RESUMO
Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Dermatite Atópica/imunologia , Epiderme/inervação , Queratina-15/metabolismo , Queratinócitos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Prurido/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Poluentes Atmosféricos/efeitos adversos , Animais , Animais Recém-Nascidos , Orientação de Axônios/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Epiderme/patologia , Regulação da Expressão Gênica , Humanos , Queratina-15/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Diferenciação Celular , Fatores Reguladores de Interferon , Plasmócitos , Animais , Camundongos , Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfócitos B/citologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Switching de Imunoglobulina/genética , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/metabolismo , Plasmócitos/imunologia , Plasmócitos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Transativadores/metabolismo , Transativadores/genéticaRESUMO
ABCC3 (also known as MRP3) is an ATP binding cassette transporter for bile acids, whose expression is downregulated in colorectal cancer through the Wnt/ß-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. Gene expression analysis of colorectal adenoma isolated from familial adenomatous polyposis patients revealed that genes related to bile acid secretion including ABCC3 were downregulated as early as at the stage of adenoma formation. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid, a secondary bile acid, respectively, in colorectal cancer cells. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that nonsteroidal anti-inflammatory drugs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of familial adenomatous polyposis. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling.
Assuntos
Neoplasias Colorretais , Ácido Desoxicólico , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Humanos , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Ácido Desoxicólico/farmacologia , Ácido Desoxicólico/metabolismo , Regulação para Baixo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genéticaRESUMO
Expression of the gene for collagen XVII (COL17A1) in tumor tissue is positively or negatively associated with patient survival depending on cancer type. High COL17A1 expression is thus a favorable prognostic marker for breast cancer but unfavorable for pancreatic cancer. This study explored the effects of COL17A1 expression on pancreatic tumor growth and their underlying mechanisms. Analysis of published single-cell RNA-sequencing data for human pancreatic cancer tissue revealed that COL17A1 was expressed predominantly in cancer cells rather than surrounding stromal cells. Forced expression of COL17A1 did not substantially affect the proliferation rate of the mouse pancreatic cancer cell lines KPC and AK4.4 in vitro. However, in mouse homograft tumor models in which KPC or AK4.4 cells were injected into syngeneic C57BL/6 or FVB mice, respectively, COL17A1 expression promoted or suppressed tumor growth, respectively, suggesting that the effect of COL17A1 on tumor growth was influenced by the tumor microenvironment. RNA-sequencing analysis of tumor tissue revealed effects of COL17A1 on gene expression profiles (including the expression of genes related to cell proliferation, the immune response, Wnt signaling, and Hippo signaling) that differed between C57BL/6-KPC and FVB-AK4.4 tumors. Our data thus suggest that COL17A1 promotes or suppresses cancer progression in a manner dependent on the interaction of tumor cells with the tumor microenvironment.
Assuntos
Neoplasias Pancreáticas , Microambiente Tumoral , Camundongos , Animais , Humanos , Microambiente Tumoral/genética , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/patologia , RNA , Colágeno Tipo XVII , Neoplasias PancreáticasRESUMO
ANO3 encodes Anoctamin-3, also known as TMEM16C, a calcium-activated chloride channel. Heterozygous variants of ANO3 can cause dystonia 24, an adult-onset focal dystonia. Some pediatric cases have been reported, but most patients were intellectually normal with some exceptions. Here, we report a two-year-old girl who showed mild to moderate developmental delay, tremor, and ataxic gait, but no obvious dystonia. Trio exome sequencing identified a heterozygous de novo missense variant NM_031418.4:c.1809T>G, p.(Asn603Lys) in the ANO3 gene. Three cases with ANO3 variants and intellectual disability have been reported, including the present case. These variants were predicted to face in the same direction on the same alpha-helix (the transmembrane 4 domain), suggesting an association between these variants and childhood-onset movement disorder with intellectual disability. In pediatric cases with developmental delay and movement disorders such as tremor and ataxia, specific variants in the transmembrane 4 domain of ANO3 may be a cause, even in the absence of dystonia.
