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BACKGROUND: Although the risk of exposure to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is higher for frontline healthcare workers, not all personnel have similar risks. Determining infection rate is difficult due to the limits on testing and the high rate of asymptomatic individuals. Detection of antibodies against SARS-CoV-2 may be useful for determining prior exposure to the virus and assessing mitigation strategies, such as isolation, masks, and other protective equipment. METHODS: An online assessment that included demographic, clinical, and exposure information and a blood sample was collected from 20 614 participants out of ~43 000 total employees at Beaumont Health, which includes 8 hospitals distributed across the Detroit metropolitan area in southeast Michigan. The presence of anti-SARS-CoV-2 IgG was determined using the EUROIMMUN assay. RESULTS: A total of 1818 (8.8%) participants were seropositive between April 13 and May 28, 2020. Among the seropositive individuals, 44% reported that they were asymptomatic during the month prior to blood collection. Healthcare roles such as phlebotomy, respiratory therapy, and nursing/nursing support exhibited significantly higher seropositivity. Among participants reporting direct exposure to a Coronavirus Disease 2019 (COVID-19) positive individual, those wearing an N95/PAPR mask had a significantly lower seropositivity rate (10.2%) compared to surgical/other masks (13.1%) or no mask (17.5%). CONCLUSIONS: Direct contact with COVID-19 patients increased the likelihood of seropositivity among employees but study participants who wore a mask during COVID-19 exposures were less likely to be seropositive. Additionally, a large proportion of seropositive employees self-reported as asymptomatic. (Funded by Beaumont Health and by major donors through the Beaumont Health Foundation). CLINICALTRIALS.GOV NUMBER: NCT04349202.
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COVID-19 , Anticorpos Antivirais , Pessoal de Saúde , Humanos , Michigan , SARS-CoV-2RESUMO
The transcription factor NF-kappaB is a critical regulator of inflammatory and cell survival signals. Proteasomal degradation of NF-kappaB subunits plays an important role in the termination of NF-kappaB activity, and at least one of the identified ubiquitin ligases is a multimeric complex containing Copper Metabolism Murr1 Domain 1 (COMMD1) and Cul2. We report here that GCN5, a histone acetyltransferase, associates with COMMD1 and other components of the ligase, promotes RelA ubiquitination, and represses kappaB-dependent transcription. In this role, the acetyltransferase activity of GCN5 is not required. Interestingly, GCN5 binds more avidly to RelA after phosphorylation on Ser 468, an event that is dependent on IKK activity. Consistent with this, we find that both GCN5 and the IkappaB Kinase (IKK) complex promote RelA degradation. Collectively, the data indicate that GCN5 participates in the ubiquitination process as an accessory factor for a ubiquitin ligase, where it provides a novel link between phosphorylation and ubiquitination.
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Coenzimas/metabolismo , Fator de Transcrição RelA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Humanos , Quinase I-kappa B/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Estabilidade Proteica , UbiquitinaçãoRESUMO
OBJECTIVE: To compare total immunoglobulin (Ig) E assay performance characteristics between Abbott Architect and Siemens Immulite test systems. Reference intervals were also determined for both platforms in an American population of healthy adults. METHODS: Agreement of the two total IgE assays was evaluated in a cohort of 331 subjects with normal complete blood count (CBC) and comprehensive metabolic panel (CMP) results. Reference intervals were established in 302 subjects after exclusion of atopic individuals on the Abbott Architect and Siemens Immulite test systems. RESULTS: We demonstrated a 32% positive bias for total IgE quantitation on the Siemens Immulite platform compared to the Abbott Architect, despite both methods calibrated against the same WHO international reference material (75/502), Furthermore, the upper limit of the reference interval (95th percentile) was determined to be higher for the Siemens Immulite assay compared to the Abbott Architect (132 and 102 IU/mL, respectively). CONCLUSION: Despite the use of a common WHO reference material for total IgE assay calibration, significant differences in quantitation was observed between two FDA-cleared test systems. Given that, it is warranted for clinical laboratories to verify vendor established reference intervals and adjust accordingly based on internal assessment of the normal range.
