RESUMO
The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.
Assuntos
5-Aminolevulinato Sintetase , Anemia Sideroblástica , Humanos , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/deficiência , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Mutação com Perda de Função , Estabilidade Enzimática , Heme/metabolismo , Heme/química , Porfirias/genética , Porfirias/metabolismo , Modelos Moleculares , Mutação , Protoporfiria EritropoéticaRESUMO
G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.
Assuntos
Diferenciação Celular , Eritropoese , Hemina , Leucemia Eritroblástica Aguda , Correpressor 1 de Receptor Nuclear , Humanos , Células K562 , Eritropoese/genética , Leucemia Eritroblástica Aguda/patologia , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 1 de Receptor Nuclear/genética , Hemina/farmacologia , Células Eritroides/metabolismo , Células Eritroides/citologia , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Hemoglobinas/metabolismo , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Inherited iron metabolism defects are possibly missed or underdiagnosed in iron-deficient endemic settings because of a lack of awareness or a methodical screening approach. Hence, we systematically evaluated anemia cases (2019 to 2021) based on clinical phenotype, normal screening tests (high-performance liquid chromatography, α gene sequencing, erythrocyte sedimentation rate, C-reactive protein, and tissue transglutaminase), and abnormal iron profile by targeted next-generation sequencing (26-gene panel) supplemented with whole-exome sequencing, multiplex ligation probe amplification/mitochondrial DNA sequencing, and chromosomal microarray. Novel variants in ALAS2, STEAP3, and HSPA9 genes were functionally validated. A total of 290 anemia cases were screened, and 41 (14%) enrolled for genomic testing as per inclusion criteria. Comprehensive genomic testing revealed pathogenic variants in 23 of 41 cases (56%). Congenital sideroblastic anemia was the most common diagnosis (14/23; 61%), with pathogenic variations in ALAS2 (n = 6), SLC25A38 (n = 3), HSPA9 (n = 2) and HSCB, SLC19A2, and mitochondrial DNA deletion (n = 1 each). Nonsideroblastic iron defects included STEAP3-related microcytic anemia (2/23; 8.7%) and hypotransferrenemia (1/23; 4.3%). A total of 6 of 22 cases (27%) revealed a non-iron metabolism gene defect on whole-exome sequencing. Eleven novel variants (including variants of uncertain significance) were noted in 13 cases. Genotype-phenotype correlation revealed a significant association of frameshift/nonsense/splice variants with lower presentation age (0.8 months versus 9 years; P < 0.01) compared with missense variants. The systematic evaluation helped uncover an inherited iron defect in 41% (17/41) of cases, suggesting the need for active screening and awareness for these rare diseases in an iron-deficient endemic population.
Assuntos
Anemia Sideroblástica , Ferro , Humanos , Lactente , Ferro/metabolismo , Mutação , Anemia Sideroblástica/epidemiologia , Anemia Sideroblástica/genética , Anemia Sideroblástica/diagnóstico , Genômica , DNA Mitocondrial , Proteínas de Membrana Transportadoras/genética , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismoRESUMO
Photodynamic therapy (PDT) is nowadays widely employed in cancer treatment. We sought to assess the efficacy of combining PDT with anti-programmed cell death protein 1 (PD1) and to investigate the associated mechanisms in nonsmall cell lung cancer (NSCLC). We established a xenograft tumor model in C57BL/6J mice using Lewis lung carcinoma (LLC) cells, recorded tumor growth, and quantified reactive oxygen species (ROS) levels using a ROS detection kit. Pathological changes were assessed through H&E staining, while immunofluorescence (IF) was used to determine the expression of CD8 and Foxp3. Transcriptomic analysis was conducted, analyzing differential expressed genes (DEGs) among control, PDT, and PDT combined with anti-PD1 (PDT+anti-PD1) groups. Functional enrichment analysis via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed. The Cancer Genome Atlas (TCGA) database was utilized to analyze the expression of aminolevulinate