RESUMO
Juvenile-onset recurrent respiratory papillomatosis (JRRP) is a rare and debilitating childhood disease that presents with recurrent growth of papillomas in the upper airway. Two common human papillomaviruses (HPVs), HPV-6 and -11, are implicated in most cases, but it is still not understood why only a small proportion of children develop JRRP following exposure to these common viruses. We report 2 siblings with a syndromic form of JRRP associated with mild dermatologic abnormalities. Whole-exome sequencing of the patients revealed a private homozygous mutation in NLRP1, encoding Nucleotide-Binding Domain Leucine-Rich Repeat Family Pyrin Domain-Containing 1. We find the NLRP1 mutant allele to be gain of function (GOF) for inflammasome activation, as demonstrated by the induction of inflammasome complex oligomerization and IL-1ß secretion in an overexpression system. Moreover, patient-derived keratinocytes secrete elevated levels of IL-1ß at baseline. Finally, both patients displayed elevated levels of inflammasome-induced cytokines in the serum. Six NLRP1 GOF mutations have previously been described to underlie 3 allelic Mendelian diseases with differing phenotypes and modes of inheritance. Our results demonstrate that an autosomal recessive, syndromic form of JRRP can be associated with an NLRP1 GOF mutation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Mutação com Ganho de Função , Homozigoto , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções Respiratórias/genética , Infecções Respiratórias/patologia , Pré-Escolar , Citocinas/metabolismo , Feminino , Humanos , Lactente , Inflamassomos , Queratinócitos/citologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Masculino , Proteínas NLR , Linhagem , Irmãos , SíndromeRESUMO
The protein-coding exome of a patient with a monogenic disease contains about 20,000 variants, only one or two of which are disease causing. We found that 58% of rare variants in the protein-coding exome of the general population are located in only 2% of the genes. Prompted by this observation, we aimed to develop a gene-level approach for predicting whether a given human protein-coding gene is likely to harbor disease-causing mutations. To this end, we derived the gene damage index (GDI): a genome-wide, gene-level metric of the mutational damage that has accumulated in the general population. We found that the GDI was correlated with selective evolutionary pressure, protein complexity, coding sequence length, and the number of paralogs. We compared GDI with the leading gene-level approaches, genic intolerance, and de novo excess, and demonstrated that GDI performed best for the detection of false positives (i.e., removing exome variants in genes irrelevant to disease), whereas genic intolerance and de novo excess performed better for the detection of true positives (i.e., assessing de novo mutations in genes likely to be disease causing). The GDI server, data, and software are freely available to noncommercial users from lab.rockefeller.edu/casanova/GDI.
Assuntos
Exoma , Doenças Genéticas Inatas/genética , Humanos , Curva ROCRESUMO
Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis.
RESUMO
NF-kappaB transcription factors are key regulators of cellular proliferation and frequently contribute to oncogenesis. The herpesviral oncoprotein Tio, which promotes growth transformation of human T cells in a recombinant herpesvirus saimiri background, potently induces canonical NF-kappaB signaling through membrane recruitment of the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, we show that, in addition to Tio-TRAF6 interaction, the Tio-induced canonical NF-kappaB signal requires the presence of the regulatory subunit of the inhibitor of kappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO), and the activity of its key kinase, IKKbeta, to up-regulate expression of endogenous cellular inhibitor of apoptosis 2 (cIAP2) and interleukin 8 (IL-8) proteins. Dependent on TRAF6 and NEMO, Tio enhances the expression of the noncanonical NF-kappaB proteins, p100 and RelB. Independent of TRAF6 and NEMO, Tio mediates stabilization of the noncanonical kinase, NF-kappaB-inducing kinase (NIK). Concomitantly, Tio induces efficient processing of the p100 precursor molecule to its active form, p52, as well as DNA binding of nuclear p52 and RelB. In human T cells transformed by infection with a Tio-recombinant virus, sustained expression of p100, RelB, and cIAP2 depends on IKKbeta activity, yet processing to p52 remains largely unaffected by IKKbeta inhibition. However, long term inhibition of IKKbeta disrupts the continuous growth of the transformed cells and induces cell death. Hence, the Tio oncoprotein triggers noncanonical NF-kappaB signaling through NEMO-dependent up-regulation of p100 precursor and RelB, as well as through NEMO-independent generation of p52 effector.
