RESUMO
During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.
Assuntos
COVID-19/virologia , Fusão de Membrana , Mutação , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/epidemiologia , Cricetinae , Células Gigantes/metabolismo , Células Gigantes/virologia , Masculino , Mesocricetus , Filogenia , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Virulência/genética , Replicação ViralRESUMO
IMPORTANCE: Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.
Assuntos
Genoma Viral , Mutação , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , COVID-19/virologia , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Genoma Viral/genéticaRESUMO
BACKGROUND: Schnitzler syndrome is a rare disorder with chronic urticaria, and there is no report summarizing the current status in Japan. METHODS: A nationwide survey of major dermatology departments in Japan was conducted in 2019. We further performed a systematic search of PubMed and Ichushi-Web, using the keywords "Schnitzler syndrome" and "Japan" then contacted the corresponding authors or physicians for further information. RESULTS: Excluding duplicates, a total of 36 clinically diagnosed cases were identified from 1994 through the spring of 2022, with a male to female ratio of 1:1. The median age of onset was 56.5 years. It took 3.3 years from the first symptom, mostly urticaria, to reach the final diagnosis. The current status of 30 cases was ascertained; two patients developed B-cell lymphoma. SchS treatment was generally effective with high doses of corticosteroids, but symptoms sometimes recurred after tapering. Colchicine was administered in 17 cases and was effective in 8, but showed no effect in the others. Tocilizumab, used in six cases, improved laboratory abnormalities and symptoms, but lost its efficacy after several years. Rituximab, used in five cases, was effective in reducing serum IgM levels or lymphoma mass, but not in inflammatory symptoms. Four cases were treated with IL-1 targeting therapy, either anakinra or canakinumab, and achieved complete remission, except one case with diffuse large B-cell lymphoma. CONCLUSIONS: Since Schnitzler syndrome is a rare disease, the continuous collection and long-term follow-up of clinical information is essential for its appropriate treatment and further understanding of its pathophysiology.
Assuntos
Urticária Crônica , Síndrome de Schnitzler , Urticária , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Síndrome de Schnitzler/diagnóstico , Síndrome de Schnitzler/tratamento farmacológico , Urticária/diagnóstico , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Urticária Crônica/tratamento farmacológico , Japão/epidemiologiaRESUMO
The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a GFP reporter under the HIV-1 promoter and an mKO2 reporter under the internal EF1α promoter. This viral construct enables direct identification of HIV-1 both productively and latently infected cells. In this study we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using Cap Analysis of Gene Expression (CAGE).We deep sequenced CAGE tags in uninfected, latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus producing cells had differentially expressed TSSs related to T-cell activation and apoptosis when compared to uninfected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to uninfected cells. Among these, SPP1 and APOE were down-regulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have anti-viral properties. Components of the PI3K/mTOR pathway, MLST8, 4EBP and RPS6, were significant TSSs in productively infected cells, and S6K phosphorylation was increased compared to latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent.Importance: Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used CAGE to compare the transcriptional landscape of latently infected cells with that of non-infected or productively infected cells. We found that latently infected cells and non-infected cells show quite similar transcriptional profiles. Our data suggest that T-cells cannot recognize incoming viral components nor the integrated HIV-1 genome when infection remains latent. These findings should guide future research into widening our approaches to identify and target latent HIV-1 infected cells.
RESUMO
APOBEC3B (A3B) is a cytosine deaminase that converts cytosine to uracil in single-stranded DNA. Cytosine-to-thymine and cytosine-to-guanine base substitution mutations in trinucleotide motifs (APOBEC mutational signatures) were found in various cancers including lymphoid hematological malignancies such as multiple myeloma and A3B has been shown to be an enzymatic source of mutations in those cancers. Although the importance of A3B is being increasingly recognized, it is unclear how A3B expression is regulated in cancer cells as well as normal cells. To answer these fundamental questions, we analyzed 1276 primary myeloma cells using single-cell RNA-sequencing (scRNA-seq) and found that A3B was preferentially expressed at the G2/M phase, in sharp contrast to the expression patterns of other APOBEC3 genes. Consistently, we demonstrated that A3B protein was preferentially expressed at the G2/M phase in myeloma cells by cell sorting. We also demonstrated that normal blood cells expressing A3B were also enriched in G2/M-phase cells by analyzing scRNA-seq data from 86,493 normal bone marrow mononuclear cells. Furthermore, we revealed that A3B was expressed mainly in plasma cells, CD10+ B cells and erythroid cells, but not in granulocyte-macrophage progenitors. A3B expression profiling in normal blood cells may contribute to understanding the defense mechanism of A3B against viruses, and partially explain the bias of APOBEC mutational signatures in lymphoid but not myeloid malignancies. This study identified the cells and cellular phase in which A3B is highly expressed, which may help reveal the mechanisms behind carcinogenesis and cancer heterogeneity, as well as the biological functions of A3B in normal blood cells.