Assuntos
Distonia , Deficiência Intelectual , Pré-Escolar , Feminino , Humanos , Anoctaminas/genética , Canais de Cloreto/genética , Deficiências do Desenvolvimento/genética , Distonia/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , TremorRESUMO
BACKGROUND: DNA replisome is a molecular complex that plays indispensable roles in normal DNA replication. IMAGE-I syndrome is a DNA replisome-associated genetic disease caused by biallelic mutations in the gene encoding DNA polymerase epsilon catalytic subunit 1 (POLE). However, the underlying molecular mechanisms remain largely unresolved. METHODS: The clinical manifestations in two patients with IMAGE-I syndrome were characterised. Whole-exome sequencing was performed and altered mRNA splicing and protein levels of POLE were determined. Subcellular localisation, cell cycle analysis and DNA replication stress were assessed using fibroblasts and peripheral blood from the patients and transfected cell lines to determine the functional significance of POLE mutations. RESULTS: Both patients presented with growth retardation, adrenal insufficiency, immunodeficiency and complicated diffuse large B-cell lymphoma. We identified three novel POLE mutations: namely, a deep intronic mutation, c.1226+234G>A, common in both patients, and missense (c.2593T>G) and in-frame deletion (c.711_713del) mutations in each patient. The unique deep intronic mutation produced aberrantly spliced mRNAs. All mutants showed significantly reduced, but not null, protein levels. Notably, the mutants showed severely diminished nuclear localisation, which was rescued by proteasome inhibitor treatment. Functional analysis revealed impairment of cell cycle progression and increase in the expression of phospho-H2A histone family member X in both patients. CONCLUSION: These findings provide new insights regarding the mechanism via which POLE mutants are highly susceptible to proteasome-dependent degradation in the nucleus, resulting in impaired DNA replication and cell cycle progression, a characteristic of DNA replisome-associated diseases.
RESUMO
Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.
Assuntos
Catarata/genética , Epiderme/patologia , Hipotricose/genética , Transferases Intramoleculares/genética , Cristalino/patologia , Adolescente , Animais , Catarata/congênito , Catarata/patologia , Colesterol/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Epiderme/enzimologia , Saúde Holística , Humanos , Hipotricose/congênito , Hipotricose/patologia , Transferases Intramoleculares/metabolismo , Lanosterol/análise , Lanosterol/metabolismo , Cristalino/enzimologia , Masculino , Camundongos , Camundongos Knockout , Mutação , Linhagem , Sebo/química , Sequenciamento do ExomaRESUMO
Most cancer cells show chromosomal instability (CIN), a condition in which chromosome missegregation occurs at high rates. Growing evidence suggests that CIN is not just a consequence of, but a driving force for, oncogenic transformation, although the relationship between CIN and tumorigenesis has not been fully elucidated. Here we found that conventional two-dimensional (2D) culture of HeLa cells, a cervical cancer-derived cell line, was a heterogenous population containing cells with different CIN levels. Although cells with high-CIN levels (high-CIN cells) grew more slowly compared with cells with low-CIN levels (low-CIN cells) in 2D monolayer culture, they formed tumors in nude mice and larger spheres in three-dimensional (3D) culture, which was more representative of the in vivo environment. The duration of mitosis was longer in high-CIN cells, reflecting their higher mitotic defects. Single-cell genome sequencing revealed that high-CIN cells exhibited a higher karyotype heterogeneity compared with low-CIN cells. Intriguingly, the karyotype heterogeneity was reduced in the spheres formed by high-CIN cells, suggesting that cells with growth advantages were selected, although genomic copy number changes specific for spheres were not identified. When we examined gene expression profiles, genes related to the K-ras signaling were upregulated, while those related to the unfolded protein response were downregulated in high-CIN cells in 3D culture compared with 2D culture, suggesting the relevance of these genes for their survival. Our data suggested that, although CIN is disadvantageous in monolayer culture, it promotes the selection of cells with growth advantages under in vivo environments, which may lead to tumorigenesis.
Assuntos
Instabilidade Cromossômica , Mitose , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Instabilidade Cromossômica/genética , Células HeLa , Humanos , Camundongos , Camundongos NusRESUMO
Noonan syndrome (NS) is characterized by distinctive craniofacial appearance, short stature, and congenital heart disease. Approximately 80% of individuals with NS harbor mutations in genes whose products are involved in the RAS/mitogen-activating protein kinase (MAPK) pathway. However, the underlying genetic causes in nearly 20% of individuals with NS phenotype remain unexplained. Here, we report four de novo RRAS2 variants in three individuals with NS. RRAS2 is a member of the RAS subfamily and is ubiquitously expressed. Three variants, c.70_78dup (p.Gly24_Gly26dup), c.216A>T (p.Gln72His), and c.215A>T (p.Gln72Leu), have been found in cancers; our functional analyses showed that these three changes induced elevated association of RAF1 and that they activated ERK1/2 and ELK1. Notably, prominent activation of ERK1/2 and ELK1 by p.Gln72Leu associates with the severe phenotype of the individual harboring this change. To examine variant pathogenicity in vivo, we generated zebrafish models. Larvae overexpressing c.70_78dup (p.Gly24_Gly26dup) or c.216A>T (p.Gln72His) variants, but not wild-type RRAS2 RNAs, showed craniofacial defects and macrocephaly. The same dose injection of mRNA encoding c.215A>T (p.Gln72Leu) caused severe developmental impairments and low dose overexpression of this variant induced craniofacial defects. In contrast, the RRAS2 c.224T>G (p.Phe75Cys) change, located on the same allele with p.Gln72His in an individual with NS, resulted in no aberrant in vitro or in vivo phenotypes by itself. Together, our findings suggest that activating RRAS2 mutations can cause NS and expand the involvement of RRAS2 proto-oncogene to rare germline disorders.