synthase gene (ALAS2), integrin alpha10 (ITGA10), ATP1A2, a disintegrin and metalloprotease 12 (ADAM12), and Lox1 in lung adenocarcinoma and adjacent tissues, with concurrent immune infiltration analysis. Quantitative real-time polymerase chain reaction and western blot were employed to measure mRNA and protein expression levels. Treatment with PDT combined with anti-PD1 significantly inhibited tumor growth and increased the number of CD8+ cells while decreasing Foxp3+ cells. Immune infiltration results presented ALAS2, ADAM12, and ITGA10 were associated with various types of T cells or macrophages. Additionally, the expression levels of EGFR, ERK, and PI3K/Akt were suppressed after PDT with anti-PD1 treatment. Our findings collectively suggest that PDT combined with anti-PD1 treatment could enhance the infiltration of CD8+ T cells, suppressing tumor growth, and this effect was associated with ALAS2, ITGA10, and ADAM12. The underlying mechanism might be linked to EGFR, ERK, and PI3K/Akt signaling. Overall, this study provides valuable insights into the application of PDT combined with anti-PD1 treatment in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Fotoquimioterapia , Humanos , Camundongos , Animais , Camundongos Endogâmicos C57BL , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linfócitos T CD8-Positivos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Espécies Reativas de Oxigênio , Neoplasias Pulmonares/tratamento farmacológico , Receptores ErbB , Fatores de Transcrição Forkhead , Imunidade , 5-Aminolevulinato SintetaseRESUMO
Congenital sideroblastic anemia (CSA) is a group of disorders caused by different genetic mutations that result in low iron utilization and ineffective erythropoiesis. Current treatments are limited, and some patients do not respond to vitamin B6 therapy. Luspatercept is a novel erythropoietic maturation agent approved for adult ß-thalassemia and Myelodysplastic syndromes with ring sideroblasts (MDS-RS) associated with ineffective erythropoiesis. Here we report 2 patients with CSA due to mutations in ALAS2 and SLC25A38 genes who became unresponsive after a period of treatment with vitamin B6 and iron chelators but achieved transfusion independence and a markedly reduced spleen after combination with luspatercept.
Assuntos
Receptores de Activinas Tipo II , Anemia Sideroblástica , Doenças Genéticas Ligadas ao Cromossomo X , Proteínas Recombinantes de Fusão , Adulto , Humanos , 5-Aminolevulinato Sintetase , Receptores de Activinas Tipo II/efeitos adversos , Anemia Sideroblástica/tratamento farmacológico , Anemia Sideroblástica/genética , Anemia Sideroblástica/congênito , Fragmentos Fc das Imunoglobulinas/efeitos adversos , Proteínas Recombinantes de Fusão/efeitos adversos , Vitamina B 6RESUMO
Cold agglutinin disease (CAD) is a rare B-cell lymphoproliferative disorder of the bone marrow, manifested by autoimmune hemolytic anemia caused by binding of monoclonal IgM autoantibodies to the I antigen. Underlying genetic changes have previously been reported, but their impact on gene expression profile has been unknown. Here, we define differentially expressed genes in CAD B cells. To unravel downstream alteration in cellular pathways, gene expression by RNA sequencing was undertaken. Clonal B-cell samples from 12 CAD patients and IgM-expressing memory B cells from 4 healthy individuals were analyzed. Differential expression analysis and filtering resulted in 93 genes with significant differential expression. Top upregulated genes included SLC4A1, SPTA1, YBX3, TESC, HBD, AHSP, TRAF1, HBA2, RHAG, CA1, SPTB, IL10, UBASH3B, ALAS2, HBA1, CRYM, RGCC, KANK2, and IGHV4-34. They were upregulated at least 8-fold, while complement receptor 1 (CR1/CD35) was downregulated 11-fold in clonal CAD B cells compared to control B cells. Flow cytometry analyses further confirmed reduced CR1 (CD35) protein expression by clonal CAD IgM+ B cells compared to IgM+ memory B cells in controls. CR1 (CD35) is an important negative regulator of B-cell activation and differentiation. Therefore, reduced CR1 (CD35) expression may increase activation, proliferation, and antibody production in CAD-associated clonal B cells.