Assuntos
Divisão Celular/genética , Citidina Desaminase/genética , Fase G2/genética , Antígenos de Histocompatibilidade Menor/genética , Linfócitos B/metabolismo , Células Cultivadas , Células Eritroides/metabolismo , Fase G1/genética , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Neprilisina/metabolismo , Plasmócitos/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA-Seq , Fase S/genética , Análise de Célula ÚnicaRESUMO
Few studies have examined the prognostic impact of blood markers [other than the five factors in the enhanced International Prognostic Index (NCCN-IPI)] in elderly patients with diffuse large B cell lymphoma (DLBCL). We retrospectively analyzed 391 DLBCL patients receiving rituximab plus anthracycline-containing chemotherapy to examine the prognostic impact of simple blood markers. The NCCN-IPI was more accurate for discriminating prognoses than the original IPI. Multivariate analysis identified platelet count (<100,000/µl) and albumin (<3.5 g/dl) levels as significantly associated with lower overall survival (OS), independently of the NCCN-IPI. These parameters stratified patients into three risk groups: platelet-albumin (PA) score low (platelet count ≥100,000/µl, albumin ≥3.5 g/dl, n = 243); intermediate (platelet count <100,000/µl, albumin ≥3.5 g/dl or platelet count ≥100,000/µl, albumin <3.5 g/dl, n = 125); and high (platelet count <100,000/µl, albumin <3.5 g/dl, n = 23). The 5-year OS rates were 81.5, 48.6, and 20.2 %, respectively (p < 0.001). Notably, most patients with a low platelet count (n = 30) were stratified into the high-risk subgroup, suggesting that platelet count was prognostic for high-risk patients with a dismal outcome. In elderly patients (n = 291), the prognostic value of the NCCN-IPI might be diminished because the low-risk category was excluded; however, the PA score was predictive of survival: the 5-year OS rates for PA score low (n = 171), intermediate (n = 101), and high (n = 19) groups were 77.6, 47.9, and 19.0 %, respectively (p < 0.001). Platelet count and albumin levels are useful prognostic factors, and their combined use can predict survival, even in elderly patients.
Assuntos
Linfoma Difuso de Grandes Células B/sangue , Linfoma Difuso de Grandes Células B/diagnóstico , Albumina Sérica/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antraciclinas/administração & dosagem , Biomarcadores/sangue , Feminino , Seguimentos , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas/métodos , Prognóstico , Estudos Retrospectivos , Rituximab/administração & dosagem , Adulto JovemRESUMO
APOBEC3B (A3B) is a DNA cytosine deaminase and catalyzes cytosine deamination, resulting in mutations in genomic DNA. A3B is aberrantly expressed in a variety of cancers and considered to be a source of genomic mutations that contribute to cancer progression and metastasis. However, the mechanisms through which A3B expression is dysregulated in cancer cells are not fully elucidated. Here we report that the classical NF-κB pathway plays a crucial role in the transcriptional regulation of A3B in various cancer cells, including lymphoid malignancies. PMA, a strong activator of PKC, induces A3B at both mRNA and protein levels in cancer cell lines, and specific inhibitors of both PKC and IKK downregulate A3B expression. Using luciferase reporter and EMSA assays, we identify 3 NF-κΒ binding sites in the A3B promoter and reveal that NF-κB p65/p50 and p65/c-Rel heterodimers are important for A3B transcription. These results suggest that the classical NF-κB pathway is responsible for activation of A3B mRNA expression and further imply that inhibition of PKC and IKK might augment cancer treatment by reducing cancer progression and metastasis through downregulation of A3B expression.
