RESUMO
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.
Assuntos
Anticorpos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo , Animais , Linhagem Celular , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Proteínas Formadoras de Poros Nucleares/química , Biblioteca de Peptídeos , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiae , Schizosaccharomyces , Ubiquitina/química , UbiquitinaçãoRESUMO
Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.
Assuntos
Cisteína Endopeptidases/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Animais , Morte Celular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Feminino , Inflamação/genética , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Poliubiquitina/química , Poliubiquitina/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/metabolismo , UbiquitinaçãoRESUMO
Chronic viruses such as herpes simplex virus 1 (HSV-1) evade the hosts' immune system by inducing the exhaustion of antiviral T cells. In the present study, we found that exhausted HSV-specific CD8+ T cells, with elevated expression of programmed death ligand-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) receptors were frequent in symptomatic patients, with a history of numerous episodes of recurrent corneal herpetic disease, compared to asymptomatic patients who never had corneal herpetic disease. Subsequently, using a rabbit model of recurrent ocular herpes, we found that the combined blockade of PD-1 and LAG-3 pathways with antagonist antibodies significantly restored the function of tissue-resident antiviral CD8+ TRM cells in both the cornea and the trigeminal ganglia (TG). An increased number of functional tissue-resident HSV-specific CD8+ TRM cells in latently infected rabbits was associated with protection against recurrent herpes infection and disease. Compared to the PD-1 or LAG-3 blockade alone, the combined blockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional Ki-67+, IFN-γ+, CD107+, and CD8+ T cells. Moreover, using the human leukocyte antigen (HLA) transgenic rabbit model, we found that dual blockade of PD-1 and LAG-3 reinforced the effect of a multiepitope vaccine in boosting the frequency of HSV-1-specific CD8+ TRM cells and reducing disease severity. Thus, both the PD-1 and the LAG-3 exhaustion pathways play a fundamental role in ocular herpes T cell immunopathology and provide important immune checkpoint targets to combat ocular herpes.IMPORTANCE HSV-specific tissue-resident memory CD8+ TRM cells play a critical role in preventing virus reactivation from latently infected TG and subsequent virus shedding in tears that trigger the recurrent corneal herpetic disease. In this report, we determined how the dual blockade of PD-1 and LAG-3 immune checkpoints, combined with vaccination, improved the function of CD8+ TRM cells associated with a significant reduction in recurrent ocular herpes in HLA transgenic (Tg) rabbit model. The combined blockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional CD8+ TRM cells that infiltrated both the cornea and the TG. The preclinical findings using the established HLA Tg rabbit model of recurrent herpes highlight that blocking immune checkpoints combined with a T cell-based vaccine would provide an important strategy to combat recurrent ocular herpes in the clinic.
Assuntos
Antígenos CD/imunologia , Herpesvirus Humano 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Adulto , Animais , Antígenos CD/metabolismo , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Córnea/virologia , Epitopos de Linfócito T/imunologia , Feminino , Antígenos HLA/metabolismo , Antígeno HLA-A2/imunologia , Vacinas contra o Vírus do Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Antígenos de Histocompatibilidade/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunização/métodos , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Coelhos , Gânglio Trigeminal/virologia , Vacinação/métodos , Eliminação de Partículas Virais/imunologia , Eliminação de Partículas Virais/fisiologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Circulating conventional memory CD8+ T cells (i.e., the CD8+ effector memory T [TEM] cell and CD8+ central memory T [TCM] cell subsets) and the noncirculating CD8+ tissue-resident memory T (TRM) cell subset play a critical role in mucosal immunity. Mucosal chemokines, including the recently discovered CXCL17, are also important in mucosal immunity because they are homeostatically expressed in mucosal tissues. However, whether the CXCL17 chemokine contributes to the mobilization of memory CD8+ T cell subsets to access infected mucosal tissues remains to be elucidated. In this study, we report that after intravaginal HSV type 1 infection of B6 mice, we detected high expression levels of CXCL17 and increased numbers of CD44highCD62LlowCD8+ TEM and CD103highCD8+ TRM cells expressing CXCR8, the cognate receptor of CXCL17, in the vaginal mucosa (VM) of mice with reduced genital herpes infection and disease. In contrast to wild-type B6 mice, the CXCL17-/- mice developed 1) fewer CXCR8+CD8+ TEM and TRM cells associated with more virus replication in the VM and more latency established in dorsal root ganglia, and 2) reduced numbers and frequencies of functional CD8+ T cells in the VM. These findings suggest that the CXCL17/CXCR8 chemokine pathway plays a crucial role in mucosal vaginal immunity by promoting the mobilization of functional protective CD8+ TEM and CD8+ TRM cells, within this site of acute and recurrent herpes infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiocinas CXC/imunologia , Herpes Genital/imunologia , Imunidade nas Mucosas/imunologia , Vagina/imunologia , Animais , Quimiotaxia de Leucócito/imunologia , Feminino , Memória Imunológica/imunologia , Camundongos , Subpopulações de Linfócitos T/imunologiaRESUMO
Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that infects the cornea, causing potentially blinding herpetic disease. A clinical herpes vaccine is still lacking. In the present study, a novel prime/pull vaccine was tested in a human leukocyte antigen (HLA) transgenic rabbit model of ocular herpes (HLA Tg rabbits). Three peptide epitopes were selected, from the HSV-1 membrane glycoprotein C (UL44400-408), the DNA replication binding helicase (UL9196-204), and the tegument protein (UL25572-580), all preferentially recognized by CD8+ T cells from "naturally protected" HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who never had recurrent corneal herpetic disease). HLA Tg rabbits were immunized with a mixture of these three ASYMP CD8+ T cell peptide epitopes (UL44400-408, UL9196-204, and UL25572-580), which were delivered subcutaneously with CpG2007 adjuvant (prime). Fifteen days later, half of the rabbits received a topical ocular treatment with a recombinant neurotropic adeno-associated virus type 8 (AAV8) vector expressing the T cell-attracting CXCL10 chemokine (pull). The frequency and function of HSV-specific CD8+ T cells induced by the prime/pull vaccine were assessed in the peripheral blood, cornea, and trigeminal ganglion (TG). Compared to the cells generated in response to peptide immunization alone, the peptide/CXCL10 prime/pull vaccine generated frequent polyfunctional gamma interferon-positive (IFN-γ+) CD107+ CD8+ T cells that infiltrated both the cornea and TG. CD8+ T cell mobilization into the cornea and TG of prime/pull-vaccinated rabbits was associated with a significant reduction in corneal herpesvirus infection and disease following an ocular HSV-1 (strain McKrae) challenge. These findings draw attention to the novel prime/pull vaccine strategy for mobilizing antiviral CD8+ T cells into tissues to protect against herpesvirus infection and disease.IMPORTANCE There is an urgent need for a vaccine against widespread herpes simplex virus infections. The present study demonstrates that immunization of HLA transgenic rabbits with a peptide/CXCL10 prime/pull vaccine triggered mobilization of HSV-specific CD8+ T cells locally into the cornea and TG, the sites of acute and latent herpesvirus infections, respectively. Mobilization of antiviral CD8+ T cells into the cornea and TG of rabbits that received the prime/pull vaccine was associated with protection against ocular herpesvirus infection and disease following an ocular HSV-1 challenge. These results highlight the importance of the prime/pull vaccine strategy to bolster the number and function of protective CD8+ T cells within infected tissues.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiocina CXCL10/metabolismo , Córnea/imunologia , Vacinas contra o Vírus do Herpes Simples/imunologia , Ceratite Herpética/prevenção & controle , Subpopulações de Linfócitos T/imunologia , Gânglio Trigeminal/imunologia , Animais , Animais Geneticamente Modificados , Quimiocina CXCL10/administração & dosagem , Modelos Animais de Doenças , Epitopos/imunologia , Antígenos HLA/genética , Antígenos HLA/metabolismo , Vacinas contra o Vírus do Herpes Simples/administração & dosagem , Humanos , Interferon gama/análise , Ceratite Herpética/patologia , Ceratite Herpética/virologia , Proteína 1 de Membrana Associada ao Lisossomo/análise , Coelhos , Simplexvirus/imunologia , Simplexvirus/isolamento & purificação , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Carga ViralRESUMO
BACKGROUND: Association between low counts of herpesvirus-specific T cells and subsequent relapse of hematologic malignancy has been shown in two retrospective studies. METHODS: Here we present results of a prospective validation study. Multiple subsets of Epstein-Barr virus (EBV)-specific T cells were measured in 69 patients on day 56 and 84, using intracellular flow cytometry after incubation of blood mononuclear cells (MNCs) with EBV peptides or lysate. RESULTS: All EBV T-cell subsets measured, both on day 56 and 84, were lower in patients who did versus did not subsequently relapse. This was most significant for day 56 EBV lysate-stimulated CD8 T cells producing interferon-gamma. Patients with day 56 counts of this subset >5/µL had a significantly lower likelihood of relapse compared with those with ≤5/µL (subhazard ratio, 5.7; Pâ¯=â¯0.007). Similar significant associations were shown for a total of seven EBV T-cell subsets on day 56 and nine subsets on day 84. However, sensitivity and specificity of relapse prediction using the count of any subset was low (area under the curve of receiver-operator characteristic curve was <0.8). DISCUSSION: In conclusion, the association between EBV T-cell counts and subsequent relapse is valid. However, its clinical utility appears to be limited.