RESUMEN
A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.
Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Anticuerpos Anti-VIH , Proteína gp41 de Envoltorio del VIH , Infecciones por VIH , VIH-1 , Macaca mulatta , Animales , Humanos , Proteína gp41 de Envoltorio del VIH/inmunología , Anticuerpos Anti-VIH/inmunología , Ratones , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , VIH-1/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Vacunación , Anticuerpos ampliamente neutralizantes/inmunología , Linfocitos B/inmunología , Nanopartículas/química , Femenino , Regiones Determinantes de Complementariedad/inmunología , Epítopos/inmunologíaRESUMEN
Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.
Asunto(s)
Afinidad de Anticuerpos , Linfocitos B , Centro Germinal , Anticuerpos Anti-VIH , VIH-1 , Centro Germinal/inmunología , Animales , Ratones , Humanos , Linfocitos B/inmunología , VIH-1/inmunología , Anticuerpos Anti-VIH/inmunología , Afinidad de Anticuerpos/inmunología , Anticuerpos Neutralizantes/inmunología , Infecciones por VIH/inmunología , Vacunas contra el SIDA/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Técnicas de Sustitución del Gen , Ratones Transgénicos , Anticuerpos ampliamente neutralizantes/inmunología , Ratones Endogámicos C57BLRESUMEN
Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.
Asunto(s)
Vacunas contra el SIDA , Anticuerpos ampliamente neutralizantes , Centro Germinal , Anticuerpos Anti-VIH , VIH-1 , Inmunización Secundaria , Nanopartículas , Vacunas de ARNm , Animales , Humanos , Ratones , Vacunas contra el SIDA/inmunología , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Reacciones Cruzadas , Técnicas de Sustitución del Gen , Centro Germinal/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , VIH-1/genética , Liposomas , Células B de Memoria/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/genética , Hipermutación Somática de Inmunoglobulina , Vacunas de ARNm/inmunología , Femenino , Ratones Endogámicos C57BLRESUMEN
Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.
Asunto(s)
Anticuerpos ampliamente neutralizantes , Nanopartículas , ARN Mensajero , Animales , Ratones , Humanos , ARN Mensajero/inmunología , ARN Mensajero/genética , Nanopartículas/química , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Lípidos/inmunología , Infecciones por VIH/inmunología , Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , VIH-1/inmunología , Femenino , Anticuerpos Monoclonales , LiposomasRESUMEN
Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus de la Influenza A , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae , Vacunación , Animales , Ratones , Humanos , Anticuerpos Antivirales/inmunología , Vacunas contra la Influenza/inmunología , Virus de la Influenza A/inmunología , Anticuerpos Neutralizantes/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Sustitución de Aminoácidos , Linfocitos B/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Anticuerpos ampliamente neutralizantes/inmunologíaRESUMEN
Antibodies are produced at high rates to provide immunoprotection, which puts pressure on the B cell translational machinery. Here, we identified a pattern of codon usage conserved across antibody genes. One feature thereof is the hyperutilization of codons that lack genome-encoded Watson-Crick transfer RNAs (tRNAs), instead relying on the posttranscriptional tRNA modification inosine (I34), which expands the decoding capacity of specific tRNAs through wobbling. Antibody-secreting cells had increased I34 levels and were more reliant on I34 for protein production than naïve B cells. Furthermore, antibody I34-dependent codon usage may influence B cell passage through regulatory checkpoints. Our work elucidates the interface between the tRNA pool and protein production in the immune system and has implications for the design and selection of antibodies for vaccines and therapeutics.
Asunto(s)
Anticuerpos , Formación de Anticuerpos , Linfocitos B , Uso de Codones , Cadenas Pesadas de Inmunoglobulina , Inosina , ARN de Transferencia , Formación de Anticuerpos/genética , Codón/genética , Inosina/genética , Inosina/metabolismo , ARN de Transferencia/genética , Anticuerpos/genética , Humanos , Linfocitos B/inmunología , Cadenas Pesadas de Inmunoglobulina/genéticaRESUMEN
Oral cancer is primarily squamous-cell carcinoma with a 5-year survival rate of approximately 50%. Lysyl oxidase (LOX) participates in collagen and elastin maturation. The propeptide of LOX is released as an 18 kDa protein (LOX-PP) in the extracellular environment by procollagen C-proteinases and has tumor-inhibitory properties. A polymorphism in the propeptide region of LOX (rs1800449, G473A) results in a single amino acid substitution of Gln for Arg. Here we investigated the frequency of rs1800449 in OSCC employing TCGA database resources and determined the kinetics and severity of precancerous oral lesion development in wildtype and corresponding knockin mice after exposure to 4-nitroquinoline oxide (4 NQO) in drinking water. Data show that the OSCC is more common in humans carrying the variant compared to the wildtype. Knockin mice are more susceptible to lesion development. The immunohistochemistry of LOX in mouse tissues and in vitro studies point to a negative feedback pathway of wildtype LOX-PP on LOX expression that is deficient in knockin mice. Data further demonstrate modulations of T cell phenotype in knockin mice toward a more tumor-permissive condition. Data provide initial evidence for rs1800449 as an oral cancer susceptibility biomarker and point to opportunities to better understand the functional mechanism of LOX-PP cancer inhibitory activity.