Assuntos
Anemia Hemolítica Autoimune , Humanos , Anemia Hemolítica Autoimune/genética , Anemia Hemolítica Autoimune/metabolismo , Regulação para Baixo , Receptores de Complemento 3b/genética , Linfócitos B , Imunoglobulina M , Perfilação da Expressão Gênica , Proteínas Sanguíneas/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismoRESUMO
OBJECTIVE: High-throughput sequencing was used to screen expressing differences of miRNA, lncRNA, and mRNA in CD19+ B peripheral blood samples of newly diagnosed immune thrombocytopenia (ITP) patients and healthy controls. The study aimed to explore the regulatory role of ceRNA network in the pathogenesis of dysfunctional CD19 + B lymphocytes of ITP patients. METHODS: CD19+ B lymphocytes were isolated from peripheral blood samples of ITP patients and their healthy counterparts. High-throughput sequencing was used to screen for the expression of miRNA, lncRNA, and mRNA of ITP patients and healthy controls, which were analysed by the ceRNA network. Moreover, qPCR was used to verify the differential expression of miRNA, lncRNA, and mRNA in ITP patients and healthy controls. The correlation between differentially expressed miRNA, lncRNA, mRNA, and B lymphocyte subsets was also analysed. RESULTS: The CD19+ B lymphocytes of 4 newly diagnosed ITP patients and 4 healthy controls were sequenced and analysed. There were 65 differentially expressed lncRNA and 149 mRNA forming a ceRNA network showed that 12 lncRNA and 136 differentially expressed mRNA were closely associated. Similarly, miR-144-3p, miR-374c-3p, and miR-451a were highly expressed in ITP patients, as confirmed by qPCR, which was consistent with the high-throughput sequence results. LOC102724852 and CCL20 were highly expressed in ITP patients, while LOC105378901, LOC112268311, ALAS2, and TBC1D3F were not as compared to healthy controls, which was consistent with the high-throughput sequence results. In addition, the expression of miR-374c-3p, LOC112268311, LOC105378901, and CXCL3 were correlated with the percentage of B lymphocyte subsets. CONCLUSIONS: The ceRNA network of miRNA, lncRNA, and mRNA in peripheral CD19 + B lymphocytes plays an essential role in the pathogenesis of ITP.
Assuntos
MicroRNAs , Púrpura Trombocitopênica Idiopática , RNA Longo não Codificante , Trombocitopenia , Humanos , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/genética , RNA Longo não Codificante/genética , MicroRNAs/genética , Linfócitos B , RNA Mensageiro/genética , Antígenos CD19/genética , Redes Reguladoras de Genes , 5-Aminolevulinato Sintetase/genéticaRESUMO
BACKGROUND: : Erythroid cells play important roles in hemostasis and disease. However, there is still significant knowledge gap regarding stress erythropoiesis. METHODS: : Two single-cell RNAseq datasets of erythroid cells on GEO with accession numbers GSE149938 and GSE184916 were obtained. The datasets from two sources, bone marrow and peripheral blood were analyzed using Seurat v4.1.1, and other tools in R. QC metrics were performed, data were normalized and scaled. Principal components that capture the variation of the data were determined. In clustering the cells, KNN graph was constructed and Louvain algorithm was applied to optimize the standard modularity function. Clusters were defined via differential expression of features. RESULTS: We identified 9 different cell types, with a particular cluster representing the stress erythroids. The clusters showed differentially expressed genes as observed from the gene signature plot. The stress erythroid cluster differentially expressed some genes including ALAS2, HEMGN, and GUK1. CONCLUSION: The erythroid population was found to be heterogeneous, with a distinct sub-cell type constituting the stress erythroids; this may have important implications for our knowledge of steady-state and stress erythropoiesis, and the markers found in this cluster may prove useful for future research into the dynamics of stress erythroid progenitor cell differentiation.