Assuntos
Citidina Desaminase/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Menor/genética , NF-kappa B/metabolismo , Transdução de Sinais , Sítios de Ligação , Linhagem Celular Tumoral , Citidina Desaminase/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Células MCF-7 , Antígenos de Histocompatibilidade Menor/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
A 19-year-old woman complaining of fever and a sore throat was diagnosed with very severe aplastic anemia (AA) by bone marrow examination at a local hospital. Despite administration of antibiotics and granulocyte-colony stimulating factor to treat the soft tissue infection in her neck, her neutrophil count showed no increase. Because emergent allogeneic stem cell transplantation (SCT) was necessary, she was referred to our hospital. On admission, computed tomography revealed right-sided severe pharyngitis and lymphadenitis causing tracheal stenosis, and emergent intubation was required the next day. Granulocyte transfusion therapy (GTX) from related donors coupled with broad-spectrum antibiotic administration controlled the otherwise overwhelming infection. The patient received allogeneic peripheral blood SCT using a reduced-intensity conditioning regimen. After allogeneic SCT, successful engraftment was obtained. She was discharged from the hospital 59 days after allogeneic SCT. She remains alive and well, as of the latest follow up. This case clearly demonstrates that GTX is useful for controlling severe infection and enables patients with severe AA to receive allogeneic SCT safely.
Assuntos
Anemia Aplástica/terapia , Transplante de Células-Tronco Hematopoéticas , Anemia Aplástica/patologia , Transfusão de Componentes Sanguíneos , Feminino , Granulócitos , Humanos , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
Schnitzler syndrome (SchS) is a rare autoinflammatory disease characterized by bone pain, recurrent fever, leukocytosis, and elevated C-reactive protein, along with an urticaria-like rash and monoclonal immunoglobulin (Ig)M or IgG gammopathy. Notably, the condition is distinguished by a relatively persistent recurrent urticarial-like rash. Histopathological features observed in the skin comprise diffuse neutrophil infiltration into the dermis, absence of dermal edema, and vascular wall degeneration, all of which classify SchS as a neutrophilic urticarial dermatosis (NUD). Accumulated histological data from skin biopsies of patients with NUD have revealed a sensitive histopathological marker for NUD, acknowledged as neutrophilic epitheliotropism, which has been proposed as reflecting an autoinflammatory condition. In this report, we present three SchS patients: two men (ages 55 and 68) and a woman (age 75), all displaying neutrophilic epitheliotropism in their skin biopsy specimens. Additionally, a review of eight previously reported SchS cases in Japan identified neutrophilic epithliotropism in five cases. These findings suggest that the inclination of neutrophils toward the epithelial tissue could aid in confirming diagnoses of NUD in most cases that need to be differentiated from conventional urticaria. Consequently, we emphasize that acknowledging neutrophilic epithelial predilection as a hallmark of NUD is critical for expediting early diagnosis and appropriate treatment for SchS.
Assuntos
Exantema , Síndrome de Schnitzler , Urticária , Masculino , Feminino , Humanos , Idoso , Síndrome de Schnitzler/diagnóstico , Japão , Urticária/diagnóstico , Urticária/patologia , Pele/patologia , Exantema/patologiaAssuntos
Sarcoma de Células Dendríticas Foliculares/genética , Infecções por HIV/genética , Linfoma Difuso de Grandes Células B/genética , Segunda Neoplasia Primária/genética , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Sarcoma de Células Dendríticas Foliculares/metabolismo , Sarcoma de Células Dendríticas Foliculares/patologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Segunda Neoplasia Primária/metabolismo , Segunda Neoplasia Primária/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
DNA cytosine deaminase APOBEC3B (A3B) is an endogenous source of mutations in many human cancers, including multiple myeloma. A3B proteins form catalytically inactive high molecular mass (HMM) complexes in nuclei, however, the regulatory mechanisms of A3B deaminase activity in HMM complexes are still unclear. Here, we performed mass spectrometry analysis of A3B-interacting proteins from nuclear extracts of myeloma cell lines and identified 30 putative interacting proteins. These proteins are involved in RNA metabolism, including RNA binding, mRNA splicing, translation, and regulation of gene expression. Except for SAFB, these proteins interact with A3B in an RNA-dependent manner. Most of these interacting proteins are detected in A3B HMM complexes by density gradient sedimentation assays. We focused on two interacting proteins, ILF2 and SAFB. We found that overexpressed ILF2 enhanced the deaminase activity of A3B by 30%, while SAFB did not. Additionally, siRNA-mediated knockdown of ILF2 suppressed A3B deaminase activity by 30% in HEK293T cell lysates. Based on these findings, we conclude that ILF2 can interact with A3B and enhance its deaminase activity in HMM complexes.