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Imunoglobulina E , Humanos , Imunoglobulina E/sangue , Adulto , Valores de Referência , Feminino , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Idoso , Voluntários Saudáveis , Adolescente , Imunoensaio/normas , Imunoensaio/métodos , Reprodutibilidade dos Testes , CalibragemRESUMO
BACKGROUND: Serologic analysis is an important tool towards assessing the humoral response to COVID-19 infection and vaccination. Numerous serologic tests and platforms are currently available to support this line of testing. Two broad antibody testing categories are point-of-care lateral flow immunoassays and semi-quantitative immunoassays performed in clinical laboratories, which typically require blood collected from a finger-stick and a standard venipuncture blood draw, respectively. This study evaluated the use of dried blood spot (DBS) collections as a sample source for COVID-19 antibody testing using an automated clinical laboratory test system. METHODS: Two hundred and ninety-four participants in the BLAST COVID-19 seroprevalence study (NCT04349202) were recruited at the time of a scheduled blood draw to have an additional sample taken via finger stick as a DBS collection. Using the EUROIMMUN assay to assess SARS-CoV-2 anti-spike IgG status, DBS specimens were tested on 7, 14, 21, and 28 days post- collection and compared to the reference serum sample obtained from a blood draw for the BLAST COVID-19 study. RESULTS: SARS-CoV-2 anti-spike IgG status from DBS collections demonstrated high concordance with serum across all time points (7-28 days). However, the semi-quantitative value from DBS collections was lower on average than that from serum, resulting in increased uncertainty around the equivocal-to-positive analytical decision point. CONCLUSIONS: DBS collections can be substituted for venipuncture when assaying for COVID-19 IgG antibody, with samples being stable for at least 28 days at room temperature. Finger-stick sampling can therefore be advantageous for testing large populations for SARS-CoV-2 antibodies without the need for phlebotomists or immediate processing of samples. We have high confidence in serostaus determination from DBS collections, although the reduced semi-quantitative value may cause some low-level positives to fall into the equivocal or even negative range.
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COVID-19 , Humanos , Anticorpos Antivirais , COVID-19/diagnóstico , Teste Sorológico para COVID-19 , Teste para COVID-19 , Teste em Amostras de Sangue Seco , Imunoglobulina G , Flebotomia , SARS-CoV-2 , Sensibilidade e Especificidade , Estudos SoroepidemiológicosRESUMO
Humoral and cellular immune responses to SARS-CoV-2 and other coronaviruses in lung transplant recipients are unknown. We measured antibodies and T cell responses against the SARS-CoV-2 spike S2 and nucleocapsid antigens and spike antigens from common respiratory coronaviruses (229E, NL63, OC43, and HKU1) after vaccination or infection of LTxRs. 148 LTxRs from single center were included in this study: 98 after vaccination and 50 following SARS-CoV-2 infection. Antibodies were quantified by enzyme-linked immunosorbent assay. The frequency of T cells secreting IL2, IL4, IL10, IL17, TNFα, and IFNγ were enumerated by enzyme-linked immunospot assay. Our results have shown the development of antibodies to SARS-CoV-2 spike protein in infected LTxRs (39/50) and vaccinated LTxRs (52/98). Vaccinated LTxRs had higher number of T cells producing TNFα but less cells producing IFNγ than infected LTxRs in response to the nucleocapsid antigen and other coronavirus spike antigens. We didn't find correlation between the development of antibodies and cellular immune responses against the SARS-CoV-2 spike protein after vaccination. Instead, LTxRs have pre-existing cellular immunity to common respiratory coronaviruses, leading to cross-reactive immunity against SARS-CoV-2 which likely will provide protection against SARS-Cov-2 infection.