Assuntos
Células Eritroides , Análise da Expressão Gênica de Célula Única , Humanos , Células Precursoras Eritroides , Algoritmos , Diferenciação Celular , Proteínas Nucleares , 5-Aminolevulinato SintetaseRESUMO
BACKGROUND: Feature selection is a critical step for translating advances afforded by systems-scale molecular profiling into actionable clinical insights. While data-driven methods are commonly utilized for selecting candidate genes, knowledge-driven methods must contend with the challenge of efficiently sifting through extensive volumes of biomedical information. This work aimed to assess the utility of large language models (LLMs) for knowledge-driven gene prioritization and selection. METHODS: In this proof of concept, we focused on 11 blood transcriptional modules associated with an Erythroid cells signature. We evaluated four leading LLMs across multiple tasks. Next, we established a workflow leveraging LLMs. The steps consisted of: (1) Selecting one of the 11 modules; (2) Identifying functional convergences among constituent genes using the LLMs; (3) Scoring candidate genes across six criteria capturing the gene's biological and clinical relevance; (4) Prioritizing candidate genes and summarizing justifications; (5) Fact-checking justifications and identifying supporting references; (6) Selecting a top candidate gene based on validated scoring justifications; and (7) Factoring in transcriptome profiling data to finalize the selection of the top candidate gene. RESULTS: Of the four LLMs evaluated, OpenAI's GPT-4 and Anthropic's Claude demonstrated the best performance and were chosen for the implementation of the candidate gene prioritization and selection workflow. This workflow was run in parallel for each of the 11 erythroid cell modules by participants in a data mining workshop. Module M9.2 served as an illustrative use case. The 30 candidate genes forming this module were assessed, and the top five scoring genes were identified as BCL2L1, ALAS2, SLC4A1, CA1, and FECH. Researchers carefully fact-checked the summarized scoring justifications, after which the LLMs were prompted to select a top candidate based on this information. GPT-4 initially chose BCL2L1, while Claude selected ALAS2. When transcriptional profiling data from three reference datasets were provided for additional context, GPT-4 revised its initial choice to ALAS2, whereas Claude reaffirmed its original selection for this module. CONCLUSIONS: Taken together, our findings highlight the ability of LLMs to prioritize candidate genes with minimal human intervention. This suggests the potential of this technology to boost productivity, especially for tasks that require leveraging extensive biomedical knowledge.
Assuntos
Relevância Clínica , Mineração de Dados , Humanos , Perfilação da Expressão Gênica , Conhecimento , Idioma , 5-Aminolevulinato SintetaseRESUMO
BACKGROUND: Hereditary hemolytic anemia (HHA) refers to a heterogeneous group of genetic disorders that share one common feature: destruction of circulating red blood cells (RBCs). The destruction of RBCs may be due to membranopathies, enzymopathies, or hemoglobinopathies. Because these are genetic disorders, incorporation of next-generation sequencing (NGS) has facilitated the diagnostic process of HHA. METHOD: Genetic data from 29 patients with suspected hereditary anemia in a tertiary hospital were retrospectively reviewed to evaluate the efficacy of NGS on hereditary anemia diagnosis. Targeted NGS was performed with custom probes for 497 genes associated with hematologic disorders. After genomic DNA was extracted from peripheral blood, prepared libraries were hybridized with capture probes and sequenced using NextSeq 550Dx (Illumina, San Diego, CA, USA). RESULT: Among the 29 patients, ANK1 variants were detected in five, four of which were pathogenic or likely pathogenic variants. SPTB variants were detected in six patients, five of which were classified as pathogenic or likely pathogenic variants. We detected g6pd pathogenic and spta1 likely pathogenic variants in two patients and one patient, respectively. Whole-gene deletions in both HBA1 and HBA2 were detected in two patients, while only HBA2 deletion was detected in one patient. One likely pathogenic variant in PLKR was detected in one patient, and one likely pathogenic variant in ALAS2 was detected in another. CONCLUSION: Here, NGS played a critical role in definitive diagnosis in 18 out of 29 patients (62.07%) with suspected HHA. Thus, its incorporation into the diagnostic workflow is crucial.
