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1.
Nat Commun ; 12(1): 4991, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404810

RESUMO

Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the ß-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.


Assuntos
Edição de Genes , Hemoglobinas/genética , Hemoglobinas/metabolismo , Sistemas CRISPR-Cas , Cromatina , Cromossomos , Proteínas de Ligação a DNA/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Expressão Gênica , Globinas , Humanos , Mutação , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Globinas beta/genética
2.
Nat Commun ; 12(1): 4922, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389729

RESUMO

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Globinas/genética , Células-Tronco Hematopoéticas/metabolismo , Perda de Heterozigosidade/genética , Deleção de Sequência , Células Cultivadas , Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Metilação de DNA , Expressão Gênica , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Fator de Crescimento Insulin-Like II/genética , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética
3.
Genes (Basel) ; 12(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208174

RESUMO

There are many co-regulated genes in eukaryotic cells. The coordinated activation or repression of such genes occurs at specific stages of differentiation, or under the influence of external stimuli. As a rule, co-regulated genes are dispersed in the genome. However, there are also gene clusters, which contain paralogous genes that encode proteins with similar functions. In this aspect, they differ significantly from bacterial operons containing functionally linked genes that are not paralogs. In this review, we discuss the reasons for the existence of gene clusters in vertebrate cells and propose that clustering is necessary to ensure the possibility of selective activation of one of several similar genes.


Assuntos
Evolução Molecular , Família Multigênica , Animais , Caderinas/genética , Caderinas/metabolismo , Células Eritroides/metabolismo , Globinas/genética , Globinas/metabolismo , Humanos
4.
Mol Ther ; 29(6): 1933-1934, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-33961803
6.
J Biol Chem ; 296: 100291, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33453283

RESUMO

Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell-type-specific expression, regulation, and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that-in addition to the testes-ADGB is prominently expressed in the female reproductive tract, lungs, and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays, and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Cílios/genética , Fatores de Transcrição Forkhead/genética , Globinas/genética , Fatores de Transcrição de Fator Regulador X/genética , Transcriptoma , Animais , Sítios de Ligação , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Bovinos , Cílios/metabolismo , Elementos Facilitadores Genéticos , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Globinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Pulmão/citologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Células MCF-7 , Masculino , Anotação de Sequência Molecular , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição de Fator Regulador X/metabolismo , Análise de Sequência de RNA , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
7.
Dev Cell ; 56(4): 478-493.e11, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33476555

RESUMO

The human genome harbors 14,000 duplicated or retroposed pseudogenes. Given their functionality as regulatory RNAs and low conservation, we hypothesized that pseudogenes could shape human-specific phenotypes. To test this, we performed co-expression analyses and found that pseudogene exhibited tissue-specific expression, especially in the bone marrow. By incorporating genetic data, we identified a bone-marrow-specific duplicated pseudogene, HBBP1 (η-globin), which has been implicated in ß-thalassemia. Extensive functional assays demonstrated that HBBP1 is essential for erythropoiesis by binding the RNA-binding protein (RBP), HNRNPA1, to upregulate TAL1, a key regulator of erythropoiesis. The HBBP1/TAL1 interaction contributes to a milder symptom in ß-thalassemia patients. Comparative studies further indicated that the HBBP1/TAL1 interaction is human-specific. Genome-wide analyses showed that duplicated pseudogenes are often bound by RBPs and less commonly bound by microRNAs compared with retropseudogenes. Taken together, we not only demonstrate that pseudogenes can drive human evolution but also provide insights on their functional landscapes.