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COVID-19 , SARS-CoV-2 , Humanos , Transplantados , Fator de Necrose Tumoral alfa , Anticorpos , Imunidade Celular , ELISPOT , Anticorpos AntiviraisRESUMO
Cullin RING ligases (CRLs), the most prolific class of ubiquitin ligase enzymes, are multimeric complexes that regulate a wide range of cellular processes. CRL activity is regulated by CAND1 (Cullin-associated Nedd8-dissociated protein 1), an inhibitor that promotes the dissociation of substrate receptor components from the CRL. We demonstrate here that COMMD1 (copper metabolism MURR1 domain-containing 1), a factor previously found to promote ubiquitination of various substrates, regulates CRL activation by antagonizing CAND1 binding. We show that COMMD1 interacts with multiple Cullins, that the COMMD1-Cul2 complex cannot bind CAND1, and that, conversely, COMMD1 can actively displace CAND1 from CRLs. These findings highlight a novel mechanism of CRL activation and suggest that CRL regulation may underlie the pleiotropic activities of COMMD1.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Culina/metabolismo , Complexos Multiproteicos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Culina/genética , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos/genética , Ligação Proteica/fisiologia , Fatores de Transcrição/genéticaRESUMO
Objective: We aimed to identify risk factors for hospital admission and severe disease among fully vaccinated (FV) individuals with COVID-19. Further, we investigated if risk factors for hospitalization and severe disease are similar between unvaccinated (UV) and vaccinated individuals. Methods: This was a multicenter, observational cohort analysis from a large regional healthcare system in metro Detroit using electronic health record data to evaluate risk factors for hospitalization and severe COVID-19 disease. Vaccination data were retrieved using electronic medical records linked to our statewide immunization database. Consecutive adult FV and UV patients with a primary admission diagnosis of COVID-19 were included in the comparative analysis. Partially vaccinated patients and patients who had received a booster dose were excluded. The primary outcome of this study was hospital admission and severe disease inclusive of intensive care unit (ICU) admission, mechanical ventilation, or death. Results: Between December 15, 2020 and December 19, 2021, 20,584 emergency department visits met our inclusion criteria. Among these, 2005 (9.7%) visits consisted of FV individuals, 18,579 (90.3%) were UV, and 40.3% of UV and 52.7% of FV required hospitalization with similar (12.7% and 12.6%, respectively) rates of severe disease. Hospitalized UV patients with severe disease were younger than their FV counterparts (49.5% <65 years vs. 13.5% p < 0.001). Risk factors for severe disease on UV and FV included age ≥65 years (UV: adjusted odds ratio [aOR] 1.49, 95% confidence interval [CI] 1.28-1.73, p < 0.001 and FV: aOR 2.50, 95% CI 1.44-4.36 p = 0.001) and weighted Elixhauser score >10 (UV: aOR 9.11, 95% CI 6.92-12.00, p < 0.001 and FV: aOR 6.04, 95% CI 2.68-13.26, p < 0.001). However, only on UV status, body mass index (BMI) ≥30 kg/m2 was associated with increased odds of severe disease (aOR 2.59, 95% CI 2.09-3.22, p < 0.001). Conclusions: FV patients with breakthrough SARS-CoV-2 infection who require hospitalization and have severe disease are older and have more medical comorbidities compared to UV patients. When comparing risk factors for severe disease between UV and FV individuals, FV status is particularly associated with reduced risk among patients with a BMI ≥30 kg/m2 and a moderate number of medical comorbidities, regardless of age, highlighting the importance of vaccination in these particularly vulnerable groups.
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INTRODUCTION: Serological testing is an important tool to assist with assessing the immune response to SARS-CoV-2 infections, the causative agent of COVID-19. A quantitative assay was recently developed by Abbott Laboratories to measures antibodies against the receptor binding domain of the spike protein. In addition to assessing disease prevalence, this assay is useful towards determining the scale and duration of the humoral response to infection and vaccination. Here we evaluated the clinical and analytical performance of the quantitative Abbott AdviseDx SARS-CoV-2 IgG II assay and characterized the longitudinal dynamics of the IgG response against SARS-CoV-2 in 402 infected individuals up to 322 days post-symptom onset. METHODS: To assess test sensitivity, 1257 serum specimens derived from 402 patients positive for SARS-CoV-2 by RT-PCR were analyzed on the Abbott Alinity platform. To evaluate test specificity, 394 specimens were tested from patients who were symptomatic but PCR negative for SARS-CoV-2, as well as 305 archived pre-pandemic samples. To further characterize test performance metrics, we evaluated assay precision and linearity. RESULTS: The Abbott AdviseDx SARS-CoV-2 IgG II assay exhibited diagnostic specificity of 99.02% using 305 pre - COVID-19 serum specimens and 98.73% using 394 PCR negative specimens. Using 1257 sequential serum samples collected from PCR-confirmed individuals, clinical test sensitivity of the assay was 39.7% at 3-7 days, 75.9% at 8-14 days, 95.6% at 15-21 days, and 98.7% at 4-5 weeks post-symptom