). Ablation of these neurons in females greatly diminishes sexual receptivity. Strikingly, the corresponding ablation in males reduces mating and aggression. Our findings reveal the functions of a molecularly defined, sexually dimorphic neuronal population in the brain. Moreover, we show that sexually dimorphic neurons can control distinct sex-typical behaviors in both sexes.
Asunto(s)
Agresión/fisiología , Hipotálamo/metabolismo , Neuronas/metabolismo , Caracteres Sexuales , Conducta Sexual , Animales , Femenino , Hipotálamo/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Progesterona/análisis , Receptores de Progesterona/metabolismo , Conducta Sexual AnimalRESUMEN
Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here, we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these, we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro- and catalytic domains for three different matrix metalloproteases (MMPs), MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impair cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2 (EphA2), that is known to transform it from a tumor suppressor to an oncoprotein. We believe that the EDS method greatly facilitates the generation of antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications.
Asunto(s)
Epítopos , Epítopos/inmunología , Humanos , Proteolisis , Unión Proteica , Ingeniería de Proteínas/métodos , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz/inmunología , Anticuerpos/inmunología , Biblioteca de PéptidosRESUMEN
The caspases are unique proteases that mediate the major morphological changes of apoptosis and various other cellular remodeling processes. As we catalog and study the myriad proteins subject to cleavage by caspases, we are beginning to appreciate the full functional repertoire of these enzymes. Here, we examine current knowledge about caspase cleavages: what kinds of proteins are cut, in what contexts, and to what end. After reviewing basic caspase biology, we describe the technologies that enable high-throughput caspase substrate discovery and the datasets they have yielded. We discuss how caspases recognize their substrates and how cleavages are conserved among different metazoan organisms. Rather than comprehensively reviewing all known substrates, we use examples to highlight some functional impacts of caspase cuts during apoptosis and differentiation. Finally, we discuss the roles caspase substrates can play in medicine. Though great progress has been made in this field, many important areas still await exploration.
Asunto(s)
Apoptosis/fisiología , Caspasas/química , Caspasas/metabolismo , Diferenciación Celular/fisiología , Animales , Caspasas/clasificación , Caspasas/genética , Dimerización , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Modelos Moleculares , Conformación Proteica , Transducción de Señal/fisiología , Especificidad por SustratoRESUMEN
Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.
Asunto(s)
Arginina Deiminasa Proteína-Tipo 4 , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Arginina Deiminasa Proteína-Tipo 4/química , Humanos , Dominio Catalítico , Microscopía por Crioelectrón , Modelos Moleculares , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/metabolismo , Artritis Reumatoide/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Hidrolasas/metabolismo , Hidrolasas/química , Desiminasas de la Arginina Proteica/metabolismo , Desiminasas de la Arginina Proteica/químicaRESUMEN
Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.
Asunto(s)
Hidrolasas Diéster Fosfóricas , Anticuerpos de Dominio Único , Hidrolasas Diéster Fosfóricas/metabolismo , Monoéster Fosfórico Hidrolasas , Microscopía por CrioelectrónRESUMEN
BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.
Asunto(s)
Apoptosis , Proteína X Asociada a bcl-2 , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Humanos , Apoptosis/efectos de los fármacos , Cisteína/química , Cisteína/metabolismo , Animales , Aldehídos/química , Aldehídos/farmacología , Modelos Moleculares , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismoRESUMEN
Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.
Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Ingeniería de Proteínas , Caspasa 6/metabolismo , Línea Celular , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Leupeptinas/farmacología , Complejo de la Endopetidasa Proteasomal , Inhibidores de ProteasomaRESUMEN
Combining multiple therapeutic strategies in NRAS/BRAF mutant melanoma-namely MEK/BRAF kinase inhibitors, immune checkpoint inhibitors (ICIs), and targeted immunotherapies-may offer an improved survival benefit by overcoming limitations associated with any individual therapy. Still, optimal combination, order, and timing of administration remains under investigation. Here, we measure how MEK inhibition (MEKi) alters anti-tumor immunity by utilizing quantitative immunopeptidomics to profile changes in the peptide major histocompatibility molecules (pMHC) repertoire. These data reveal a collection of tumor antigens whose presentation levels are selectively augmented following therapy, including several epitopes present at over 1,000 copies per cell. We leveraged the tunable abundance of MEKi-modulated antigens by targeting four epitopes with pMHC-specific T cell engagers and antibody drug conjugates, enhancing cell killing in tumor cells following MEK inhibition. These results highlight drug treatment as a means to enhance immunotherapy efficacy by targeting specific upregulated pMHCs and provide a methodological framework for identifying, quantifying, and therapeutically targeting additional epitopes of interest.
Asunto(s)
Melanoma , Quinasas de Proteína Quinasa Activadas por Mitógenos , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Antígenos de Neoplasias/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , EpítoposRESUMEN
The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.
Asunto(s)
Anticuerpos/metabolismo , Inmunoconjugados/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Especificidad de Anticuerpos , HumanosRESUMEN
The selenoprotein glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid peroxides into nontoxic lipid alcohols. GPX4 has emerged as a promising therapeutic target for cancer treatment, but some cancer cells are resistant to ferroptosis triggered by GPX4 inhibition. Using a chemical-genetic screen, we identify LRP8 (also known as ApoER2) as a ferroptosis resistance factor that is upregulated in cancer. Loss of LRP8 decreases cellular selenium levels and the expression of a subset of selenoproteins. Counter to the canonical hierarchical selenoprotein regulatory program, GPX4 levels are strongly reduced due to impaired translation. Mechanistically, low selenium levels result in ribosome stalling at the inefficiently decoded GPX4 selenocysteine UGA codon, leading to ribosome collisions, early translation termination and proteasomal clearance of the N-terminal GPX4 fragment. These findings reveal rewiring of the selenoprotein hierarchy in cancer cells and identify ribosome stalling and collisions during GPX4 translation as ferroptosis vulnerabilities in cancer.
Asunto(s)
Ferroptosis , Selenio , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Ribosomas/metabolismo , Selenio/metabolismo , Selenio/farmacología , Selenoproteínas/genéticaRESUMEN
Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, coculturing primary CD8+ T cells with Tregs only modestly affected the CD8+ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function.
Asunto(s)
Proteoma , Linfocitos T Reguladores , Linfocitos T CD8-positivos , Humanos , Hipoxia , Microambiente TumoralRESUMEN
Since the discovery of oncogenes, there has been tremendous interest to understand their mechanistic basis and to develop broadly actionable therapeutics. Some of the most frequently activated oncogenes driving diverse cancers are c-MYC, EGFR, HER2, AKT, KRAS, BRAF, and MEK. Using a reductionist approach, we explored how cellular proteomes are remodeled in isogenic cell lines engineered with or without these driver oncogenes. The most striking discovery for all oncogenic models was the systematic downregulation of scores of antiviral proteins regulated by type 1 interferon. These findings extended to cancer cell lines and patient-derived xenograft models of highly refractory pancreatic cancer and osteosarcoma driven by KRAS and MYC oncogenes. The oncogenes reduced basal expression of and autocrine stimulation by type 1 interferon causing remarkable convergence on common phenotypic and functional profiles. In particular, there was dramatically lower expression of dsRNA sensors including DDX58 (RIG-I) and OAS proteins, which resulted in attenuated functional responses when the oncogenic cells were treated with the dsRNA mimetic, polyI:C, and increased susceptibility to infection with an RNA virus shown using SARS-CoV-2. Our reductionist approach provides molecular and functional insights connected to immune evasion hallmarks in cancers and suggests therapeutic opportunities.
