RESUMEN
Ubiquitination is a posttranslational modification that is crucial for the dynamic regulation of diverse signaling pathways. To enhance our understanding of ubiquitination-mediated signaling, we generated a new class of bispecific antibodies that combine recognition of ubiquitination substrates and specific polyubiquitin linkages. RIP1-K63 and RIP1-linear (Lin) linkage polyubiquitin bispecific antibodies detected linkage-specific ubiquitination of the proinflammatory kinase RIP1 in cells and in tissues and revealed RIP1 ubiquitination by immunofluorescence. Similarly, ubiquitination of the RIP1-related kinase RIP2 with K63 or linear linkages was specifically detected with the RIP2-K63 and RIP2-Lin bispecific antibodies, respectively. Furthermore, using the RIP2-K63 and RIP2-Lin bispecific antibodies, we found prominent K63-linked and linear RIP2 ubiquitination in samples from patients with ulcerative colitis and Crohn's disease. We also developed a bispecific antibody (K63-Lin) that simultaneously recognizes K63-linked and linear ubiquitination of components of various signaling pathways. Together, these bispecific antibodies represent a new class of reagents with the potential to be developed for the detection of inflammatory biomarkers.
Asunto(s)
Anticuerpos Biespecíficos , Ubiquitina , Humanos , Anticuerpos Biespecíficos/metabolismo , Poliubiquitina/metabolismo , Transducción de Señal/fisiología , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
As central effectors of the adaptive immune response, immunoglobulins, or antibodies, provide essential protection from pathogens through their ability to recognize foreign antigens, aid in neutralization, and facilitate elimination from the host. Mammalian immunoglobulins can be classified into five isotypes-IgA, IgD, IgE, IgG, and IgM-each with distinct roles in mediating various aspects of the immune response. Of these isotypes, IgA and IgM are the only ones capable of multimerization, arming them with unique biological functions. Increased valency of polymeric IgA and IgM provides high avidity for binding low-affinity antigens, and their ability to be transported across the mucosal epithelium into secretions by the polymeric immunoglobulin receptor allows them to play critical roles in mucosal immunity. Here we discuss the molecular assembly, structure, and function of these multimeric antibodies.
Asunto(s)
Inmunoglobulina A , Receptores de Inmunoglobulina Polimérica , Animales , Humanos , Inmunidad Mucosa , Inmunoglobulina M/química , Inmunoglobulina M/metabolismo , Mamíferos/metabolismo , Membrana Mucosa , Receptores de Inmunoglobulina Polimérica/químicaRESUMEN
Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.
Asunto(s)
Antibacterianos/farmacología , Anticuerpos Monoclonales/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/inmunología , Animales , Antibacterianos/química , Antibacterianos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Humanos , Macrófagos/microbiología , Ratones , Viabilidad Microbiana/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Prueba de Estudio Conceptual , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/metabolismo , Células RAW 264.7 , RatasRESUMEN
Immunoglobulins (Ig) A and M are the only human antibodies that form oligomers and undergo transcytosis to mucosal secretions via the polymeric Ig receptor (pIgR). When complexed with the J-chain (JC) and the secretory component (SC) of pIgR, secretory IgA and IgM (sIgA and sIgM) play critical roles in host-pathogen defense. Recently, we determined the structure of sIgA-Fc which elucidated the mechanism of polymeric IgA assembly and revealed an extensive binding interface between IgA-Fc, JC, and SC. Despite low sequence identity shared with IgA-Fc, IgM-Fc also undergoes JC-mediated assembly and binds pIgR. Here, we report the structure of sIgM-Fc and carryout a systematic comparison to sIgA-Fc. Our structural analysis reveals a remarkably conserved mechanism of JC-templated oligomerization and SC recognition of both IgM and IgA through a highly conserved network of interactions. These studies reveal the structurally conserved features of sIgM and sIgA required for function in mucosal immunity.
