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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 57-59, 2020 Jan 10.
Artigo em Chinês | MEDLINE | ID: mdl-31922598

RESUMO

OBJECTIVE: To explore the genetic basis of a child with idiopathic mental retardation. METHODS: Clinical data and peripheral blood sample of the child were collected. Genomic DNA was extracted and subjected to copy number analysis using single nucleotide polymrophism array comparative genome hybridization (SNP-aCGH) and targeted capture and next generation sequencing (NGS). RESULTS: No microdeletion/microduplication were detected by SNP-aCGH. NGS has detected homozygous c.722delA (p.Asp241fs) variant of the LISN1 gene, which is known to underlie autosomal recessive mental retardation-27 (MRT 27). Both parents are carriers of the variant, conforming to the autosomal recessive inheritance. CONCLUSION: A novel pathogenic variant of the LINS1 gene has been identified, which probably underlies the MRT 27 in the patient.


Assuntos
Deficiência Intelectual , Proteínas , Criança , Hibridização Genômica Comparativa , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Deficiência Intelectual/genética , Proteínas/genética
2.
Biosci Biotechnol Biochem ; 84(1): 154-158, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31794328

RESUMO

Malectin is a maltose-binding endoplasmic reticulum protein conserved in animals. In Arabidopsis thaliana, we identified four genes that encode malectin-like domain (MLD)- and leucine-rich repeat (LRR)-containing proteins (AtMLLRs): two were receptor-like proteins (AtMLLR1 and 2) and the other two were extracellular proteins (AtMLLR3 and 4). The promoter:G3GFP+promoter:GUS assay indicated the organ- and cell-specific expression of the AtMLLR2 and AtMLLR3 genes.Abbreviations: Cmr: chloramphenicol-resistance marker; G3GFP: G3 green fluorescent protein; GUS: ß-glucuronidase; KD: kinase domain; LRR: leucine-rich repeat; MLD: malectin-like domain; RLK: receptor-like kinase; SP: signal peptide; TMD: transmembrane domain; Tnos: nopaline synthase terminator.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Expressão Gênica , Lectinas/genética , Proteínas de Membrana/genética , Proteínas/genética , Retículo Endoplasmático/metabolismo , Glucuronidase/química , Proteínas de Fluorescência Verde/química , Leucina/genética , Microscopia de Fluorescência , Filogenia , Plantas Geneticamente Modificadas , Domínios Proteicos/genética , Coloração e Rotulagem
3.
Sheng Wu Gong Cheng Xue Bao ; 35(10): 1843-1856, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31668033

RESUMO

By constructing mutant libraries and utilizing high-throughput screening methods, directed evolution has emerged as the most popular strategy for protein design nowadays. In the past decade, taking advantages of computer performance and algorithms, computer-assisted protein design has rapidly developed and become a powerful method of protein engineering. Based on the simulation of protein structure and calculation of energy function, computational design can alter the substrate specificity and improve the thermostability of enzymes, as well as de novo design of artificial enzymes with expected functions. Recently, machine learning and other artificial intelligence technologies have also been applied to computational protein engineering, resulting in a series of remarkable applications. Along the lines of protein engineering, this paper reviews the progress and applications of computer-assisted protein design, and current trends and outlooks of the development.


Assuntos
Evolução Molecular Direcionada , Engenharia de Proteínas , Proteínas/química , Proteínas/metabolismo , Ensaios de Triagem em Larga Escala , Proteínas/genética , Especificidade por Substrato
4.
Adv Exp Med Biol ; 1163: 89-105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31707701

RESUMO

Correlation between an allosteric site and its orthosteric site refers to the phenomenon that perturbations like ligand binding, mutation, or posttranslational modifications at the allosteric site leverage variation in the orthosteric site. Understanding this kind of correlation not only helps to disclose how information is transmitted in allosteric regulation but also provides clues for allosteric drug discovery. This chapter starts with an overview of correlation studies on allosteric and orthosteric sites and then introduces recent progress in evolutionary and simulation-based dynamic studies. Discussions and perspectives on future directions are also given.