Assuntos
Eritropoese/genética , Globinas/genética , Pseudogenes , Talassemia beta/genética , Ligação Competitiva , Medula Óssea/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Células Eritroides/metabolismo , Células Eritroides/patologia , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Especificidade de Órgãos/genética , Ligação Proteica , Estabilidade Proteica , Estabilidade de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo
8.
J Biochem ; 169(6): 663-673, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-33479760

RESUMO

Tardigrades, a phylum of meiofaunal organisms, exhibit extraordinary tolerance to various environmental conditions, including extreme temperatures (-273 to 151°C) and exposure to ionizing radiation. Proteins from anhydrobiotic tardigrades with homology to known proteins from other organisms are new potential targets for structural genomics. Recently, we reported spectroscopic and structural characterization of a hexacoordinated haemoglobin (Kumaglobin [Kgb]) found in an anhydrobiotic tardigrade. In the absence of its exogenous ligand, Kgb displays hexacoordination with distal and proximal histidines. In this work, we analysed binding of the molecular oxygen ligand following reduction of haem in Kgb using a pulse radiolysis technique. Radiolytically generated hydrated electrons (eaq-) reduced the haem iron of Kgb within 20 µs. Subsequently, ferrous haem reacted with O2 to form a ferrous-dioxygen intermediate with a second-order rate constant of 3.0 × 106 M-1 s-1. The intermediate was rapidly (within 0.1 s) autooxidized to the ferric form. Redox potential measurements revealed an E'0 of -400 mV (vs. standard hydrogen electrode) in the ferric/ferrous couple. Our results suggest that Kgb may serve as a physiological generator of O2▪- via redox signalling and/or electron transfer.


Assuntos
Globinas/química , Histidina/química , Oxigênio/metabolismo , Tardígrados/metabolismo , Água/química , Animais , Transporte de Elétrons , Globinas/metabolismo , Histidina/metabolismo , Ligantes , Oxirredução , Radiólise de Impulso
9.
Free Radic Biol Med ; 162: 423-434, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33144263

RESUMO

Cytoglobin is a conserved hemoprotein ubiquitously expressed in mammalian tissues, which conducts electron transfer reactions with proposed signaling functions in nitric oxide (NO) and lipid metabolism. Cytoglobin has an E7 distal histidine (His81), which unlike related globins such as myoglobin and hemoglobin, is in equilibrium between a bound, hexacoordinate state and an unbound, pentacoordinate state. The His81 binding equilibrium appears to be allosterically modulated by the presence of an intramolecular disulfide between two cysteines (Cys38 and Cys83). The formation of this disulfide bridge regulates nitrite reductase activity and lipid binding. Herein, we attempt to clarify the effects of defined thiol oxidation states on small molecule binding of cytoglobin heme, using cyanide binding to probe the ferric state. Cyanide binding kinetics to wild-type cytoglobin reveal at least two kinetically distinct subpopulations, depending on thiol oxidation states. Experiments with covalent thiol modification by NEM, glutathione, and amino acid substitutions (C38S, C83S and H81A), indicate that subpopulations ranging from fully reduced thiols, single thiol oxidation, and intramolecular disulfide formation determine heme binding properties by modulating the histidine-heme affinity and ligand binding. The redox modulation of ligand binding is sensitive to physiological levels of hydrogen peroxide, with a functional midpoint redox potential for the native cytoglobin intramolecular disulfide bond of -189 ± 4 mV, a value within the boundaries of intracellular redox potentials. These results support the hypothesis that Cys38 and Cys83 on cytoglobin serve as sensitive redox sensors that modulate the cytoglobin distal heme pocket reactivity and ligand binding.


Assuntos
Globinas , Heme , Animais , Citoglobina/metabolismo , Globinas/genética , Heme/metabolismo , Humanos , Oxirredução , Ligação Proteica
10.
Free Radic Biol Med ; 162: 471-477, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33166649

RESUMO

Neuroglobin is the third member of the globin family to be identified in 2000 in neurons of both human and mouse nervous systems. Neuroglobin is an oxygen-binding globin found in neurons within the central nervous system as well as in peripheral neurons, that produces a protective effect against hypoxic/ischemic damage induced by promoting oxygen availability within the mitochondria. Numerous investigations have demonstrated that impaired neuroglobin functioning is implicated in the pathogenesis of multiple neurodegenerative disorders. Several in vitro and animal studies have reported the potential of neuroglobin upregulation in improving the neuroprotection through modulation of mitochondrial functions, such as ATP production, clearing reactive oxygen species (ROS), promoting the dynamics of mitochondria, and controlling apoptosis. Neuroglobin acts as a stress-inducible globin, which has been associated hypoxic/ischemic insults where it acts to protect the heart and brain, providing a wide range of applicability in the treatment of human disorders. This review article discusses normal physiological functions of neuroglobin in mitochondria-associated pathways, as well as outlining how dysregulation of neuroglobin is associated with the pathogenesis of neurodegenerative disorders.