Asunto(s)
Neoplasias de la Boca , Proteína-Lisina 6-Oxidasa , Animales , Humanos , Ratones , Carcinógenos , Colágeno/genética , Neoplasias de la Boca/genética , Polimorfismo Genético , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Ratones , Humanos , Animales , Anticuerpos Anti-VIH , Anticuerpos ampliamente neutralizantes , Anticuerpos Neutralizantes , ARN Mensajero/genética , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.
Asunto(s)
Linfocitos B , Centro Germinal , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Antígenos , Epítopos , Inmunidad Humoral , RatonesRESUMEN
Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Epítopos/inmunología , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/inmunología , Vacunas de ADN/inmunología , Traslado Adoptivo , Animales , Anticuerpos Antiprotozoarios/metabolismo , Modelos Animales de Enfermedad , Epítopos/genética , Ingeniería Genética , Humanos , Evasión Inmune , Inmunogenicidad Vacunal , Ratones , Ratones SCID , Proteínas Protozoarias/genética , Relación Estructura-Actividad , VacunaciónRESUMEN
B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.
Asunto(s)
Linfocitos B/metabolismo , Sistemas CRISPR-Cas/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Sistemas CRISPR-Cas/inmunología , Línea Celular , Técnicas de Sustitución del Gen/métodos , Centro Germinal/inmunología , Centro Germinal/metabolismo , Células HEK293 , VIH-1/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
Here, we describe a one-step, in vivo CRISPR/Cas9 nuclease-mediated strategy to generate knock-in mice. We produced knock-in (KI) mice wherein a 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease-induced double-stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double-stranded breaks are subsequently repaired via homology-directed repair by a plasmid-borne template containing the pre-arranged human immunoglobulin heavy chain. To validate our knock-in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B-cell development and performing single B-cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30-50%). In the future, we envision that such knock-in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases.
Asunto(s)
Linfocitos B/inmunología , Sistemas CRISPR-Cas , Técnicas de Sustitución del Gen/métodos , Cadenas Pesadas de Inmunoglobulina , Animales , Linfocitos B/citología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Ratones TransgénicosRESUMEN
The propeptide (LOX-PP) domain of the lysyl oxidase proenzyme was shown to inhibit the transformed phenotype of breast, lung and pancreatic cells in culture and the formation of Her2/neu-driven breast cancer in a xenograft model. A single nucleotide polymorphism (SNP, rs1800449) positioned in a highly conserved region of LOX-PP results in an Arg158Gln substitution (humans). This arginine (Arg)âglutamine (Gln) substitution profoundly impaired the ability of LOX-PP to inhibit the invasive phenotype and xenograft tumor formation. To study the effect of the SNP in vivo, here we established a knock in (KI) mouse line (LOX-PPGln mice) expressing an Arg152Gln substitution corresponding to the human Arg158Gln polymorphism. Breast cancer was induced in wild-type (WT) and LOX-PPGln female mice beginning at 6 weeks of age by treatment with 7,12-dimethylbenz(a)anthracene (DMBA) in combination with progesterone. Time course analysis of tumor development demonstrated earlier tumor onset and shorter overall survival in LOX-PPGln versus WT mice. To further compare the tumor burden in WT and LOX-PPGln mice, inguinal mammary glands from both groups of mice were examined for microscopic lesion formation. LOX-PPGln glands contained more lesions (9.6 versus 6.9 lesions/#4 bilateral). In addition, more DMBA-treated LOX-PPGln mice had increased leukocyte infiltrations in their livers and were moribund compared with DMBA-treated WT mice. Thus, these data indicate that the ArgâGln substitution in LOX-PP could be an important marker associated with a more aggressive cancer phenotype and that this KI model is ideal for further mechanistic studies regarding the tumor suppressor function of LOX-PP.