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Subpopulações de Linfócitos T/virologia , Transplante Homólogo/efeitos adversos , Adulto , Idoso , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Criopreservação , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/patologia , Feminino , Neoplasias Hematológicas/epidemiologia , Neoplasias Hematológicas/patologia , Herpesvirus Humano 4/imunologia , Humanos , Incidência , Interferon gama/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco de Sangue Periférico/métodos , Estudos Prospectivos , Curva ROC , Subpopulações de Linfócitos T/imunologia , Doadores de Tecidos , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Atopy is defined as excess allergen-specific IgE (A-IgE). IgE is produced by plasma cells that differentiate from allergen-specific B cells. B cells are known to be killed by chemotherapy; however, it is not known whether A-IgE-secreting plasma cells are killed or inhibited by chemotherapy. If yes, serum A-IgE levels would be expected to decrease after chemotherapy. OBJECTIVES: We aimed to determine whether A-IgE levels in atopic individuals (serum A-IgE ≥0.35 kUA/L) decrease into the nonatopic range (< 0.35 kUA/L) after chemotherapy. METHODS: In 105 patients undergoing chemotherapy for acute leukemia, we measured serum A-IgE before and after chemotherapy. In a subset of these patients, we also measured B cell counts before and after chemotherapy. RESULTS: Of the 105 patients, 36 were atopic. In these patients, median A-IgE level before chemotherapy was 1.6 kUA/L whereas the median level after chemotherapy was 0.6 kUA/L (p < 0.001). In 12/36 (33%) patients, A-IgE levels decreased into the nonatopic range. In nonatopic patients (n = 69), the median A-IgE level also dropped: from 0.04 kUA/L before to 0.03 kUA/L after chemotherapy (p = 0.001). Among the total patients (n = 105), the median pre:post-chemotherapy A-IgE ratio was 1.8 (2.6 in atopic and 1.5 in nonatopic patients). In contrast, the median ratio of pre:post-chemotherapy B cell counts was 87.6. CONCLUSIONS: A-IgE levels decrease after chemotherapy but markedly less than B cell counts. Thus, at least some A-IgE plasma cells appear to survive chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Linfócitos B/efeitos dos fármacos , Tratamento Farmacológico , Imunoglobulina E/sangue , Adulto , Idoso , Antineoplásicos/uso terapêutico , Linfócitos B/citologia , Humanos , Leucemia/tratamento farmacológico , Pessoa de Meia-IdadeRESUMO
Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger 4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.
Assuntos
NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Dedos de Zinco , Sítios de Ligação , Cristalografia por Raios X , Lisina/metabolismo , Modelos Moleculares , Mutação/genética , Proteínas Nucleares/química , Poliubiquitina/metabolismo , Ligação Proteica , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc.
Assuntos
Anticorpos/química , Fucose/metabolismo , Cetona Oxirredutases/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Animais , Anticorpos/genética , Anticorpos/metabolismo , Células CHO , Sistemas CRISPR-Cas , Cricetinae , Cricetulus , Fucose/química , Edição de Genes , Técnicas de Inativação de Genes , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Fibroblastos , Humanos , Camundongos , Mitose/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Paclitaxel/farmacologia , Farmacogenética , Fosforilação/efeitos dos fármacos , Poliploidia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Vincristina/farmacologiaRESUMO
Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.
Assuntos
Inibidores de Proteases/farmacologia , Ubiquitina Tiolesterase/química , Ubiquitina/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de UbiquitinaRESUMO
The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3ß to foster phosphorylation of the Wnt effector ß-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced ß-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Proteína Axina/metabolismo , Poliubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Via de Sinalização Wnt/fisiologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Proteína Axina/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Poliubiquitina/genética , Ligação Proteica , Ubiquitina-Proteína Ligases/genéticaRESUMO
Chinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high-end pH-controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production. Accumulation of SCFAs in the culture media is critical for metabolic shift toward higher specific productivity and hence titer. Here we describe knocking out BCKDHa/b genes (2XKO), which act downstream of the BCAT1, in a BAX/BAK KO CHO host cell line background to reduce accumulation of growth-inhibitory molecules in culture. Evaluation of the new 4XKO CHO cell lines in fed-batch production cultures (without HiPDOG) revealed that partial KO of BCKDHa/b genes in an apoptosis-resistant (BAX/BAK KO) background can achieve higher viabilities and mAb titers. This was evident when SCFAs were added to boost productivity as such additives negatively impacted culture viability in the WT but not BAX/BAK KO cells during batch production. Altogether, our findings suggest that SCFA addbacks can significantly increase productivity and mAb titers in the context of apoptosis-attenuated CHO cells with partial KO of BCAA genes. Such engineered CHO hosts can offer productivity advantages for expressing biotherapeutics in an industrial setting.