Asunto(s)
COVID-19 , Interferón beta , Oncogenes , Proteómica , Animales , Factores de Restricción Antivirales , COVID-19/inmunología , Carcinogénesis , Línea Celular Tumoral , Humanos , Interferón beta/inmunología , Proteínas Proto-Oncogénicas p21(ras)/genética , SARS-CoV-2RESUMEN
Therapies that boost the antitumor immune response have shown a great deal of success. Although most of these therapies have focused on enhancing T cell functions, there is a growing interest in developing therapies that can target other immune cell subsets. Like T cells, natural killer (NK) cells are cytotoxic effector cells that play a key role in the antitumor response. To advance the development of NK-based therapies, we developed a functional screen to rapidly identify antibodies that can activate NK cells. We displayed antibodies on a mammalian target cell line and probed their ability to stimulate NK cell-mediated cytotoxicity. From this screen, we identified five antibodies that bound with high affinity to NK cells and stimulated NK cell-mediated cytotoxicity and interferon-γ (IFN-γ) secretion. We demonstrate that these antibodies can be further developed into bispecific antibodies to redirect NK cell-mediated cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. While antibodies to two of the receptors, CD16 and NCR1, have previously been targeted as bispecific antibodies to redirect NK cell-mediated cytotoxicity, we demonstrate that bispecific antibodies targeting NCR3 can also potently activate NK cells. These results show that this screen can be used to directly identify antibodies that can enhance antitumor immune responses.
Asunto(s)
Anticuerpos/fisiología , Citotoxicidad Celular Dependiente de Anticuerpos/fisiología , Células Asesinas Naturales/fisiología , Afinidad de Anticuerpos , Neoplasias de la Mama , Línea Celular Tumoral , Femenino , Proteínas Ligadas a GPI , Regulación de la Expresión Génica/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/metabolismo , Linfoma de Células B/tratamiento farmacológico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Superficie Celular , Receptores de IgG , Reproducibilidad de los Resultados , Rituximab/farmacologíaRESUMEN
N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants (subtiligase-TM) to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. To facilitate exploration of our datasets, we developed a web-accessible Atlas of Subtiligase-Captured Extracellular N Termini (ASCENT; http://wellslab.org/ascent). This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.
Asunto(s)
Membrana Celular/metabolismo , Mapeo Peptídico/métodos , Péptido Sintasas/metabolismo , Subtilisinas/metabolismo , Células HEK293 , Humanos , Mutación , Péptido Sintasas/genética , Procesamiento Proteico-Postraduccional , Proteolisis , Subtilisinas/genéticaRESUMEN
MYC is a powerful transcription factor overexpressed in many human cancers including B cell and prostate cancers. Antibody therapeutics are exciting opportunities to attack cancers but require knowledge of surface proteins that change due to oncogene expression. To identify how MYC overexpression remodels the cell surface proteome in a cell autologous fashion and in different cell types, we investigated the impact of MYC overexpression on 800 surface proteins in three isogenic model cell lines either of B cell or prostate cell origin engineered to have high or low MYC levels. We found that MYC overexpression resulted in dramatic remodeling (both up- and down-regulation) of the cell surfaceome in a cell type-dependent fashion. We found systematic and large increases in distinct sets of >80 transporters including nucleoside transporters and nutrient transporters making cells more sensitive to toxic nucleoside analogs like cytarabine, commonly used for treating hematological cancers. Paradoxically, MYC overexpression also increased expression of surface proteins driving cell turnover such as TNFRSF10B, also known as death receptor 5, and immune cell attacking signals such as the natural killer cell activating ligand NCR3LG1, also known as B7-H6. We generated recombinant antibodies to these two targets and verified their up-regulation in MYC overexpression cell lines and showed they were sensitive to bispecific T cell engagers (BiTEs). Our studies demonstrate how MYC overexpression leads to dramatic bidirectional remodeling of the surfaceome in a cell type-dependent but functionally convergent fashion and identify surface targets or combinations thereof as possible candidates for cytotoxic metabolite or immunotherapy.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Linfocitos B/efectos de los fármacos , Antígenos B7/genética , Células Epiteliales/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Anticuerpos Biespecíficos/biosíntesis , Linfocitos B/inmunología , Linfocitos B/patología , Antígenos B7/antagonistas & inhibidores , Antígenos B7/inmunología , Ingeniería Celular/métodos , Línea Celular Tumoral , Citarabina/farmacología , Células Epiteliales/inmunología , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunosupresores/farmacología , Inmunoterapia/métodos , Masculino , Terapia Molecular Dirigida/métodos , Plásmidos/química , Plásmidos/metabolismo , Próstata/inmunología , Próstata/patología , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , TransfecciónRESUMEN
Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml-1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.