onset. The assay is linear across the analytical measurement range claimed by the manufacturer (22-25,000 AU/mL) and exhibited good analytical precision. The median concentration of IgG increased steadily from <22 AU/mL at 3-7 days post-symptom onset, to a peak of 14,421 AU/mL at 6-7 weeks. Although antibody concentration started to decline at 8-9 weeks following symptom onset, all patients remained seropositive during the observation period. When the positivity rate of this assay was compared with the Abbott anti-NP IgG and EUROIMMUN anti-S1 IgG tests, clinical sensitivity of the Abbott AdviseDx SARS-CoV-2 IgG II assay was the highest at all time points with the exception of 4-5 weeks after symptom onset. CONCLUSION: The Abbott AdviseDx SARS-CoV-2 IgG II assay offers high test specificity and sensitivity across a broad reportable range. We anticipate this assay will be a useful towards quantitatively assessing the humoral immune response to COVID-19 infection and vaccination.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , COVID-19/diagnóstico , Humanos , Imunoglobulina G , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Background: Antibody levels against SARS-CoV-2 can be used as an indicator of recent or past vaccination or infection. However, the prognostic value of antibodies targeting the receptor binding protein (anti-RBD) in hospitalized patients is not widely reported. Purpose: Determine prognostic impact of SARS-CoV-2 antibody quantification at the time of admission on clinical outcomes in hospitalized COVID-19 patients. Methods: We conducted a pilot observational study on patients hospitalized with SARS-CoV-2 infection to determine the prognostic impact of antibody quantitation within the first two days of admission. Anti-nucleocapsid IgG (anti-N) and Anti-RBD levels were measured. Anti-RBD level of 500 AU/mL was used as a cutoff to stratify patients. Spearman's rank Coefficient (rs) was used to demonstrate association. Results: Of the 26 patients included, those who were vaccinated more frequently tested positive for Anti-RBD (100% vs 46.2%, P = 0.005) with higher median titer level (623 vs 0, P = 0.011) compared to unvaccinated patients. Anti-N positivity was more frequently seen in unvaccinated patients (53.9% vs 7.7%, P = 0.03). Anti-RBD levels >500 were associated with lower overall hospital length of stay (LOS)(5 vs 10 days, P = 0.046). The analysis employing a Spearman Rank coefficient demonstrated a strong negative correlation between anti-S titer and LOS (rs=-.515, p = 0.007) and a moderate negative correlation with oxygen needs (rs =-.401, p = 0.042). Conclusion: Anti-RBD IgG levels were associated with lower LOS and oxygen needs during hospitalization. Further studies are needed to determine if levels on admission can be used as a prognostic indicator.
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Copper metabolism MURR1 domain1 (COMMD1) is a novel inhibitor of the transcription factors NF-kappaB and HIF-1, which play important roles in inflammation and tumor growth, respectively. In this study, we identified two highly conserved nuclear export signals (NESs) in COMMD1 and revealed that these NESs were essential and sufficient to induce maximal nuclear export of COMMD1. Inhibition of CRM1-mediated nuclear export by Leptomycin B resulted in nuclear accumulation of COMMD1. In addition, low oxygen concentrations induced the active export of COMMD1 from the nucleus in a CRM1-dependent manner. Disruption of the NESs in COMMD1 increased the repression of COMMD1 in transcriptional activity of NF-kappaB and HIF-1. In conclusion, these data indicate that COMMD1 undergoes constitutive nucleocytoplasmic transport as a novel mechanism to regulate NF-kappaB and HIF-1 signaling.
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Núcleo Celular/metabolismo , Citosol/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Cobre/metabolismo , Ácidos Graxos Insaturados , Humanos , Sinais de Exportação Nuclear , Transdução de Sinais , Fator de Transcrição RelA/metabolismoRESUMO
COMMD {COMM [copper metabolism Murr1 (mouse U2af1-rs1 region 1)] domain-containing} proteins participate in several cellular processes, ranging from NF-kappaB (nuclear factor kappaB) regulation, copper homoeostasis, sodium transport and adaptation to hypoxia. The best-studied member of this family is COMMD1, but relatively little is known about its regulation, except that XIAP [X-linked IAP (inhibitor of apoptosis)] functions as its ubiquitin ligase. In the present study, we identified that the COMM domain of COMMD1 is required for its interaction with XIAP, and other COMMD proteins can similarly interact with IAPs. Two conserved leucine repeats within the COMM domain were found to be critically required for XIAP binding. A COMMD1 mutant which was unable to bind to XIAP demonstrated a complete loss of basal ubiquitination and great stabilization of the protein. Underscoring the importance of IAP-mediated ubiquitination, we found that long-term expression of wild-type COMMD1 results in nearly physiological protein levels as a result of increased ubiquitination, but this regulatory event is circumvented when a mutant form that cannot bind XIAP is expressed. In summary, our findings indicate that COMMD1 expression is controlled primarily by protein ubiquitination, and its interaction with IAP proteins plays an essential role.