Assuntos
Sítio Alostérico , Descoberta de Drogas , Proteínas , Relação Estrutura-Atividade , Regulação Alostérica , Sítios de Ligação , Simulação por Computador , Proteínas/química , Proteínas/genética
6.
Phys Chem Chem Phys ; 21(44): 24393-24405, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31663524

RESUMO

Engineered repeat proteins have proven to be a fertile ground for studying the competition between folding, misfolding and transient aggregation of tethered protein domains. We examine the interplay between folding and inter-domain interactions of engineered FiP35 WW domain repeat proteins with n = 1 through 5 repeats. We characterize protein expression, thermal and guanidium melts, as well as laser T-jump kinetics. All experimental data is fitted by a global fitting model with two states per domain (U, N), plus a third state M to account for non-native states due to domain interactions present in all but the monomer. A detailed structural model is provided by coarse-grained simulated annealing using the AWSEM Hamiltonian. Tethered FiP35 WW domains with n = 2 and 3 domains are just slightly less stable than the monomer. The n = 4 oligomer is yet less stable, its expression yield is much lower than the monomer's, and depends on the purification tag used. The n = 5 plasmid did not express at all, indicating the sudden onset of aggregation past n = 4. Thus, tethered FiP35 has a critical nucleus size for inter-domain aggregation of n ≈ 4. According to our simulations, misfolded structures become increasingly prevalent as one proceeds from monomer to pentamer, with extended inter-domain beta sheets appearing first, then multi-sheet 'intramolecular amyloid' structures, and finally novel motifs containing alpha helices. We discuss the implications of our results for oligomeric aggregate formation and structure, transient aggregation of proteins whilst folding, as well as for protein evolution that starts with repeat proteins.


Assuntos
Proteínas/química , Cinética , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Proteínas/genética , Proteínas/metabolismo , Termodinâmica , Domínios WW
7.
BMC Bioinformatics ; 20(1): 455, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492097

RESUMO

BACKGROUND: Evolutionary information contained in the amino acid sequences of proteins specifies the biological function and fold, but exactly what information contained in the protein sequence drives both of these processes? Considerable progress has been made to answer this fundamental question, but it remains challenging to explore the potential space of cooperative interactions between amino acids. Statistical analysis plays a significant role in studying such interactions and its use has expanded in recent years to studies ranging from coevolution-guided rational protein design to protein folding in silico. RESULTS: Here we describe a computational tool named Sibe for use in studies of protein sequence, folding, and design using evolutionary coupling between amino acids as a driving factor. In this study, Sibe is used to identify positionally conserved couplings between pairwise amino acids and aid rational protein design. In this process, pairwise couplings are filtered according to the relative entropy computed from the positional conservations and grouped into several 'blocks', which could contribute to driving protein folding and design. A human ß2-adrenergic receptor (ß2AR) was used to demonstrate that those 'blocks' contribute the rational design for specifying functional residues. Sibe also provides folding modules based on both the positionally conserved couplings and well-established statistical potentials for simulating protein folding in silico and predicting tertiary structure. Our results show that statistically inferences of basic evolutionary principles, such as conservations and coupled-mutations, can be used to rapidly design a diverse set of proteins and study protein folding. CONCLUSIONS: The developed software Sibe provides a computational tool for systematical analysis from protein primary to its tertiary structure using the evolutionary couplings as a driving factor. Sibe, written in C++, accounts for compatibility with the 'big data' era in biological science, and it primarily focuses on protein sequence analysis, but it is also applicable to extend to other modeling and predictions of experimental measurements.


Assuntos
Biologia Computacional/métodos , Simulação por Computador , Engenharia de Proteínas , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , Entropia , Humanos , Mutação , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Análise de Sequência , Software
8.
Anticancer Res ; 39(9): 4637-4642, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519561

RESUMO

AIM: The aim of this study was to characterize the role of Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis chromosomal region gene 1 (AMMECR1) in human lung cancer cell lines. MATERIALS AND METHODS: AMMECR1 gene expression was evaluated in four lung cell lines, with A549 then selected for further in-depth examination. To characterize the role of AMMECR1, silencing was achieved utilizing lentivirus-mediated RNA interference, and confirmed by quantitative real-time polymerase chain reaction and western blotting. The impact of AMMECR1 silencing on cellular proliferation was assessed using Celigo-based and MTT assays. Apoptosis was determined using the annexin V-allophycocyanin single staining method. Cell-cycle arrest was assessed by flow cytometry. Finally, colony formation was assessed using Giemsa staining. RESULTS: In A549 cells, AMMECR1 silencing was found to significantly suppress cell proliferation, reduce colony formation, promote apoptosis, and arrest cells in the S and G2/M phases. CONCLUSION: AMMECR1 plays a critical role in cell proliferation, cell-cycle progression, and apoptosis of human lung cancer cells, and may serve as a potential therapeutic target for non-small-cell lung cancer.