Assuntos
Anemia Hemolítica Congênita , Humanos , Criança , Estudos Retrospectivos , Anemia Hemolítica Congênita/diagnóstico , Anemia Hemolítica Congênita/genética , Eritrócitos , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas do Citoesqueleto , 5-Aminolevulinato SintetaseRESUMO
Protoporphyrin IX (PPIX) is an intermediate in the heme biosynthesis pathway. Abnormal accumulation of PPIX due to certain pathological conditions such as erythropoietic protoporphyria and X-linked protoporphyria causes painful phototoxic reactions of the skin, which can significantly impact daily life. Endothelial cells in the skin have been proposed as the primary target for PPIX-induced phototoxicity through light-triggered generation of reactive oxygen species. Current approaches for the management of PPIX-induced phototoxicity include opaque clothing, sunscreens, phototherapy, blood therapy, antioxidants, bone marrow transplantation, and drugs that increase skin pigmentation. In this review, we discuss the present understanding of PPIX-induced phototoxicity including PPIX production and disposition, conditions that lead to PPIX accumulation, symptoms and individual differences, mechanisms, and therapeutics.
Assuntos
Células Endoteliais , Protoporfiria Eritropoética , Humanos , Células Endoteliais/metabolismo , Protoporfirinas/farmacologia , Protoporfirinas/metabolismo , Protoporfiria Eritropoética/metabolismo , Protoporfiria Eritropoética/patologia , Protoporfiria Eritropoética/terapia , 5-Aminolevulinato SintetaseRESUMO
Congenital sideroblastic anemia is characterized by anemia and intramitochondrial iron accumulation in erythroid precursors that form ring sideroblasts. The most common recessive forms are caused by sequence variations in the ALAS2 and SLC25A38 genes. In patients with transfusion-dependent and pyridoxine- resistant severe congenital sideroblastic anemia, hematopoietic stem celltransplantis the only curative option. Herein, we described successful implementations of allogeneic hematopoietic stem cell transplant in 4 Iranian children with congenital sideroblastic anemia. The patients had presented with clinical manifestations of anemia early in life, and the diagnoses of congenital sideroblastic anemia were established through blood tests and bone marrow aspiration. Congenital sideroblastic anemia was further confirmed by the identification of pathogenic variants in SLC25A38 in 2 patients. All 4 patients received allogeneic hematopoietic stem cell transplant with myeloablative conditioning regimen that included busulfan, cyclophosphamide, andrabbit antithymocyte globulin. A combination of cyclosporine A and methotrexate or mycophenolate mofetil was used for graft-versus-host disease prophylaxis. Bone marrow and peripheral blood from sibling or related donors with fully matched human leukocyte antigen profiles were applied. The outcomes of hematopoietic stem celltransplantin patients with congenital sideroblastic anemia were favorable. Three patients achieved full donor chimerism (>95%, 98%, and 100%), and the other patient showed mixed chimerism (75%). All patients remained transfusion independent. Hemato- poietic stem celltransplantis a curative treatmentthat can provide long-term survival for patients with congenital sideroblastic anemia, particularly when used in a timely manner. There remain ongoing challenges in various aspects of hematopoietic stem celltransplantin patients with congenital sideroblastic anemia, which remain to be elucidated.