Assuntos
Proteínas do Tecido Nervoso , Doenças Neurodegenerativas , Animais , Globinas/genética , Mitocôndrias , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/tratamento farmacológico , Neuroglobina
11.
Mar Genomics ; 57: 100831, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33250437

RESUMO

In the freezing waters of the Southern Ocean, Antarctic teleost fish, the Notothenioidei, have developed unique adaptations to cope with cold, including, at the extreme, the loss of hemoglobin in icefish. As a consequence, icefish are thought to be the most vulnerable of the Antarctic fish species to ongoing ocean warming. Some icefish also fail to express myoglobin but all appear to retain neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X. Despite the lack of the inducible heat shock response, Antarctic notothenioid fish are endowed with physiological plasticity to partially compensate for environmental changes, as shown by numerous physiological and genomic/transcriptomic studies over the last decade. However, the regulatory mechanisms that determine temperature/oxygen-induced changes in gene expression remain largely unexplored in these species. Proteins such as globins are susceptible to environmental changes in oxygen levels and temperature, thus playing important roles in mediating Antarctic fish adaptations. In this study, we sequenced the full-length transcripts of myoglobin, neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X from the Antarctic red-blooded notothenioid Trematomus bernacchii and the white-blooded icefish Chionodraco hamatus and evaluated transcripts levels after exposure to high temperature and low oxygen levels. Basal levels of globins are similar in the two species and both stressors affect the expression of Antarctic fish globins in brain, retina and gills. Temperature up-regulates globin expression more effectively in white-blooded than in red-blooded fish while hypoxia strongly up-regulates globins in red-blooded fish, particularly in the gills. These results suggest globins function as regulators of temperature and hypoxia tolerance. This study provides the first insights into globin transcriptional changes in Antarctic fish.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica/fisiologia , Globinas/genética , Perciformes/genética , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Globinas/química , Globinas/metabolismo , Masculino , Perciformes/metabolismo , Filogenia , Alinhamento de Sequência/veterinária
12.
Genome ; 64(4): 476-489, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33232179

RESUMO

The chicken model organism has advanced the areas of developmental biology, virology, immunology, oncology, epigenetic regulation of gene expression, conservation biology, and genomics of domestication. Further, the chicken model organism has aided in our understanding of human disease. Through the recent advances in high-throughput sequencing and bioinformatic tools, researchers have successfully identified sequences in the chicken genome that have human orthologs, improving mammalian genome annotation. In this review, we highlight the importance of chicken as an animal model in basic and pre-clinical research. We will present the importance of chicken in poultry epigenetics and in genomic studies that trace back to their ancestor, the last link between human and chicken in the tree of life. There are still many genes of unknown function in the chicken genome yet to be characterized. By taking advantage of recent sequencing technologies, it is possible to gain further insight into the chicken epigenome.


Assuntos
Galinhas/genética , Epigênese Genética , Epigenômica/métodos , Genoma , Animais , Cromatina/química , Biologia Computacional , Epigenoma , Eritrócitos , Eritropoese , Expressão Gênica , Técnicas Genéticas , Genômica , Globinas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunidade Inata , Aves Domésticas/genética , RNA não Traduzido
13.
Artigo em Inglês | MEDLINE | ID: mdl-33202310