Assuntos
Apoptose/genética , Ciclo Celular/genética , Neoplasias Pulmonares/genética , Proteínas/genética , Células A549 , Linhagem Celular Tumoral , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas/metabolismo , RNA Mensageiro/genética
9.
BMC Bioinformatics ; 20(1): 470, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31521111

RESUMO

BACKGROUND: Neurogenesis in the murine cerebral cortex involves the coordinated divisions of two main types of progenitor cells, whose numbers, division modes and cell cycle durations set up the final neuronal output. To understand the respective roles of these factors in the neurogenesis process, we combine experimental in vivo studies with mathematical modeling and numerical simulations of the dynamics of neural progenitor cells. A special focus is put on the population of intermediate progenitors (IPs), a transit amplifying progenitor type critically involved in the size of the final neuron pool. RESULTS: A multiscale formalism describing IP dynamics allows one to track the progression of cells along the subsequent phases of the cell cycle, as well as the temporal evolution of the different cell numbers. Our model takes into account the dividing apical progenitors (AP) engaged into neurogenesis, both neurogenic and proliferative IPs, and the newborn neurons. The transfer rates from one population to another are subject to the mode of division (proliferative, or neurogenic) and may be time-varying. The model outputs are successfully fitted to experimental cell numbers from mouse embryos at different stages of cortical development, taking into account IPs and neurons, in order to adjust the numerical parameters. We provide additional information on cell kinetics, such as the mitotic and S phase indexes, and neurogenic fraction. CONCLUSIONS: Applying the model to a mouse mutant for Ftm/Rpgrip1l, a gene involved in human ciliopathies with severe brain abnormalities, reveals a shortening of the neurogenic period associated with an increased influx of newborn IPs from apical progenitors at mid-neurogenesis. Our model can be used to study other mouse mutants with cortical neurogenesis defects and can be adapted to study the importance of progenitor dynamics in cortical evolution and human diseases.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Modelos Biológicos , Neurogênese , Animais , Ciclo Celular , Divisão Celular , Córtex Cerebral/fisiopatologia , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Proteínas/genética
10.
Phys Chem Chem Phys ; 21(37): 20678-20692, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31508628

RESUMO

In this work we present a high-throughput approach to the computation of absorption UV-Vis spectra tailored to mutagenesis studies. The scheme makes use of a single molecular dynamics trajectory of a reference (non-mutated) species. The shifts in absorption energy caused by a residue mutation are evaluated by building an effective potential of the environment and computing a correction term based on perturbation theory. The sampling is only performed in the phase space of the initial protein. We analyze the robustness of the method by comparing different approximations for the effective potential, the sampling of mutant residue geometries and observing the impact in the prediction of both bathocromic and hypsochromic shifts. As a test subject, we consider a red fluorescent protein variant with potential biotechnological applications.


Assuntos
Testes Genéticos/métodos , Luz , Proteínas/química , Proteínas/genética , Análise Espectral , Raios Ultravioleta , Simulação de Dinâmica Molecular , Mutação
11.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(9): 910-913, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31515788

RESUMO

OBJECTIVE: To explore the genetic basis for an infant featuring developmental delay, hand deformity and hypertonia of extremities. METHODS: Clinical data and peripheral blood samples of the proband and her parents were collected. Following DNA extraction, potential mutations were screened on an Ion PGM platform using a gene panel. Suspected mutation was verified by PCR and Sanger sequencing. RESULTS: A novel heterozygous nonsense mutation, c.2521C>T(p.R841X), was identified in the NIPBL gene. The mutation may cause premature termination of translation of the adhesion protein loading factor at 841st amino acids. The same mutation was not found in her parents and 931 healthy controls, and was absent from public databases including ExAC and 1000G. Bioinformatic analysis suggested the mutation to be disease causing. CONCLUSION: The c.2521C>T (p.R841X) mutation of the NIPBL gene probably underlies the Cornelia De Lange syndrome in the infant. Prenatal diagnosis may be provided to this family upon their subsequent pregnancy.