Asunto(s)
Inmunoglobulina A Secretora/química , Cadenas J de Inmunoglobulina/metabolismo , Inmunoglobulina M/química , Componente Secretorio/metabolismo , Línea Celular , Humanos , Inmunoglobulina A Secretora/metabolismo , Inmunoglobulina M/metabolismo , Modelos Moleculares , Conformación Proteica , Homología Estructural de Proteína , TranscitosisRESUMEN
Treatment of ocular disease is hindered by the presence of the blood-retinal barrier, which restricts access of systemic drugs to the eye. Intravitreal injections bypass this barrier, delivering high concentrations of drug to the targeted tissue. However, the recommended dosing interval for approved biologics is typically 6-12 weeks, and frequent travel to the physician's office poses a substantial burden for elderly patients with poor vision. Real-world data suggest that many patients are under-treated. Here, we investigate IgMs as a novel platform for treating ocular disease. We show that IgMs are well-suited to ocular administration due to moderate viscosity, long ocular exposure, and rapid systemic clearance. The complement-dependent cytotoxicity of IgMs can be readily removed with a P436G mutation, reducing safety liabilities. Furthermore, dodecavalent binding of IgM hexamers can potently activate pathways implicated in the treatment of progressive blindness, including the Tie2 receptor tyrosine kinase signaling pathway for the treatment of diabetic macular edema, or the death receptor 4 tumor necrosis family receptor pathway for the treatment of wet age-related macular degeneration. Collectively, these data demonstrate the promise of IgMs as therapeutic agonists for treating progressive blindness.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inmunoglobulina M/farmacología , Degeneración Macular , Cuerpo Vítreo/metabolismo , Animales , Células CHO , Cricetulus , Humanos , Inyecciones Intravítreas , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , RatasRESUMEN
Secretory immunoglobulin A (sIgA) represents the immune system's first line of defense against mucosal pathogens. IgAs are transported across the epithelium, as dimers and higher-order polymers, by the polymeric immunoglobulin receptor (pIgR). Upon reaching the luminal side, sIgAs mediate host protection and pathogen neutralization. In recent years, an increasing amount of attention has been given to IgA as a novel therapeutic antibody. However, despite extensive studies, sIgA structures have remained elusive. Here, we determine the atomic resolution structures of dimeric, tetrameric, and pentameric IgA-Fc linked by the joining chain (JC) and in complex with the secretory component of the pIgR. We suggest a mechanism in which the JC templates IgA oligomerization and imparts asymmetry for pIgR binding and transcytosis. This framework will inform the design of future IgA-based therapeutics.
Asunto(s)
Inmunoglobulina A Secretora/química , Fragmentos Fc de Inmunoglobulinas/química , Multimerización de Proteína , Humanos , Cadenas J de Inmunoglobulina/química , Receptores de Inmunoglobulina Polimérica/química , TranscitosisRESUMEN
IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/inmunología , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/genética , Perros , Femenino , Glicosilación , Semivida , Humanos , Inmunoglobulina A/genética , Inmunoglobulina G/genética , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genéticaRESUMEN
As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.
Asunto(s)
Anticuerpos/inmunología , Descubrimiento de Drogas/métodos , Mapeo Epitopo/métodos , Antígenos de Histocompatibilidad Clase I/inmunología , Epítopos/química , Epítopos/inmunología , Glicosilación , Antígenos de Histocompatibilidad Clase I/química , Humanos , Ingeniería de Proteínas/métodosRESUMEN
Posttranslational modification of cellular proteins by ubiquitin serves a variety of functions. Among the multitude of ubiquitin substrates, ubiquitin itself is the most prevalent. For many years, the direct detection of polyubiquitin chains attached to cellular substrates was not practical, with cell biologists relegated to indirect approaches involving site-directed mutagenesis or in vitro biochemistry. Recent advances in two technologies-polyubiquitin linkage-specific antibodies and mass spectrometry proteomics, have overcome that limitation. Using one or both of these, the direct analysis of polyubiquitin chain linkages on cellular substrate proteins may be performed. This paper describes the complimentary nature of linkage-specific antibodies and mass spectrometry proteomics for the characterization of complex ubiquitin signals using lessons learned in early development of both technologies.
Asunto(s)
Anticuerpos/química , Espectrometría de Masas , Poliubiquitina/química , Anticuerpos/inmunología , Anticuerpos/metabolismo , Especificidad de Anticuerpos/inmunología , Western Blotting , Inmunoprecipitación , Espectrometría de Masas/métodos , Poliubiquitina/inmunología , Poliubiquitina/metabolismo , Unión Proteica , UbiquitinaciónRESUMEN
Tumor progression locus 2 (TPL2, also known as COT or MAP3K8) is a mitogen-activated protein kinase kinase (MAP3K) activated downstream of TNFαR, IL1R, TLR, CD40, IL17R, and some GPCRs. TPL2 regulates the MEK1/2 and ERK1/2 pathways to regulate a cascade of inflammatory responses. In parallel to this, TPL2 also activates p38α and p38δ to drive the production of various inflammatory mediators in neutrophils. We discuss the implications of this finding in the context of various inflammatory diseases.