Assuntos
Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mieloma Múltiplo/genética , Mutação/genética , Proteína do Fator Nuclear 45/genética , Proteína do Fator Nuclear 45/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Proteína do Fator Nuclear 45/metabolismo , Mapas de Interação de Proteínas/genéticaRESUMO
BACKGROUND: SARS-CoV-2 Omicron variants are highly resistant to vaccine-induced immunity and human monoclonal antibodies. METHODS: We previously reported that two nanobodies, P17 and P86, potently neutralize SARS-CoV-2 VOCs. In this study, we modified these nanobodies into trimers, called TP17 and TP86 and tested their neutralization activities against Omicron BA.1 and subvariant BA.2 using pseudovirus assays. Next, we used TP17 and TP86 nanobody cocktail to treat ACE2 transgenic mice infected with lethal dose of SARS-CoV-2 strains, original, Delta and Omicron BA.1. RESULTS: Here, we demonstrate that a novel nanobody TP86 potently neutralizes both BA.1 and BA.2 Omicron variants, and that the TP17 and TP86 nanobody cocktail broadly neutralizes in vitro all VOCs as well as original strain. Furthermore, intratracheal administration of this nanobody cocktail suppresses weight loss and prolongs survival of human ACE2 transgenic mice infected with SARS-CoV-2 strains, original, Delta and Omicron BA.1. CONCLUSIONS: Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice.
Antibodies are made by the immune system to identify and inactivate infectious agents such as viruses. Alpacas produce a simple type of antibodies called nanobodies. We previously developed two nanobodies named P17 and P86 that inactivate SARS-CoV-2. In this study, we modified these nanobodies to create two nanobodies named TP17 and TP86. The cocktail of these nanobodies inactivated different types of SARS-CoV-2 viruses including Omicron BA.1 and BA.2. The cocktail also prolonged survival of mice infected with lethal doses of SARS-CoV-2.
RESUMO
We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, and those in war crises, have been problematic. Nanobodies are small, stable, customizable, and inexpensive to produce. Herein, we present a panel of nanobodies that can detect the spike proteins of five SARS-CoV-2 variants of concern (VOCs) including Omicron. Here we show via ELISA, lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays that our nanobodies can quantify the spike variants. This panel of nanobodies broadly neutralizes viral infection caused by pseudotyped and authentic SARS-CoV-2 VOCs. Structural analyses show that the P86 clone targets epitopes that are conserved yet unclassified on the receptor-binding domain (RBD) and contacts the N-terminal domain (NTD). Human antibodies rarely access both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins that go undetected by conventional antibodies.
Assuntos
COVID-19 , Anticorpos de Domínio Único , Anticorpos Antivirais , Humanos , Glicoproteínas de Membrana/metabolismo , Testes de Neutralização , SARS-CoV-2/genética , Anticorpos de Domínio Único/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral/metabolismoRESUMO
SARS-CoV-2 Lambda, a variant of interest, has spread in some South American countries; however, its virological features and evolutionary traits remain unclear. In this study, we use pseudoviruses and reveal that the spike protein of the Lambda variant is more infectious than that of other variants due to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino acid deletion in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies and further augments antibody-mediated enhancement of infection. Although this mutation generates a nascent N-linked glycosylation site, the additional N-linked glycan is dispensable for the virological property conferred by this mutation. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the RSYLTPGD246-253N mutation is closely associated with the substantial spread of the Lambda variant in South America.
Assuntos
COVID-19/imunologia , Imunidade/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Feminino , Glicosilação , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.
Assuntos
Citidina Desaminase/genética , Dano ao DNA/genética , Antígenos de Histocompatibilidade Menor/genética , Mieloma Múltiplo/genética , Anticorpos Monoclonais Humanizados/farmacologia , Bortezomib/farmacologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Lenalidomida/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Mieloma Múltiplo/radioterapia , Mutação/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Ácidos Polimetacrílicos/farmacologia , RNA Interferente Pequeno/genética , Radiação , Transdução de Sinais/efeitos dos fármacosRESUMO
The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family members have cytidine deaminase activity and can induce cytosine to uracil transition in nucleic acid. The main function of APOBEC3 (A3) proteins is to trigger an innate immune response to viral infections. Recent reports have shown that several APOBEC family proteins such as A3B can induce somatic mutations into genomic DNA and thus promote cancer development. However, the role of A3D on somatic mutations is unclear. Here, we identified the alternative splicing of A3D, and investigated each splice variant's subcellular localization and role in DNA mutagenesis. We identified four A3D variants, which all have one or two cytidine deaminase domains. The full-length form of A3D (variant 1) and truncated forms of A3D (variant 2, 6, 7) showed the ability to induce C/G to T/A transitions in foreign DNA and genomic DNA and retained antiretroviral activity. Furthermore, we demonstrated that A3D and A3B could induce deletions that are possibly repaired by microhomology-mediated end joining (MMEJ). Taken together, our experiments illustrated that alternative splicing generates functional diversity of A3D, and some variants can act as DNA mutators in genomic DNA.