Assuntos
Síndrome de Lange/genética , Diagnóstico Pré-Natal , Proteínas/genética , Proteínas de Ciclo Celular , Síndrome de Lange/diagnóstico , Feminino , Heterozigoto , Humanos , Lactente , Mutação , Gravidez
12.
Eur. j. anat ; 23(5): 361-368, sept. 2019. ilus
Artigo em Inglês | IBECS | ID: ibc-183866

RESUMO

Peg10 (paternally expressed 10) is a retrotransposon-derived gene that is highly conserved across mammalian species. Peg10 is involved in cell proliferation and differentiation, and is essential for placenta formation in mice. Although a number of studies have examined Peg10 expression in the placenta, its cellular localization in the brain is still unclear. The function of Peg10 in the brain is also unknown. Here, we examined Peg10 distribution in the mouse brain. In situ hybridization revealed intense expression of the gene in the core region of the accumbens nucleus, lateral division of the bed nucleus of the stria terminalis, medial preoptic nucleus, paraventricular nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, premammillary nucleus, central amygdaloid nucleus and lateral parabrachial nucleus. Moderate to intense expression of Peg10 was also observed in monoaminergic nuclei such as the substantia nigra, dorsal raphe nucleus and locus coeruleus. These results suggest that Peg10 may play a role in motivational processes, emotional regulation, and autonomic functions in the brain. The findings also suggest that Peg10 may have contributed to the evolution of mammals, not only by participating in placenta formation, but also by regulating parental behavior and hormonal secretions necessary for maternal responsiveness


No disponible


Assuntos
Animais , Camundongos , Cérebro/anatomia & histologia , Hipotálamo/anatomia & histologia , Sistema Límbico/anatomia & histologia , Proteínas/genética , Hibridização in Situ Fluorescente/veterinária , Hipocampo/anatomia & histologia , Diencéfalo/anatomia & histologia
13.
Nat Chem Biol ; 15(9): 872-881, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406370

RESUMO

Hundreds of cellular proteins require iron cofactors for activity, and cells express systems for their assembly and distribution. Molecular details of the cytosolic iron pool used for iron cofactors are lacking, but iron chaperones of the poly(rC)-binding protein (PCBP) family play a key role in ferrous ion distribution. Here we show that, in cells and in vitro, PCBP1 coordinates iron via conserved cysteine and glutamate residues and a molecule of noncovalently bound glutathione (GSH). Proteomics analysis of PCBP1-interacting proteins identified BolA2, which functions, in complex with Glrx3, as a cytosolic [2Fe-2S] cluster chaperone. The Fe-GSH-bound form of PCBP1 complexes with cytosolic BolA2 via a bridging Fe ligand. Biochemical analysis of PCBP1 and BolA2, in cells and in vitro, indicates that PCBP1-Fe-GSH-BolA2 serves as an intermediate complex required for the assembly of [2Fe-2S] clusters on BolA2-Glrx3, thereby linking the ferrous iron and Fe-S distribution systems in cells.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas/metabolismo , Antibacterianos/farmacologia , Proteínas de Transporte , Citosol/metabolismo , Doxiciclina/farmacologia , Compostos Férricos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Proteínas/genética , Compostos de Amônio Quaternário/farmacologia
14.
Nucleic Acids Res ; 47(18): 9666-9684, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392335

RESUMO

Break induced replication (BIR) is a double strand break repair pathway that can promote genetic instabilities similar to those observed in cancer. Instead of a replication fork, BIR is driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced kataegis in human cancers, suggesting that BIR might represent an important source of these hyper-mutagenic events.