Asunto(s)
Inflamación/metabolismo , Quinasas Quinasa Quinasa PAM/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Autoinmunidad , Humanos , Quinasas Quinasa Quinasa PAM/química , Proteínas Proto-Oncogénicas/químicaRESUMEN
Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information.
Asunto(s)
Anticuerpos Biespecíficos/análisis , Transducción de Señal , Ubiquitina/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclo Celular , Humanos , Mitosis , Biosíntesis de Proteínas , UbiquitinaciónRESUMEN
Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Asma/terapia , Células Th2/citología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Basófilos/citología , Citocinas , Modelos Animales de Enfermedad , Eosinófilos/citología , Humanos , Inmunidad Innata , Pulmón/citología , Pulmón/inmunología , Linfocitos/citología , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Ratones SCID , Ratones TransgénicosRESUMEN
Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVß3 and αVß5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVß3 and αVß5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8.
Asunto(s)
Antígenos de Superficie , Expresión Génica , Proteínas de la Leche , Animales , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/química , Antígenos de Superficie/genética , Antígenos de Superficie/aislamiento & purificación , Células CHO , Cricetinae , Cricetulus , Humanos , Ratones , Proteínas de la Leche/biosíntesis , Proteínas de la Leche/química , Proteínas de la Leche/genética , Proteínas de la Leche/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Células Sf9 , SpodopteraRESUMEN
Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.
Asunto(s)
Técnicas de Visualización de Superficie Celular/métodos , Sistema Libre de Células , Fragmentos Fab de Inmunoglobulinas , Biblioteca de Péptidos , Anticuerpos/genética , Anticuerpos Monoclonales Humanizados/genética , Antígeno Carcinoembrionario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Trastuzumab , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Antibodies that specifically recognize polyubiquitin chains containing ubiquitins linked at a particular lysine residue are powerful tools for interrogating endogenous protein modifications. Here, we describe protocols for revealing K11-, K48-, and K63-linked polyubiquitin chains by western blotting, immunoprecipitation, or immunostaining.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Poliubiquitina/química , Poliubiquitina/inmunología , Western Blotting/métodos , Inmunoprecipitación/métodos , Poliubiquitina/metabolismo , Procesamiento Proteico-Postraduccional , Enzimas Activadoras de Ubiquitina , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.
Asunto(s)
Anticuerpos/química , Fragmentos Fab de Inmunoglobulinas/química , Poliubiquitina/química , Anticuerpos/metabolismo , Técnica del Anticuerpo Fluorescente , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoprecipitación , Modelos Moleculares , Poliubiquitina/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/metabolismo , UbiquitinaciónRESUMEN
Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.
Asunto(s)
Proteínas Mutantes/química , Ubiquitina/química , Proteínas Ubiquitinadas/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Humanos , Inmunoprecipitación , Células Jurkat , Leupeptinas/farmacología , Lisina/química , Metionina/química , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/química , Inhibidores de Proteasoma , Espectrometría de Masas en Tándem , Proteínas Ubiquitinadas/química , UbiquitinaciónRESUMEN
Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Ciclo Celular/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Ciclosoma-Complejo Promotor de la Anafase , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Células HeLa , Humanos , Ubiquitina/química , Ubiquitina/inmunologíaRESUMEN
Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.
Asunto(s)
Anticuerpos/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina/metabolismo , Animales , Línea Celular , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Proteínas de Complejo Poro Nuclear/química , Biblioteca de Péptidos , Proteínas de Unión al ARN/química , Saccharomyces cerevisiae , Schizosaccharomyces , Ubiquitina/química , UbiquitinaciónRESUMEN
G protein-coupled receptors are one of the largest protein families in nature; however, the mechanisms by which they activate G proteins are still poorly understood. To identify residues on the intracellular face of the human C5a receptor that are involved in G protein activation, we performed a genetic analysis of each of the three intracellular loops and the carboxyl-terminal tail of the receptor. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The third intracellular loop contains the largest number of preserved residues (positions resistant to amino acid substitutions), followed by the second loop, the first loop, and lastly the carboxyl terminus. Surprisingly, complete removal of the carboxyl-terminal tail did not impair C5a receptor signaling. When mapped onto a three-dimensional structural model of the inactive state of the C5a receptor, the preserved residues reside on one half of the intracellular surface of the receptor, creating a potential activation face. Together these data provide one of the most comprehensive functional maps of the intracellular surface of any G protein-coupled receptor to date.