Assuntos
Processamento Alternativo/genética , Citidina Desaminase/genética , Citidina Desaminase/fisiologia , DNA/genética , Mutação/genética , Antirretrovirais , Linhagem Celular Tumoral , Células Cultivadas , Citidina Desaminase/química , Reparo do DNA por Junção de Extremidades/genética , Variação Genética , Humanos , Domínios ProteicosRESUMO
APOBEC3B cytidine deaminase (A3B) catalyzes cytosine into uracil in single-strand DNA and induces C-to-T mutations in genomic DNA of various types of tumors. Accumulation of APOBEC signature mutations is correlated with a worse prognosis for patients with breast cancer or multiple myeloma, suggesting that A3B activity might be a cause of the unfavorable DNA mutations and clonal evolution in these tumors. Phosphorylation of conserved threonine residues of other cytidine deaminases, activation induced deaminase (AID) and APOBEC3G, inhibits their activity. Here we show that protein kinase A (PKA) physically binds to A3B and phosphorylates Thr214. In vitro deaminase assays and foreign DNA editing assays in cells confirm that phosphomimetic A3B mutants, T214D and T214E, completely lose deaminase activity. Molecular dynamics simulation of A3B phosphorylation reveals that Thr214 phosphorylation disrupts binding between the phospho-A3B catalytic core and ssDNA. These mutants still inhibit retroviral infectivity at least partially, and also retain full anti-retrotransposition activity. These results imply that PKA-mediated phosphorylation inhibits A3B mutagenic activity without destructing its innate immune functions. Therefore, PKA activation could reduce further accumulation of mutations in A3B overexpressing tumors.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/genética , Antígenos de Histocompatibilidade Menor/genética , Mutação , Neoplasias/enzimologia , Fosforilação , Domínio Catalítico , Citoplasma/metabolismo , Citosina/química , DNA de Cadeia Simples/genética , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Células HeLa , Humanos , Simulação de Dinâmica Molecular , Neoplasias/genética , Treonina/químicaRESUMO
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminases have emerged as potential genomic mutators in various cancers. Multiple myeloma accumulates APOBEC signature mutations as it progresses; however, the mechanisms underlying APOBEC signature acquisition and its consequences remain elusive. In this study, we examined the significance and clinical impact of APOBEC3B (A3B) activity in multiple myeloma. Among APOBECs, only highly expressed A3B was associated with poor prognosis in myeloma patients, independent of other known poor prognostic factors. Quantitative PCR revealed that CD138-positive primary myeloma cells and myeloma cell lines exhibited remarkably high A3B expression levels. Interestingly, lentiviral A3B knockdown prevented the generation of deletion and loss-of-function mutations in exogenous DNA, whereas in control cells, these mutations accumulated with time. A3B knockdown also decreased the basal levels of γ-H2AX foci, suggesting that A3B promotes constitutive DNA double-strand breaks in myeloma cells. Importantly, among control shRNA-transduced cells, we observed the generation of clones that harboured diverse mutations in exogenous genes and several endogenous genes frequently mutated in myeloma, including TP53. Taken together, the results suggest that A3B constitutively mutates the tumour genome beyond the protection of the DNA repair system, which may lead to clonal evolution and genomic instability in myeloma.
Assuntos
Citidina Desaminase/genética , Mutação com Perda de Função , Melanoma/genética , Antígenos de Histocompatibilidade Menor/genética , Deleção de Sequência , Regulação para Cima , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Humanos , Melanoma/metabolismo , Prognóstico , Regiões Promotoras Genéticas , Sindecana-1/metabolismoRESUMO
Neurolymphomatosis is a rare entity defined as nerve infiltration by neurotropic abnormal lymphocytes which can lead to the development of neuropathy, with typical presentations including pain, hypoesthesia, paresthesis and palsy. We herein report two cases where critical bilateral vocal cord paralysis due to neurolymphomatosis in recurrent nerves occurred in refractory Burkitt lymphoma and adult T-cell lymphoma patients. High-dose methotrexate and intrathecal chemotherapy injection for the nervous lesions were ineffective, and the patients died. Neurolymphomatosis of the recurrent nerve is an emergent and difficult complication and should be suspected when sudden onset of aphasia, hoarseness or shortness of breath is found in refractory lymphoma patients.