Assuntos
Cromossomos/genética , Citidina Desaminase/genética , Reparo do DNA/genética , Proteínas/genética , Recombinação Genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Humanos , Mutagênese/genética , Mutação , Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
15.
DNA Cell Biol ; 38(10): 1030-1039, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368785

RESUMO

Both endogenous and exogenous factors can cause DNA damage that compromises genomic integrity and cell viability. A proper DNA damage response (DDR) plays a role in maintaining genome stability and preventing tumorigenesis. DNA double-strand breaks (DSBs) are the most toxic DNA lesion, whose response is dominated by the ataxia-telangiectasia mutated (ATM) protein kinase. After being activated by the sensor Mre11-Rad50-Nbs1 (MRN) complex or acetyltransferase Tip60, ATM rapidly phosphorylates downstream targets to launch DDR signaling when DNA is damaged. However, the exact mechanism of DDR is complex and ambiguous. Ufmylation, one type of ubiquitin-like modification, proceeds mainly through a three-step enzymatic reaction to help ubiquitin-fold modifier 1 (Ufm1), attach to substrates with ubiquitin-like modifier-activating enzyme 5 (Uba5), Ufm1-conjugating enzyme 1 (Ufc1) and Ufm1-specific ligase 1 (Ufl1). Although ubiquitination is essential to the DSBs response, the potential function of ufmylation in DDR is largely unknown. Herein, we review the relationship between ufmylation and DDR to elucidate the function and mechanism of ufmylation in DDR, which would reveal the pathogenesis of some diseases and provide new guidance to create a therapeutic method.


Assuntos
Doenças Cardiovasculares/metabolismo , Quebras de DNA de Cadeia Dupla , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Esquizofrenia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Reparo do DNA , Genoma Humano , Instabilidade Genômica , Humanos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Proteínas/genética , Esquizofrenia/genética , Esquizofrenia/patologia , Transdução de Sinais , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
16.
J Chem Phys ; 151(4): 041101, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370538

RESUMO

We chemically characterize the symmetries underlying the exact solutions of a stochastic negatively self-regulating gene. The breaking of symmetry at a low molecular number causes three effects. Two branches of the solution exist, having high and low switching rates, such that the low switching rate branch approaches deterministic behavior and the high switching rate branch exhibits sub-Fano behavior. The average protein number differs from the deterministically expected value. Bimodal probability distributions appear as the protein number becomes a readout of the ON/OFF state of the gene.


Assuntos
Proteínas/genética , Cinética , Teoria Quântica , Soluções , Processos Estocásticos
17.
Medicine (Baltimore) ; 98(33): e16899, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31415434

RESUMO

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive multisystem disorder characterized by oculocutaneous albinism (OCA) and bleeding diathesis, although it displays both genetic and phenotypic heterogeneity. Several genetic subtypes of HPS have been identified in human; however, the characterizations of HPS type 4 (HPS-4) genotype and phenotype remain unclear. This study was aimed to identify gene mutation responsible for HPS-4 with pulmonary fibrosis (PF).Two Chinese siblings in their 50 s afflicted with OCA and progressive dyspnea were recruited and underwent clinical and genetic examinations. In both patients, chest high-resolution computerized tomography showed severe interstitial PF in bilateral lung fields, and the pulmonary function test indicated restrictive lung disease. A novel homozygous frameshift mutation (NM_022081: c.630dupC; p.A211fs) in the HPS4 gene was identified by whole-exome sequencing analysis followed by Sanger DNA sequencing, and it segregated with the phenotypes. The c.630dupC mutation was not found in unaffected healthy controls. The patients were considered as HPS-4 with interstitial PF and eventually died of respiratory failure.This is the first report on the genotype and clinical phenotype of HPS-4 in China. Our results demonstrate the association between a novel frameshift mutation in HPS4 and severe PF with poor prognosis in HPS is presented.


Assuntos
Mutação da Fase de Leitura , Síndrome de Hermanski-Pudlak/genética , Fibrose Pulmonar Idiopática/genética , Proteínas/genética , Adulto , China , Testes Genéticos , Humanos , Fibrose Pulmonar Idiopática/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , Irmãos
18.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(4): 1013-1019, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31418350