Asunto(s)
Enzima Convertidora de Angiotensina 2/química , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Anticuerpos de Cadena Única/química , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Chlorocebus aethiops , Microscopía por Crioelectrón , Células HEK293 , Humanos , Modelos Moleculares , Biblioteca de Péptidos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2 , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células VeroRESUMEN
The nearly 600 proteases in the human genome regulate a diversity of biological processes, including programmed cell death. Comprehensive characterization of protease signaling in complex biological samples is limited by available proteomic methods. We have developed a general approach for global identification of proteolytic cleavage sites using an engineered enzyme to selectively biotinylate free protein N termini for positive enrichment of corresponding N-terminal peptides. Using this method to study apoptosis, we have sequenced 333 caspase-like cleavage sites distributed among 292 protein substrates. These sites are generally not predicted by in vitro caspase substrate specificity but can be used to predict other physiological caspase cleavage sites. Structural bioinformatic studies show that caspase cleavage sites often appear in surface-accessible loops and even occasionally in helical regions. Strikingly, we also find that a disproportionate number of caspase substrates physically interact, suggesting that these dimeric proteases target protein complexes and networks to elicit apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Proteínas/análisis , Proteínas/metabolismo , Proteómica , Antineoplásicos Fitogénicos/farmacología , Caspasas/química , Etopósido/farmacología , Humanos , Células Jurkat , Unión Proteica , Especificidad por SustratoRESUMEN
The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of N-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK, and AKT. We find that each oncogene has somewhat different surfaceomes, but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting the strong dependence of the oncogene on the MAPK pathway to propagate signaling. Cell surface protein capture is mediated by covalent tagging of surface glycans, yet current methods do not afford sequencing of intact glycopeptides. Thus, we complement the surfaceome data with whole cell glycoproteomics enabled by a recently developed technique called activated ion electron transfer dissociation (AI-ETD). We found massive oncogene-induced changes to the glycoproteome and differential increases in complex hybrid glycans, especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems-level view of how specific driver oncogenes remodel the surfaceome and the glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.
Asunto(s)
Neoplasias de la Mama/genética , Glicoproteínas de Membrana/genética , Proteoma/genética , Proteómica , Biomarcadores de Tumor/genética , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/genética , Femenino , Glicoproteínas/genética , Humanos , Quinasas Quinasa Quinasa PAM/genética , Proteína Oncogénica v-akt/genética , Oncogenes/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptor ErbB-2/genética , Transducción de Señal/genéticaRESUMEN
The field of chemical modification of proteins has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently, we have developed oxaziridine chemistry for highly selective modification of methionine called redox-activated chemical tagging (ReACT) but have not broadly tested the molecular parameters for efficient and stable protein modification. Here we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, used for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and excellent reaction efficiencies with a panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than 10-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, presumably because of their reduced access to water. There was also a 10-fold variation in stability depending on the nature of the oxaziridine, which was determined to be inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs were sufficient to support their use as antibody drug conjugates and potent in a breast cancer mouse xenograft model over a month. These studies provide key parameters for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation to native or engineered methionines.