RESUMO

The globin gene repertoire of gnathostome vertebrates is dictated by differential retention and loss of nine paralogous genes: androglobin, neuroglobin, globin X, cytoglobin, globin Y, myoglobin, globin E, and the α- and ß-globins. We report the globin gene repertoire of three orders of modern amphibians: Anura, Caudata, and Gymnophiona. Combining phylogenetic and conserved synteny analysis, we show that myoglobin and globin E were lost only in the Batrachia clade, but retained in Gymnophiona. The major amphibian groups also retained different paralogous copies of globin X. None of the amphibian presented αD-globin gene. Nevertheless, two clades of ß-globins are present in all amphibians, indicating that the amphibian ancestor possessed two paralogous proto ß-globins. We also show that orthologs of the gene coding for the monomeric hemoglobin found in the heart of Rana catesbeiana are present in Neobatrachia and Pelobatoidea species we analyzed. We suggest that these genes might perform myoglobin- and globin E-related functions. We conclude that the repertoire of globin genes in amphibians is dictated by both retention and loss of the paralogous genes cited above and the rise of a new globin gene through co-option of an α-globin, possibly facilitated by a prior event of transposition.


Assuntos
Anfíbios/genética , Globinas/genética , Animais , Evolução Molecular , Filogenia , Sintenia
14.
Biochim Biophys Acta Mol Cell Res ; 1868(3): 118931, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33340546

RESUMO

The CCDC26 gene is considered to encode a functional noncoding RNA associated with acute myeloid leukemia and other cancers. However, investigations into the physiological roles of CCDC26 are rare. Previously, we reported that CCDC26 regulated proliferation and cell death of leukemia cells through KIT, a receptor tyrosine kinase, by using K562 leukemia cells and their derivative CCDC26-knockdown (KD) cells. Here we propose a new role of CCDC26 in the differentiation of erythroid cells. We showed that expression of embryonic (ε- and ζ-) globins was markedly upregulated in CCDC26-KD cells compared with K562 control cells during hemin-induced differentiation. In contrast, expression of fetal-type γ-globin, a major globin expressed in original K562 cells, was decreased. These changes in the expression of globin genes mainly took place at the transcriptional level, with significant suppression of transcription of adult (ß-, δ-) globins in CCDC26-KD cells. Re-introduction of exogenous CCDC26 into the CCDC26-KD cells recovered low-level expression of the embryonal globins. These results suggest CCDC26 has a role in switching transcription of globin genes in the differentiation of erythroid cells. The expression profile of the CCDC26-KD cells and control cells suggests FOG-2, a transcriptional modulator, as a candidate for a mediator of the CCDC26-associated regulation. We showed that both embryonic globins were transcriptionally activated in FOG-2-KD K562 cells. The KIT inhibitor ISCK03 suppressed the production of hemoglobin in K562 cells but did not affect transcription of globin genes. To summarize, FOG-2, but not KIT, is responsible for globin transcriptional regulation by CCDC26.


Assuntos
Proteínas de Ligação a DNA/genética , Células Eritroides/citologia , Globinas/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , Diferenciação Celular/efeitos dos fármacos , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Globinas/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Imidazóis/farmacologia , Células K562 , Sulfonamidas/farmacologia
15.
BMC Evol Biol ; 20(1): 165, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33371890

RESUMO

BACKGROUND: How vascular systems and their respiratory pigments evolved is still debated. While many animals present a vascular system, hemoglobin exists as a blood pigment only in a few groups (vertebrates, annelids, a few arthropod and mollusk species). Hemoglobins are formed of globin sub-units, belonging to multigene families, in various multimeric assemblages. It was so far unclear whether hemoglobin families from different bilaterian groups had a common origin. RESULTS: To unravel globin evolution in bilaterians, we studied the marine annelid Platynereis dumerilii, a species with a slow evolving genome. Platynereis exhibits a closed vascular system filled with extracellular hemoglobin. Platynereis genome and transcriptomes reveal a family of 19 globins, nine of which are predicted to be extracellular. Extracellular globins are produced by specialized cells lining the vessels of the segmental appendages of the worm, serving as gills, and thus likely participate in the assembly of a previously characterized annelid-specific giant hemoglobin. Extracellular globin mRNAs are absent in smaller juveniles, accumulate considerably in growing and more active worms and peak in swarming adults, as the need for O2 culminates. Next, we conducted a metazoan-wide phylogenetic analysis of globins using data from complete genomes. We establish that five globin genes (stem globins) were present in the last common ancestor of bilaterians. Based on these results, we propose a new nomenclature of globins, with five clades. All five ancestral stem-globin clades are retained in some spiralians, while some clades disappeared early in deuterostome and ecdysozoan evolution. All known bilaterian blood globin families are grouped in a single clade (clade I) together with intracellular globins of bilaterians devoid of red blood. CONCLUSIONS: We uncover a complex "pre-blood" evolution of globins, with an early gene radiation in ancestral bilaterians. Circulating hemoglobins in various bilaterian groups evolved convergently, presumably in correlation with animal size and activity. However, all hemoglobins derive from a clade I globin, or cytoglobin, probably involved in intracellular O2 transit and regulation. The annelid Platynereis is remarkable in having a large family of extracellular blood globins, while retaining all clades of ancestral bilaterian globins.