Assuntos
Mutação com Ganho de Função , Mutação em Linhagem Germinativa , Proteínas de Membrana/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Síndrome de Noonan/etiologia , Peixe-Zebra/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Exoma , Feminino , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Síndrome de Noonan/patologia , Fenótipo , Conformação Proteica , Proto-Oncogene Mas , Homologia de Sequência , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Paucity of interlobular bile ducts (PILBD) is a heterogeneous disorder classified into two categories, syndromic and non-syndromic bile duct paucity. Syndromic PILBD is characterized by the presence of clinical manifestations of Alagille syndrome. Non-syndromic PILBD is caused by multiple diseases, such as metabolic and genetic disorders, infectious diseases, and inflammatory and immune disorders. We evaluated a family with a dominantly inherited PILBD, who presented with cholestasis at 1-2 months of age but spontaneously improved by 1 year of age. Next-generation sequencing analysis revealed a heterozygous CACYBP/SIP p.E177Q pathogenic variant. Calcyclin-binding protein and Siah1 interacting protein (CACYBP/SIP) form a ubiquitin ligase complex and induce proteasomal degradation of non-phosphorylated ß-catenin. Immunohistochemical analysis revealed a slight decrease in CACYBP and ß-catenin levels in the liver of patients in early infancy, which almost normalized by 13 months of age. The CACYBP/SIP p.E177Q pathogenic variant may form a more active or stable ubiquitin ligase complex that enhances the degradation of ß-catenin and delays the maturation of intrahepatic bile ducts. Our findings indicate that accurate regulation of the ß-catenin concentration is essential for the development of intrahepatic bile ducts and CACYBP/SIP pathogenic variant is a novel cause of PILDB.
Assuntos
Síndrome de Alagille , Proteínas de Ligação ao Cálcio , beta Catenina , Ductos Biliares Intra-Hepáticos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Humanos , Lactente , Recém-Nascido , Ubiquitina-Proteína Ligases , beta Catenina/metabolismoRESUMO
The splicing of microexons (very small exons) is frequently dysregulated in the brain of individuals with autism spectrum disorder. However, little is known of the patterns, regulatory mechanisms and roles of microexon splicing in cancer. We here examined the transcriptome-wide profile of microexon splicing in matched colorectal cancer (CRC) and normal tissue specimens. Out of 1492 microexons comprising 3 to 15 nucleotides, 21 (1%) manifested differential splicing between CRC and normal tissue. The 21 genes harboring the differentially spliced microexons were enriched in gene ontology terms related to cell adhesion and migration. RNA interference-mediated knockdown experiments identified two splicing factors, RBFOX2 and PTBP1, as regulators of microexon splicing in CRC cells. RBFOX2 and PTBP1 were found to directly bind to microexon-containing pre-mRNAs and to control their splicing in such cells. Differential microexon splicing was shown to be due, at least in part, to altered expression of RBFOX2 and PTBP1 in CRC tissue compared to matched normal tissue. Finally, we found that changes in the pattern of microexon splicing were associated with CRC metastasis. Our data thus suggest that altered expression of RBFOX2 and PTBP1 might influence CRC metastasis through the regulation of microexon splicing.
Assuntos
Processamento Alternativo , Neoplasias Colorretais/genética , Éxons/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Células HCT116 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Immunoblotting , Metástase Neoplásica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND & AIMS: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca2+-selective ion channel, in an international cohort of patients and in mice. METHODS: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6mut/mut mice. RESULTS: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10-8). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10-8). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10-9 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10-5). TRPV6mut/mut mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis. CONCLUSIONS: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca2+ balance in pancreatic cells. TRPV6 regulates Ca2+ homeostasis and pancreatic inflammation.