Asunto(s)
Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/genética , Carcinógenos/toxicidad , Proteínas de la Matriz Extracelular/genética , Polimorfismo de Nucleótido Simple/genética , Proteína-Lisina 6-Oxidasa/genética , Animales , Biomarcadores de Tumor/genética , Carcinogénesis/inducido químicamente , Carcinogénesis/genética , Línea Celular Tumoral , Genes Supresores de Tumor/efectos de los fármacos , Xenoinjertos , Ratones , Ratones Endogámicos C57BLRESUMEN
The lysyl oxidase proenzyme propeptide region (LOX-PP) is a tumor suppressor protein whose mechanism of action is not completely understood. Here, the Ubiquitously expressed Transcript (UXT) was identified in a yeast two-hybrid assay with LOX-PP as bait and confirmed as a novel LOX-PP associating protein. UXT, a prefoldin-like protein, is ubiquitous in human and mouse. Since UXT modulates androgen receptor transcriptional activity in prostate cancer, we studied its role in breast cancer. Breast tumors and derived cell lines overexpressed UXT. UXT was able to associate with the estrogen receptor alpha (ER) and decrease its transcriptional activity and target gene expression. Conversely, UXT knockdown increased ER element-dependent transcriptional activity. Ectopic LOX-PP relocalized UXT to the cytoplasm and decreased its stability. UXT ubiquitination and depletion in the presence of LOX-PP was rescued by a proteasomal inhibitor. In summary, proteasome-mediated turnover of UXT upon interaction with LOX-PP releases repression of ER transcriptional activity. J. Cell. Biochem. 118: 2347-2356, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Tumoral , Receptor alfa de Estrógeno/genética , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Células MCF-7 , Ratones , Chaperonas Moleculares/genética , Proteínas de Neoplasias/genética , Unión Proteica , Proteína-Lisina 6-Oxidasa/genética , Transducción de Señal/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6-10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6-10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.
Asunto(s)
Neoplasias de la Mama/patología , Proteína Sustrato Asociada a CrK/fisiología , Proteínas de Neoplasias/fisiología , Secuencias de Aminoácidos , Animales , Neoplasias de la Mama/genética , Proteína Tirosina Quinasa CSK , Línea Celular Tumoral , Proteína Sustrato Asociada a CrK/química , Proteína Sustrato Asociada a CrK/genética , Progresión de la Enfermedad , Femenino , Genes Reporteros , Xenoinjertos , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Metástasis de la Neoplasia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/genética , Fosforilación , Fosfotirosina/metabolismo , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Chronic HBV infection is the leading cause of liver cirrhosis and hepatic cancer, but the individual responses toward HBV infection are highly variable, ranging from asymptomatic to chronic active hepatitis B inflammation. In this study, we hypothesized that the different individual responses to HBV infection was associated with differences in HBV-specific CD8(+) T cell-mediated inflammation and cytotoxicity. Blood samples were collected from subjects with asymptomatic HBV-infection, subjects undergoing active chronic HBV flares (active CHB), and subjects with HBV-infected hepatocellular carcinoma (HBV-HCC). By tetramer staining, we found that all three groups had similar frequencies of HBVspecific CD8(+) T cells. However, after HBV peptide stimulation, the HBV-specific CD8(+) T cells in asymptomatic subjects had significantly stronger interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and CD107a expression than those in active CHB and HBV-HCC patients. Examination of surface marker expression revealed that the PD-1(-)Tim-3(-) double-negative cell population was the main contributor to HBV-specific inflammation. In active CHB patients and HBV-HCC patients, however, the frequencies of activated PD-1(-)Tim-3(-) cells were significantly reduced. Moreover, the serum HBV DNA titer was not correlated with the frequencies of HBV-specific CD8(+) T cells but was inversely correlated with the frequencies of IFN-g-expressing and CD107a-express cells in response to HBV stimulation. Together, our data demonstrated that the status of HBVspecific CD8(+) T cell exhaustion was associated with different clinical outcomes of chronic HBV infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Adulto , Anciano , Citotoxicidad Inmunológica , ADN Viral/sangre , Progresión de la Enfermedad , Epítopos de Linfocito T/inmunología , Femenino , Citometría de Flujo , Hepatitis B Crónica/sangre , Hepatitis B Crónica/virología , Humanos , Mediadores de Inflamación/inmunología , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Proteína 1 de la Membrana Asociada a los Lisosomas/biosíntesis , Proteína 1 de la Membrana Asociada a los Lisosomas/inmunología , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunología , Carga Viral , Adulto JovenRESUMEN
CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas ras/metabolismo , Dominios Homologos src , Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Sitios de Unión , Biología Computacional , Cristalografía por Rayos X , Proteínas del Citoesqueleto/química , Células HEK293 , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Elevated levels of p130(Cas) (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130(Cas) protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130(Cas)-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130(Cas) exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130(Cas) on cell biology and therefore will be the target of future studies.
Asunto(s)
Proteína Sustrato Asociada a CrK/genética , Proteína Sustrato Asociada a CrK/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación Enzimológica de la Expresión Génica , Empalme Alternativo , Animales , Secuencia de Bases , Adhesión Celular , Movimiento Celular , Exones , Fibroblastos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Invasividad Neoplásica , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Dominios Homologos srcRESUMEN
The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the "Cbl-interacting protein of 85-kDa" (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.