RESUMO
Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.
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Introduction The Routine Opioid Outcome Monitoring (ROOM) Tool was developed for use in community pharmacies in Australia. It facilitates pharmacists' screening and brief interventions regarding an individual's opioid use for chronic pain. At our academic teaching hospital, the ROOM Tool was adapted to incorporate a communication tool that includes a pharmacist's assessment and recommendations for primary care providers. This modified ROOM Tool was implemented as part of usual care in our outpatient pharmacies; however, the value to primary care providers is unknown. Aim The aim of this study was to determine primary care provider perspectives on the modified ROOM Tool. Methods Focus groups were conducted with primary care providers from an Academic Family Health Team. The focus group encompassed topics related to the positive and negative aspects of the modified ROOM Tool in supporting the care of patients using opioids for chronic pain. Qualitative content analysis of transcripts was performed to identify themes. Results Three focus groups were conducted with a total of six participants. Four themes emerged: (i) Facilitators to using the tool, (ii) Barriers to using the tool, (iii) Recommendations for improvement, (iv) Impact of the tool on patient care and safety. Discussion The ROOM Tool paired with the communication tool supports collaboration between pharmacists and primary care providers. The communication tool standardises the approach for communicating the pharmacist's assessment and recommendations. Recommendations to refine this modified ROOM Tool may increase its utility to primary care providers and enhance the impact on patient care and safety.
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Chinese hamster ovary (CHO) cells are commonly used for the expression of therapeutic proteins. To increase the titer output of CHO production cultures either specific productivity (Qp), growth, or both need to be increased. Generally, Qp and growth are inversely correlated and cell lines with high Qp have slower growth and vice versa. During the cell line development (CLD) process, the faster-growing cells tend to take over the culture and represent the majority of the isolated clones post single cell cloning. In this study, combinations of regulated and constitutive expression systems were used to supertransfect targeted integration (TI) cell lines expressing the same antibody either constitutively or under-regulated expression. Clone screening with a hybrid expression system (inducible + constitutive) allowed identification and selection of higher titer clones under uninduced conditions, without a negative impact on cell growth during clone selection and expansion. Induction of the regulated promoter(s) during the production phase increased the Qp without negatively affecting growth, resulting in approximately twofold higher titers (from 3.5 to 6-7 g/L). This was also confirmed using a 2-site TI host where the gene of interest was expressed inducibly from Site 1 and constitutively from Site 2. Our findings suggest that such a hybrid expression CLD system can be used to increase production titers, providing a novel approach for expression of therapeutic proteins with high titer market demands.
Assuntos
Anticorpos , Cricetinae , Animais , Células CHO , Cricetulus , Células Clonais , Proliferação de Células/genética , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: Psychedelic-assisted psychotherapy with psilocybin is an emerging therapy with great promise for depression, and modern psychedelic therapy (PT) methods incorporate music as a key element. Music is an effective emotional/hedonic stimulus that could also be useful in assessing changes in emotional responsiveness following PT. METHODS: Brain responses to music were assessed before and after PT using functional Magnetic Resonance Imaging (fMRI) and ALFF (Amplitude of Low Frequency Fluctuations) analysis methods. Nineteen patients with treatment-resistant depression underwent two treatment sessions involving administration of psilocybin, with MRI data acquired one week prior and the day after completion of psilocybin dosing sessions. RESULTS: Comparison of music-listening and resting-state scans revealed significantly greater ALFF in bilateral superior temporal cortex for the post-treatment music scan, and in the right ventral occipital lobe for the post-treatment resting-state scan. ROI analyses of these clusters revealed a significant effect of treatment in the superior temporal lobe for the music scan only. Voxelwise comparison of treatment effects showed relative increases for the music scan in the bilateral superior temporal lobes and supramarginal gyrus, and relative decreases in the medial frontal lobes for the resting-state scan. ALFF in these music-related clusters was significantly correlated with intensity of subjective effects felt during the dosing sessions. LIMITATIONS: Open-label trial. Relatively small sample size. CONCLUSIONS: These data suggest an effect of PT on the brain's response to music, implying an elevated responsiveness to music after psilocybin therapy that was related to subjective drug effects felt during dosing.