Assuntos
Idade de Início , Canais de Cálcio/genética , Pancreatite Crônica/genética , Canais de Cátion TRPV/genética , Adolescente , Adulto , Idoso , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Mutação INDEL , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Pâncreas/patologia , Pancreatite Crônica/patologia , Polimorfismo de Nucleotídeo Único , Canais de Cátion TRPV/metabolismo , Sequenciamento do Exoma , Adulto JovemRESUMO
Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment and have been shown to promote cancer aggressiveness. In our previous study, analysis of expression profiles obtained from paired CAFs and normal fibroblasts from colorectal cancer (CRC) tissue revealed that gene sets related to the Wnt signaling pathway were highly enriched in colorectal CAFs. Furthermore, among the components of the ß-catenin-independent Wnt pathway, Wnt5a was highly expressed in CAFs. Since Wnt5a is considered to be a regulator of CRC progression in CAFs, we performed immunohistochemical analysis on Wnt5a in 171 patients who underwent surgery for CRC. Positive staining for Wnt5a was often found in cancer stroma, particularly in fibromatous areas, although the immunoreactivity for Wnt5a was weak in cancer cells. Wnt5a status in CAFs was significantly associated with tumor size, depth of invasion, lymphatic and vascular invasion, lymph node metastasis, TNM stage, and recurrence. Subsequent in vitro analyses using human recombinant Wnt5a protein revealed that cancer cell proliferation and migration were significantly increased by stimulation with Wnt5a. Our findings suggest that Wnt5a-derived CAFs play a crucial role in CRC progression and have potential as a target of anti-cancer therapies.
Assuntos
Fibroblastos Associados a Câncer/patologia , Neoplasias Colorretais/patologia , Proteína Wnt-5a/análise , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Células Tumorais Cultivadas , Proteína Wnt-5a/genéticaRESUMO
Heat shock protein family B member 8, encoded by HSPB8, is an essential component of the chaperone-assisted selective autophagy complex, which maintains muscle function by degrading damaged proteins in the cells. Mutations in HSPB8 have been reported to cause Charcot-Marie-Tooth type 2L, distal hereditary motor neuropathy IIa, and rimmed vacuolar myopathies (RVM). In this study, we identified a novel heterozygous frameshift variant c.525_529del in HSPB8 in a large Japanese family with RVM, using whole exome sequencing. Three affected individuals had severe respiratory failure, which has not been addressed by previous studies. Muscle atrophy in the paraspinal muscles was also a clinical feature of the individuals affected with RVM in this study. The frameshift mutation was located in the last coding exon, and the mutated protein was predicted to harbor an isoleucine-leucine-valine (ILV) sequence, which corresponds to the IXI/V (isoleucine, X amino acids, and isoleucine or valine) motif. The IXI/V motif is essential for assembly into larger oligomers in other small heat shock proteins and all frameshift mutants of HSPB8 were predicted to share the ILV sequence in the C-terminal extension. The in silico prediction tools showed low protein solubility and increased aggregation propensity for the region around the ILV sequence. The IXI/V motif might be associated with the pathogenesis of HSPB8-related RVM.
Assuntos
Miopatias Distais/genética , Predisposição Genética para Doença , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Atrofia Muscular/genética , Adulto , Miopatias Distais/diagnóstico , Miopatias Distais/patologia , Feminino , Deleção de Genes , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Atrofia Muscular/diagnóstico , Atrofia Muscular/patologia , Músculos Paraespinais/patologia , Sequenciamento do ExomaRESUMO
Nephrotic syndrome (NS) is a renal disease characterized by severe proteinuria and hypoproteinemia. Although several single-gene mutations have been associated with steroid-resistant NS, causative genes for steroid-sensitive NS (SSNS) have not been clarified. While seeking to identify causative genes associated with SSNS by whole-exome sequencing, we found compound heterozygous variants/mutations (c.524T>C; p.I175T and c.662G>A; p.R221H) of the interleukin-1 receptor accessory protein (IL1RAP) gene in two siblings with SSNS. The siblings' parents are healthy, and each parent carries a different heterozygous IL1RAP variant/mutation. Since IL1RAP is a critical subunit of the functional interleukin-1 receptor (IL-1R), we investigated the effect of these variants on IL-1R subunit function. When stimulated with IL-1ß, peripheral blood mononuclear cells from the siblings with SSNS produced markedly lower levels of cytokines compared with cells from healthy family members. Moreover, IL-1R with a variant IL1RAP subunit, reconstituted on a hematopoietic cell line, had impaired binding ability and low reactivity to IL-1ß. Thus, the amino acid substitutions in IL1RAP found in these NS patients are dysfunctional variants/mutations. Furthermore, in the kidney of Il1rap-/- mice, the number of myeloid-derived suppressor cells, which require IL-1ß for their differentiation, was markedly reduced although these mice did not show significantly increased proteinuria in acute nephrotic injury with lipopolysaccharide treatment. Together, these results identify two IL1RAP variants/mutations in humans for the first time and suggest that IL1RAP might be a causative gene for familial NS.