Assuntos
Anelídeos/classificação , Anelídeos/genética , Evolução Molecular , Globinas/genética , Animais , Genoma/genética , Hemoglobinas/genética
16.
BMC Genomics ; 21(1): 890, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33308163

RESUMO

BACKGROUND: There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 µg of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated with DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation. RESULTS: Probe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3' region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R > 0.9) than RNAse-H enzymatic depletion (R > 0.85). CONCLUSION: In this study, our results showed that 1 µg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures.


Assuntos
Poli A , RNA , Perfilação da Expressão Gênica , Globinas/genética , RNA Mensageiro/genética , Análise de Sequência de RNA
17.
J Am Chem Soc ; 142(46): 19558-19569, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33136379

RESUMO

The maleimide group is a widely used reagent for bioconjugation of peptides, proteins, and oligonucleotides employing Michael addition and Diels-Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that PdII complexes can mediate the efficient removal of various succinimide derivatives in aqueous conditions. Succinimide removal by PdII was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segment ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a succinimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.


Assuntos
Complexos de Coordenação/química , Cisteína/química , Paládio/química , Peptídeos/química , Proteínas/síntese química , Succinimidas/química , Catálise , Reação de Cicloadição , Dissulfetos/química , Globinas/síntese química , Inteínas , Maleimidas/química , Técnicas de Síntese em Fase Sólida , Tiazolidinas/química , Tiorredoxinas/síntese química , Ubiquitina/química , Ubiquitina Tiolesterase/química
18.
Yonsei Med J ; 61(12): 1064-1067, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33251782

RESUMO

Hemoglobin M (HbM) is a group of abnormal hemoglobin variants that form methemoglobin, which leads to cyanosis and hemolytic anemia. HbM-Milwaukee-2 is a rare variant caused by the point mutation CAC>TAC on codon 93 of the hemoglobin subunit beta (HBB) gene, resulting in the replacement of histidine by tyrosine. We here report the first Korean family with HbM-Milwaukee-2, whose diagnosis was confirmed by gene sequencing. A high index of suspicion for this rare Hb variant is necessary in a patient presenting with cyanosis since childhood, along with methemoglobinemia and a family history of cyanosis.


Assuntos
Cianose/etiologia , Globinas/genética , Hemoglobina M , Metemoglobinemia/congênito , Adolescente , Criança , Cianose/genética , Feminino , Globinas/química , Hemoglobina M/genética , Hemoglobinas Anormais/genética , Humanos , Masculino , Metemoglobina/análise , Metemoglobina/genética , Metemoglobinemia/diagnóstico , Metemoglobinemia/genética , Mutação Puntual , República da Coreia , Análise de Sequência de DNA
19.
Redox Biol ; 37: 101687, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32863222