RESUMO

OBJECTIVE: To investigate the effect of silencing LNK gene on the expression of EPO and EPOR in acute myeloid leukemia cells (THP-1). METHODS: THP-1 cells were cultured. The lentivirus was used as a vector to silence the LNK gene stably. After 72 hours of infection, GFP expression level was detected by the fluorescent inverted microscopy. The lentiviral Infection efficiencies were monitored by flow cytometry. The LNK silencing effect was confirmed. The mRNA expressions of EPO and EPOR were detected by RT-PCR. The protein levels of LNK, EPO and EPOR were detected by Western blot. RESULTS: At the time-point of 72 hours after lentivirus infection, the expression level of GFP was above 85% detected by fluorescent inverted microscopy. The infection efficiency was above 99% by flow cytometry. mRNA expressions of LNK, EPO and EPOR in LNK silencing group were signifycantly lower than those in control group (P<0.05). The protein levels of LNK, EPO and EPOR in LNK silencing group were significantly lower than those in the control group (P<0.05). CONCLUSION: THP-1 cell line of LNK gene silencing has been successfully established,the LNK gene has been silenced, the expression of EPO and EPOR decrease, indicating that LNK may participate in the regulation of EPO and EPOR.


Assuntos
Proteínas/genética , Western Blotting , Eritropoetina , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Receptores da Eritropoetina , Células THP-1
19.
Genome Biol ; 20(1): 149, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366358

RESUMO

The human reference genome is still incomplete, especially for those population-specific or individual-specific regions, which may have important functions. Here, we developed a HUman Pan-genome ANalysis (HUPAN) system to build the human pan-genome. We applied it to 185 deep sequencing and 90 assembled Han Chinese genomes and detected 29.5 Mb novel genomic sequences and at least 188 novel protein-coding genes missing in the human reference genome (GRCh38). It can be an important resource for the human genome-related biomedical studies, such as cancer genome analysis. HUPAN is freely available at http://cgm.sjtu.edu.cn/hupan/ and https://github.com/SJTU-CGM/HUPAN .


Assuntos
Genoma Humano , Software , Grupo com Ancestrais do Continente Africano/genética , Grupo com Ancestrais do Continente Asiático/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas/genética , Análise de Sequência de DNA
20.
BMC Evol Biol ; 19(1): 158, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31362700

RESUMO

BACKGROUND: There is wide agreement that only a subset of the twenty standard amino acids existed prebiotically in sufficient concentrations to form functional polypeptides. We ask how this subset, postulated as {A,D,E,G,I,L,P,S,T,V}, could have formed structures stable enough to found metabolic pathways. Inspired by alphabet reduction experiments, we undertook a computational analysis to measure the structural coding behavior of sequences simplified by reduced alphabets. We sought to discern characteristics of the prebiotic set that would endow it with unique properties relevant to structure, stability, and folding. RESULTS: Drawing on a large dataset of single-domain proteins, we employed an information-theoretic measure to assess how well the prebiotic amino acid set preserves fold information against all other possible ten-amino acid sets. An extensive virtual mutagenesis procedure revealed that the prebiotic set excellently preserves sequence-dependent information regarding both backbone conformation and tertiary contact matrix of proteins. We observed that information retention is fold-class dependent: the prebiotic set sufficiently encodes the structure space of α/ß and α + ß folds, and to a lesser extent, of all-α and all-ß folds. The prebiotic set appeared insufficient to encode the small proteins. Assessing how well the prebiotic set discriminates native vs. incorrect sequence-structure matches, we found that α/ß and α + ß folds exhibit more pronounced energy gaps with the prebiotic set than with nearly all alternatives. CONCLUSIONS: The prebiotic set optimally encodes local backbone structures that appear in the folded environment and near-optimally encodes the tertiary contact matrix of extant proteins. The fold-class-specific patterns observed from our structural analysis confirm the postulated timeline of fold appearance in proteogenesis derived from proteomic sequence analyses. Polypeptides arising in a prebiotic environment will likely form α/ß and α + ß-like folds if any at all. We infer that the progressive expansion of the alphabet allowed the increased conformational stability and functional specificity of later folds, including all-α, all-ß, and small proteins. Our results suggest that prebiotic sequences are amenable to mutations that significantly lower native conformational energies and increase discrimination amidst incorrect folds. This property may have assisted the genesis of functional proto-enzymes prior to the expansion of the full amino acid alphabet.


Assuntos
Aminoácidos/metabolismo , Origem da Vida , Proteínas/química , Sequência de Aminoácidos , Método de Monte Carlo , Mutagênese/genética , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas/genética
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