Assuntos
Proteína Acessória do Receptor de Interleucina-1/genética , Síndrome Nefrótica/genética , Esteroides/efeitos adversos , Animais , Pré-Escolar , Feminino , Variação Genética , Humanos , Recém-Nascido , Proteína Acessória do Receptor de Interleucina-1/sangue , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Síndrome Nefrótica/induzido quimicamente , Síndrome Nefrótica/tratamento farmacológico , Irmãos , Esteroides/uso terapêuticoRESUMO
Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.
Assuntos
Genoma Humano/genética , Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Linhagem Celular Transformada , Fibroblastos/metabolismo , Glicólise/fisiologia , Humanos , Biblioteca de Peptídeos , Proteínas Recombinantes/análiseRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
c-Jun-amino-terminal kinase-interacting protein 3 (JIP3), encoded by MAPK8IP3, is an adaptor protein of the kinesin-1 complex and essential for axonal transport in neurons. However, an association between MAPK8IP3 variants and human disease has not been established. We identified 5 individuals from four families with recurrent de novo variants c.1732C>T (p.Arg578Cys) and c.3436C>T (p.Arg1146Cys) in MAPK8IP3. The core phenotype includes spastic diplegia, intellectual disability, cerebral atrophy, and corpus callosum hypoplasia. Zebrafish embryos overexpressing human mutant JIP3 showed axon varicosities of the posterior lateral line nerve, suggesting an adverse effect on the developing axons. Our results suggest that MAPK8IP3 variants cause a neurodevelopmental disease. ANN NEUROL 2019;85:927-933.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Variação Genética/genética , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/diagnóstico por imagem , Doenças do Sistema Nervoso/genética , Fenótipo , Adolescente , Adulto , Animais , Pré-Escolar , Feminino , Humanos , Masculino , Peixe-ZebraRESUMO
BTB and CNC homology 2 (Bach2) is a transcriptional repressor that is required for the formation of the germinal center (GC) and reactions, including class switch recombination and somatic hypermutation of Ig genes in B cells, within the GC. Although BCR-induced proliferation is essential for GC reactions, the function of Bach2 in regulating B cell proliferation has not been elucidated. In this study, we demonstrate that Bach2 is required to sustain high levels of B cell proliferation in response to BCR signaling. Following BCR engagement in vitro, B cells from Bach2-deficient (Bach2-/-) mice showed lower incorporation of BrdU and reduced cell cycle progression compared with wild-type cells. Bach2-/- B cells also underwent increased apoptosis, as evidenced by an elevated frequency of sub-G1 cells and early apoptotic cells. Transcriptome analysis of BCR-engaged B cells from Bach2-/- mice revealed reduced expression of the antiapoptotic gene Bcl2l1 encoding Bcl-xL and elevated expression of cyclin-dependent kinase inhibitor (CKI) family genes, including Cdkn1a, Cdkn2a, and Cdkn2b Reconstitution of Bcl-xL expression partially rescued the proliferation defect of Bach2-/- B cells. Chromatin immunoprecipitation experiments showed that Bach2 bound to the CKI family genes, indicating that these genes are direct repression targets of Bach2. These findings identify Bach2 as a requisite factor for sustaining high levels of BCR-induced proliferation, survival, and cell cycle progression, and it promotes expression of Bcl-xL and repression of CKI genes. BCR-induced proliferation defects may contribute to the impaired GC formation observed in Bach2-/- mice.
Assuntos
Linfócitos B/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Ativação Linfocitária/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proliferação de Células , Proteínas Inibidoras de Quinase Dependente de Ciclina/imunologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/imunologiaRESUMO
PURPOSE: Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia. METHODS: Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants. RESULTS: The highest blood galactose levels observed in each patient were 17.3-41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between ß- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients' peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins. CONCLUSION: Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.