Assuntos
Anemia Sideroblástica , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Criança , Humanos , 5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/diagnóstico , Anemia Sideroblástica/genética , Anemia Sideroblástica/congênito , Ciclosporina , Irã (Geográfico) , Condicionamento Pré-TransplanteRESUMO
In heart failure, the biological and clinical connection between abnormal iron homeostasis, myocardial function, and prognosis is known; however, the expression profiles of iron-linked genes both at myocardial tissue and single-cell level are not well defined. Through publicly available bulk and single-nucleus RNA sequencing (RNA-seq) datasets of left ventricle samples from adult non-failed (NF) and dilated cardiomyopathy (DCM) subjects, we aim to evaluate the altered iron metabolism in a diseased condition, at the whole cardiac tissue and single-cell level. From the bulk RNA-seq data, we found 223 iron-linked genes expressed at the myocardial tissue level and 44 differentially expressed between DCM and NF subjects. At the single-cell level, at least 18 iron-linked expressed genes were significantly regulated in DCM when compared to NF subjects. Specifically, the iron metabolism in DCM cardiomyocytes is altered at several levels, including: (1) imbalance of Fe3+ internalization (SCARA5 down-regulation) and reduction of internal conversion from Fe3+ to Fe2+ (STEAP3 down-regulation), (2) increase of iron consumption to produce hemoglobin (HBA1/2 up-regulation), (3) higher heme synthesis and externalization (ALAS2 and ABCG2 up-regulation), (4) lower cleavage of heme to Fe2+, biliverdin and carbon monoxide (HMOX2 down-regulation), and (5) positive regulation of hepcidin (BMP6 up-regulation).
Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Adulto , Humanos , Cardiomiopatia Dilatada/metabolismo , Miocárdio/metabolismo , Regulação para Baixo , Miócitos Cardíacos/metabolismo , Insuficiência Cardíaca/metabolismo , 5-Aminolevulinato Sintetase/genética , Receptores Depuradores Classe A/genéticaRESUMO
5-Aminolevulinic acid synthase (ALAS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of heme biosynthesis in α-proteobacteria and several non-plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C-terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C-terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C-terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C-terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel ß-sheet important to Fold-Type I PLP-dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C-terminus has a homolog-specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms.
Assuntos
5-Aminolevulinato Sintetase , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , 5-Aminolevulinato Sintetase/química , Fosfato de Piridoxal/química , Domínio Catalítico , Heme/químicaRESUMO
Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5'-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR-/- hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5'TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.
Assuntos
Interferons , Mitocôndrias , Camundongos , Animais , Mitocôndrias/metabolismo , Vírus do Sarampo , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , DNA MitocondrialRESUMO
ALAS1 is a member of the α-oxoamine synthase family, which is the first rate-limiting enzyme for heme synthesis and is important for maintaining intracellular heme levels. In the ovary, ALAS1 is associated with the regulation of ovulation-related mitochondrial P450 cytochromes, steroid metabolism, and steroid hormone production. However, there are few studies on the relationship between ALAS1 and reproductive traits in goats. In this study, a mutation located in the promoter region of ALAS1 (g.48791372C>A) was found to be significantly (p < 0.05) associated with the kidding number of Yunshang black goats. Specifically, the mean kidding number in the first three litters and the kidding numbers of all three litters were significantly (p < 0.05) higher in individuals with the CA genotype or AA genotype than in those with the CC genotype. To further investigate the regulatory mechanism of ALAS1, the expression of ALAS1 in goat ovarian tissues with different genotypes was verified by real-time quantitative PCR. The results showed that the expression of ALAS1 was significantly higher in the ovaries of individuals with AA genotype than those with AC and CC genotypes (p < 0.01), and the expression trend of transcription factor ASCL2 was consistent with ALAS1. Additionally, the ALAS1 g.48791372C>A mutation created a new binding site for the transcription factor ASCL2. The luciferase activity assay indicated that the mutation increased the promoter activity of ALAS1. Overexpression of the transcription factor ASCL2 induced increased expression of ALAS1 in goat granulosa cells (p < 0.05). The opposite trend was shown for the inhibition of ASCL2 expression. The results of real-time quantitative PCR, EdU and Cell Counting Kit-8 assays indicated that the transcription factor ASCL2 increased the proliferation of goat granulosa cells by mediating the expression of ALAS1. In conclusion, the transcription factor ASCL2 positively regulated the transcriptional activity and expression levels of ALAS1, altering granulosa cell proliferation and the kidding number in goats.