Assuntos
NF-kappa B/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteína 3 com Repetições IAP de Baculovírus , Sítios de Ligação , Proliferação de Células , Humanos , Quinase I-kappa B/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-8/metabolismo , Células Jurkat , Modelos Biológicos , Proteínas Oncogênicas Virais/fisiologia , Transdução de Sinais , Linfócitos T/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelB/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of TMC6 (encoding EVER1) or TMC8 (EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human ß-papillomaviruses (ß-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the CIB1 gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to ß-HPVs of EV patients.
Assuntos
Betapapillomavirus/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Epidermodisplasia Verruciforme/imunologia , Imunidade Inata , Queratinócitos/imunologia , Proteínas de Membrana/imunologia , Complexos Multiproteicos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Adesão Celular/imunologia , Movimento Celular/imunologia , Epidermodisplasia Verruciforme/patologia , Feminino , Papillomavirus Humano 16/imunologia , Humanos , Queratinócitos/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/imunologiaRESUMO
Human T cells can be transformed and expanded with herpesvirus saimiri (HVS). HVS-transformed T cells from patients have facilitated the study of a broad range of primary immunodeficiencies (PID) in which T-cell development or function is altered. However, the utility of HVS-transformed T cells for genetic studies has been limited by technical challenges in the expression of exogenous genes, including wild-type or mutant alleles. A novel, gamma retrovirus-based method for the simple and reliable transduction, purification, and study of HVS-transformed T cells is described. © 2016 by John Wiley & Sons, Inc.
Assuntos
Vetores Genéticos/genética , Herpesvirus Saimiriíneo 2/genética , Linfócitos T/metabolismo , Transdução Genética/métodos , Transgenes , Animais , Linhagem Celular Transformada , Ordem dos Genes , Técnicas de Transferência de Genes , Humanos , Plasmídeos/genética , Retroviridae/genéticaAssuntos
DNA/genética , Epidermodisplasia Verruciforme/genética , Proteínas de Membrana/genética , Mutação , Adolescente , Adulto , Criança , Códon sem Sentido , Análise Mutacional de DNA , Epidermodisplasia Verruciforme/metabolismo , Feminino , Heterogeneidade Genética , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Linhagem , Adulto JovemRESUMO
Members of the nuclear factor κB (NF-κB) family of transcription factors regulate many cellular functions. Activation of NF-κB signaling is commonly classified as occurring through canonical or noncanonical pathways. Most NF-κB-inducing stimuli, including the viral oncoprotein Tio, lead to a concerted activation of both NF-κB pathways; however, extensive crosstalk at multiple levels between these signaling cascades restricts the ability to discriminate between the canonical and the noncanonical effects. We showed that noncanonical NF-κB activation by Tio depends on a distinct sequence motif that directly recruits tumor necrosis factor receptor-associated factor 3 (TRAF3). Through its TRAF3-binding motif, Tio triggered a ubiquitin-independent depletion of TRAF3 from the cytosol, which prevented TRAF3 from inhibiting signaling through the noncanonical NF-κB cascade. Furthermore, the Tio-TRAF3 interaction did not affect components of the canonical NF-κB signaling pathway or the expression of target genes; thus, Tio induced noncanonical NF-κB independently of crosstalk with the canonical pathway. Together, these data identify a distinct molecular mechanism of noncanonical NF-κB activation that should enable studies into the particular functions of this pathway.
Assuntos
NF-kappa B/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Rhadinovirus/metabolismo , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo , Motivos de Aminoácidos , Linhagem Celular Transformada , Regulação da Expressão Gênica/genética , Humanos , Células Jurkat , NF-kappa B/genética , Proteínas Oncogênicas Virais/genética , Rhadinovirus/genética , Fator 3 Associado a Receptor de TNF/genética , Ubiquitinação/genéticaRESUMO
The potential of Herpesvirus saimiri (HVS) subgroups A, B and C and Herpesvirus ateles (HVA) to transform primary T cells to permanent growth in vitro is restricted by the primate host species and by viral variability represented by distinct viral oncoproteins. We now addressed the relation between the transforming potential of the different viruses and the signaling pathways activated by transiently expressed oncoproteins. Marmoset lymphocytes were transformed by all HVS subgroups as well as HVA, while transformation of human cells was restricted to HVS-C and, unexpectedly, HVA. NF-κB and Src-family kinase (SFK) activity was required for survival of all transformed lymphocytes. Accordingly, NF-κB was induced by oncoproteins of all viruses. In contrast, SFK-related signaling was detectable only for oncoproteins of HVS-C and HVA. Thus, the restricted transformation of human lymphocytes likely correlates with the specific SFK targeting by these oncoproteins. These results will enable further studies into novel SFK effector mechanisms relevant for T-cell proliferation.