RESUMO

Vertebrate hemoglobin (Hb) and myoglobin (Mb) were among the first proteins whose structures and sequences were determined over 50 years ago. In the subsequent pregenomic period, numerous related proteins came to light in plants, invertebrates and bacteria, that shared the myoglobin fold, a signature sequence motif characteristic of a 3-on-3 α-helical sandwich. Concomitantly, eukaryote and bacterial globins with a truncated 2-on-2 α-helical fold were discovered. Genomic information over the last 20 years has dramatically expanded the list of known globins, demonstrating their existence in a limited number of archaeal genomes, a majority of bacterial genomes and an overwhelming majority of eukaryote genomes. In vertebrates, 6 additional globin types were identified, namely neuroglobin (Ngb), cytoglobin (Cygb), globin E (GbE), globin X (GbX), globin Y (GbY) and androglobin (Adgb). Furthermore, functions beyond the familiar oxygen transport and storage have been discovered within the vertebrate globin family, including NO metabolism, peroxidase activity, scavenging of free radicals, and signaling functions. The extension of the knowledge on globin functions suggests that the original roles of bacterial globins must have been enzymatic, involved in defense against NO toxicity, and perhaps also as sensors of O2, regulating taxis away or towards high O2 concentrations. In this review, we aimed to discuss the evolution and remarkable functional diversity of vertebrate globins with particular focus on the variety of non-canonical expression sites of mammalian globins and their according impressive variability of atypical functions.


Assuntos
Evolução Molecular , Genômica , Globinas , Animais , Citoglobina , Globinas/genética , Neuroglobina , Oxigênio , Vertebrados
20.
Rev. cuba. hematol. inmunol. hemoter ; 36(3): e1099, jul.-set. 2020. graf
Artigo em Espanhol | LILACS, CUMED | ID: biblio-1156438

RESUMO

Introducción: Los defectos genéticos en la molécula de hemoglobina se dividen en aquellos que tienen una tasa reducida de producción de una o más cadenas de globina, las talasemias; y en los que se producen cambios estructurales que conducen a inestabilidad o transporte anormal de oxígeno. Objetivo: Explicar los diferentes mecanismos por los cuales ocurren las talasemias y otras alteraciones en la síntesis de las cadenas de globina, así como las características moleculares, fisiopatogénicas y los cambios hematológicos. Métodos: Se realizó una revisión de la literatura, en inglés y español, a través del sitio web PubMed y el motor de búsqueda Google académico de artículos publicados en los últimos 10 años. Se hizo un análisis y resumen de la bibliografía revisada. Análisis y síntesis de la información: Las talasemias son un grupo heterogéneo de defectos genéticos en la síntesis de hemoglobina, que causa una disminución en la tasa de producción de una o más cadenas de la molécula. De acuerdo a la cadena de globina que presenta el defecto se dividen en α-β-, δβ- o γδβ-talasemias. Conclusiones: Las talasemias y las hemoglobinopatías son las enfermedades hemolíticas hereditarias más comunes en muchas partes del mundo, caracterizadas por complejas interacciones entre anemia, eritropoyesis ineficaz y alteraciones del metabolismo del hierro(AU)


Introduction: Genetic disorders in the hemoglobin molecule are divided into those that have a reduced rate of production of one or more globin chains, thalassemias; and those in which structural changes occur that lead to instability or abnormal oxygen transport. Objective: To explain the different mechanisms by which thalassemias and other alterations in the synthesis of globin chains occur, as well as molecular, physiopathogenic and hematological changes. Methods: A review of the literature in English and Spanish was carried out through the PubMed website and the Google Scholar search engine, searching for articles published in the last ten years. The revised bibliography was analyzed and summarized. Information analysis and synthesis: Thalassemias make up a heterogeneous group of genetic defects in the synthesis of hemoglobin, which causes a decrease in the rate of production of one or more chains of the molecule. According to the globin chain that presents the defect, they are divided into α-β-, δβ- or γδβ-thalassemias. Conclusions: Thalassemias and hemoglobinopathies are the most common hereditary hemolytic diseases in many parts of the world. They are characterized by complex interactions between anemia, ineffective erythropoiesis, and alterations in iron metabolism(AU)


Assuntos
Humanos , Globinas , Eritropoese , Hemoglobinopatias/genética , Doenças Genéticas Inatas/epidemiologia
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