Assuntos
5-Aminolevulinato Sintetase , Cabras , Fatores de Transcrição , Animais , Feminino , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Proliferação de Células , Cabras/genética , Cabras/metabolismo , Heme , Fatores de Transcrição/metabolismoRESUMO
Iron supplementation is not considered as a doping method; however, it can affect the levels of several biomarkers of the hematologic module of the athlete biological passport (ABP), such as the reticulocyte percentage (%RET) and hemoglobin (HGB) level. Thus, iron injection could be a confounding factor in antidoping analyses. Previous studies have suggested that the HGB level and the expression levels of reticulocyte-related-mRNAs, such as 5'-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1), could be promising biomarkers for the ABP and detectable in dried blood spots (DBSs). Therefore, in this study, we examined the impact of iron injection on the levels of these potential biomarkers in DBSs. Reticulocyte-related-mRNAs analyses were performed by RT-qPCR. Ferritin level in DBS was measured with enzyme-linked immunosorbent assay (ELISA) method. Notably, there were no significant effects of iron supplementation on the levels of ALAS2 and CA1 mRNAs but by contrast, the %RET and immature reticulocyte fraction (IRF) measured in whole blood increased significantly following iron injection. As expected, iron supplementation increased the ferritin level significantly in both serum and DBS samples. In conclusion, these findings reinforce the specificity of reticulocyte-related mRNAs in DBSs as biomarkers of blood doping to target in antidoping analyses.
Assuntos
Dopagem Esportivo , Humanos , Dopagem Esportivo/métodos , Reticulócitos/metabolismo , Ferro , Biomarcadores , Ferritinas , Hemoglobinas/análise , 5-Aminolevulinato SintetaseRESUMO
INTRODUCTION AND HYPOTHESIS: The pathogenesis of pelvic organ prolapse (POP) remains unknown. Herein, we aim to reveal the molecular profile of POP by transcriptomic and metabolomic analysis. METHODS: We selected 12 samples of uterosacral ligaments (USLs) from 6 POP patients and 6 controls for transcriptomic and metabolomic analyses. Differentially expressed genes (DEGs) were identified using the R package edgeR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using clusterProfiler, and a protein-protein interaction (PPI) network was constructed using STRING and visualized in Cytoscape. Metabolomic profiling was performed by a liquid chromatography-tandem mass spectrometry system. RESULTS: Transcriptomic analysis identified 487 DEGs between the POP and control groups. Functional enrichment analysis revealed that they were mostly related to immune response terms, including "adaptive immune response," "T cell differentiation," and "T cell activation." In addition, PTPRC, LCK, CD247, IL2RB, CD2, CXR5, JUN, CD3E, IL2RG, and PRF1 were the 10 nodes with the highest node degrees in the PPI network. Metabolomic profiling revealed 290 differentially expressed metabolites, which significantly enriched in "glycerophospholipid metabolism," "nicotinate and nicotinamide metabolism," "glycine, serine, and threonine metabolism," "arginine and proline metabolism," "pyrimidine metabolism," and "purine metabolism." Finally, integrated analysis revealed that the DEGs involved in these significantly enriched metabolic pathways included NT5C1A, GMPR, SDS, ALAS2, CARNS1, PYCR1, P4HA3, PGS1, and NMRK2. CONCLUSIONS: Our findings demonstrate that the immune response and metabolic regulatory pathways are intertwined in POP and might provide new therapeutic targets.