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Proteínas de Transporte/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Sequência Conservada , Regulação da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
BACKGROUND: Antibody testing has recently emerged as an option to assist with determining exposure to SARS-CoV-2, the causative agent of COVID-19. Elucidation of the kinetics and duration of the humoral response is important for clinical management and interpreting results from serological surveys. OBJECTIVES: Here we evaluated the clinical performance of Abbott SARS-CoV-2 IgM and IgG assays, as well as the longitudinal dynamics of the antibody response in symptomatic COVID-19 patients. STUDY DESIGN AND RESULTS: The diagnostic specificity was 100 % for IgM and 99.67 % for IgG using 300 pre-COVID-19 serum specimens. Using 1349 sequential serum samples collected up to 168 days post symptom onset from 427 PCR-confirmed individuals, clinical test sensitivity of the SARS-CoV-2 IgM assay was 24.6 % at ≤7 days, 75.3 % at 8-14 days, 95.0 % at 15-21 days, and 96.0 % at 4-5 weeks (peak test sensitivity). The median duration of time for IgM seroconversion was 10 days. IgM levels declined steadily 4-5 weeks after symptom onset, and the positive rate dropped to 30.8 % at >3 months. The diagnostic sensitivity for the SARS-CoV-2 IgG assay post symptom onset was 23.2 % at ≤7 days, 69.5 % at 8-14 days, 93.6 % at 15-21 days, and 99.6 % at 4-5 weeks (peak test sensitivity). The median duration of time for IgG seroconversion was 11.5 days. During the convalescent phase of the infection, a decline in the IgG level was observed in patients who were followed for >100 days. Despite that decline, 92.3 % of the patient cohort remained IgG positive 3-6 months following symptom onset. CONCLUSIONS: This study demonstrates the Abbott IgM assay against SARS-CoV-2 is detected slightly earlier compared to IgG, with both tests exhibiting excellent overall sensitivity and specificity. In symptomatic patients who test negative by PCR for a SARS-CoV-2 infection, assessing IgM and IgG antibodies can aid in supporting a diagnosis of COVID-19.
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Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19 , COVID-19/diagnóstico , Imunidade Humoral , Imunoglobulina G/sangue , Imunoglobulina M/sangue , COVID-19/imunologia , Estudos de Coortes , Humanos , Estudos Longitudinais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeAssuntos
COVID-19/patologia , Exossomos/metabolismo , Transplante de Pulmão , Glicoproteína da Espícula de Coronavírus/metabolismo , Doenças Assintomáticas , Western Blotting , COVID-19/virologia , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Subunidades Proteicas/metabolismo , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
NF-κB is a master regulator of inflammation and has been implicated in the pathogenesis of immune disorders and cancer. Its regulation involves a variety of steps, including the controlled degradation of inhibitory IκB proteins. In addition, the inactivation of DNA-bound NF-κB is essential for its regulation. This step requires a factor known as copper metabolism Murr1 domain-containing 1 (COMMD1), the prototype member of a conserved gene family. While COMMD proteins have been linked to the ubiquitination pathway, little else is known about other family members. Here we demonstrate that all COMMD proteins bind to CCDC22, a factor recently implicated in X-linked intellectual disability (XLID). We showed that an XLID-associated CCDC22 mutation decreased CCDC22 protein expression and impaired its binding to COMMD proteins. Moreover, some affected individuals displayed ectodermal dysplasia, a congenital condition that can result from developmental NF-κB blockade. Indeed, patient-derived cells demonstrated impaired NF-κB activation due to decreased IκB ubiquitination and degradation. In addition, we found that COMMD8 acted in conjunction with CCDC22 to direct the degradation of IκB proteins. Taken together, our results indicate that CCDC22 participates in NF-κB activation and that its deficiency leads to decreased IκB turnover in humans, highlighting an important regulatory component of this pathway.
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Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cromossomos Humanos X , Displasia Ectodérmica/metabolismo , Ligação Genética , Células HEK293 , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Inflamação , Microscopia de Fluorescência , Mutação , Inibidor de NF-kappaB alfa , Neoplasias/metabolismo , Estrutura Terciária de Proteína , Ubiquitina/metabolismoAssuntos
Linfócitos T/imunologia , Transferência Adotiva , Animais , Antígenos de Neoplasias , Vacinas Anticâncer/farmacologia , Imunoterapia , Ativação Linfocitária , Linfopenia/imunologia , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Irradiação Corporal TotalRESUMO
Ubiquitination can have profound effects on the stability and function of cellular proteins. Mass spectrometry (MS) can be used to map the specific amino acid residues that are conjugated to ubiquitin in a target protein. However, the purification required for proteomic analysis can be challenging. In this paper, we describe a bimolecular affinity purification scheme for the isolation of a specific ubiquitinated protein in which affinity moieties are fused to ubiquitin and to a target protein of interest. After ubiquitin conjugation in vivo, the protein target acquires two affinity tags, allowing the specific purification of its ubiquitin-modified forms. To prevent deubiquitination after lysis or the copurification of interacting cofactors, this procedure is performed after protein denaturation using polyhistidine and biotinylation tags. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified in large amounts of sufficient purity for MS analysis and for mapping of ubiquitin acceptor sites.
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Cromatografia de Afinidade/métodos , Espectrometria de Massas em Tandem , Ubiquitina/química , Proteínas Ubiquitinadas/química , Linhagem Celular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Humanos , Mapeamento de Interação de Proteínas/métodos , Proteômica , Proteínas Ubiquitinadas/isolamento & purificação , Proteínas Ubiquitinadas/metabolismo , UbiquitinaçãoRESUMO
The tandem affinity purification (TAP) procedure was pioneered in yeast for the purpose of purifying and characterizing protein complexes. While affinity purification is relatively easy to perform, nonspecific protein interactions can plague the identification of true interacting partners of the given bait utilized in the purification. To alleviate this problem, two sequential affinity purification steps are employed in the TAP procedure. Since its inception in yeast, TAP has gone through many adaptations and has been employed multiple times in diverse organisms, including mammalian systems. In all these approaches, two out of many possible affinity moieties are employed and are usually expressed as a fusion polypeptide in the amino or carboxyl-terminal region of the protein bait. In this protocol, we describe a variation on the TAP procedure in which the affinity moieties are placed on two different proteins of a molecular complex to isolate or detect components present in the complex. This variation, which we refer to as bimolecular affinity purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known components.
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Cromatografia de Afinidade/métodos , Proteínas/metabolismo , Linhagem Celular , Clonagem Molecular , Humanos , Proteínas/isolamento & purificação , Controle de QualidadeRESUMO
The COMM domain containing (COMMD) family of proteins represents a recently discovered set of evolutionarily conserved factors characterized by the presence of a defining carboxy-terminal motif. In vertebrates, there are ten members of the family, and among their emerging functions the control of the transcription factor NFkappaB has been most extensively studied. NFkappaB plays a critical role in a number of homeostatic processes in multicellular organisms, including the regulation of immunity and cell survival. COMMD proteins inhibit NFkappaB mediated gene expression, and recent mechanistic studies have revealed that COMMD1 controls the ubiquitination of NFkappaB subunits, an event linked to transcriptional termination. COMMD1 binds to a multimeric ubiquitin ligase containing Elongins B/C, Cul2 and SOCS1 (ECS( SOCS1)). In this complex, COMMD1 facilitates the binding of NFkappaB subunits to the ligase, thereby promoting their ubiquitination and degradation. Additional insights gained from these studies indicate that COMMD proteins likely play a broader role in cellular homeostasis through their participation in the ubiquitination pathway.
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Família Multigênica , NF-kappa B/fisiologia , Proteínas/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte , Ciclo Celular/fisiologia , Humanos , Ligação Proteica/fisiologia , Proteínas/genética , Transdução de Sinais/genéticaRESUMO
NF-kappaB is a pleiotropic transcription factor involved in multiple processes, including inflammation and oncogenesis. We have previously reported that COMMD1 represses kappaB-dependent transcription by negatively regulating NF-kappaB-chromatin interactions. Recently, ubiquitination of NF-kappaB subunits has been similarly implicated in the control of NF-kappaB recruitment to chromatin. We report here that COMMD1 accelerates the ubiquitination and degradation of NF-kappaB subunits through its interaction with a multimeric ubiquitin ligase containing Elongins B and C, Cul2 and SOCS1 (ECS(SOCS1)). COMMD1-deficient cells demonstrate stabilization of RelA, greater nuclear accumulation of RelA after TNF stimulation, de-repression of several kappaB-responsive genes, and enhanced NF-kappaB-mediated cellular responses. COMMD1 binds to Cul2 in a stimulus-dependent manner and serves to facilitate substrate binding to the ligase by stabilizing the interaction between SOCS1 and RelA. Our data uncover that ubiquitination and degradation of NF-kappaB subunits by this COMMD1-containing ubiquitin ligase is a novel and critical mechanism of regulation of NF-kappaB-mediated transcription.