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1.
Mol Biol (Mosk) ; 55(3): 412-421, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34097676

RESUMO

Molecular profiling of tumors may provide promising options for personalized treatment. We have examined the spectrum of germline and somatic mutations in 23 breast cancers (ВС) of various molecular subtypes, including tumors 1) with expression of estrogen, progesterone and/or epidermal growth factor receptor HER2/neu, and 2) with a triple negative phenotype. Genomic DNA specimens were isolated from archived tumor and normal tissue samples and subjected to targeted sequencing of the coding regions of 25 cancer-associated genes with a mean coverage of x 1000. In the triple negative subtype of ВС, the pathogenic germline mutations BRCA1 c.66_67delAG (185delAG) and BRCA1 c.3226_3227AG (3347delAG) were detected, while the germline mutation BRCA2 658_659del (886delGT) was found in patients with positive receptor staining. Mutations in BRCAl/2 were overrepresented by frequency (80%), pointing at common loss of heterozygosity affecting the normal allele. Somatic mutations in the TP53 gene were found in 7/10 (70%) patients with the triple negative subtype of ВС and in 3/13 (23%) in the group with positive receptor staining. Additionally, in both groups of patients, somatic mutations of the PTEN, MSH2, MSH6, and MUTYH genes were detected.


Assuntos
Neoplasias da Mama , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Feminino , Predisposição Genética para Doença , Células Germinativas , Mutação em Linhagem Germinativa , Humanos , Mutação
2.
Nat Commun ; 12(1): 3418, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103502

RESUMO

The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.


Assuntos
Cryptococcus/efeitos dos fármacos , Farmacorresistência Fúngica , Flucitosina/farmacologia , Cromossomos Fúngicos/genética , Células Clonais , Cryptococcus/genética , Cryptococcus/crescimento & desenvolvimento , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Duplicação Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Supressores , Variação Genética , Genoma Fúngico , Espaço Intracelular/metabolismo , Testes de Sensibilidade Microbiana , Mutação/genética , Reprodutibilidade dos Testes , Uridina Difosfato Ácido Glucurônico/metabolismo
3.
Nat Commun ; 12(1): 3431, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103499

RESUMO

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.


Assuntos
Genética Reversa , SARS-CoV-2/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlorocebus aethiops , Culicidae/virologia , Furina/metabolismo , Genoma Viral , Células HEK293 , Humanos , Camundongos , Mutação/genética , Células NIH 3T3 , Reação em Cadeia da Polimerase , Células RAW 264.7 , Receptores Virais/metabolismo , Células Vero , Proteínas Virais/química , Replicação Viral
4.
Nat Commun ; 12(1): 3457, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103505

RESUMO

Bacillus subtilis is a soil bacterium that is competent for natural transformation. Genetically distinct B. subtilis swarms form a boundary upon encounter, resulting in killing of one of the strains. This process is mediated by a fast-evolving kin discrimination (KD) system consisting of cellular attack and defence mechanisms. Here, we show that these swarm antagonisms promote transformation-mediated horizontal gene transfer between strains of low relatedness. Gene transfer between interacting non-kin strains is largely unidirectional, from killed cells of the donor strain to surviving cells of the recipient strain. It is associated with activation of a stress response mediated by sigma factor SigW in the donor cells, and induction of competence in the recipient strain. More closely related strains, which in theory would experience more efficient recombination due to increased sequence homology, do not upregulate transformation upon encounter. This result indicates that social interactions can override mechanistic barriers to horizontal gene transfer. We hypothesize that KD-mediated competence in response to the encounter of distinct neighbouring strains could maximize the probability of efficient incorporation of novel alleles and genes that have proved to function in a genomically and ecologically similar context.


Assuntos
Bacillus subtilis/genética , Transferência Genética Horizontal , Adaptação Fisiológica , Membrana Celular/metabolismo , DNA Bacteriano/genética , Genoma Bacteriano , Mutação/genética , Nucleotídeos/genética , Recombinação Genética/genética , Estresse Fisiológico , Transformação Genética , Regulação para Cima
5.
Nat Commun ; 12(1): 3440, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103529

RESUMO

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.


Assuntos
Fator de Iniciação 2B em Eucariotos/metabolismo , Estresse Fisiológico , Fosfatos Açúcares/metabolismo , Regulação Alostérica , Sítios de Ligação , Sequência Conservada , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/ultraestrutura , Evolução Molecular , Guanosina Difosfato/metabolismo , Células HEK293 , Humanos , Leucoencefalopatias/patologia , Ligantes , Metaboloma , Modelos Moleculares , Mutação/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Fosfatos Açúcares/química
6.
Nat Commun ; 12(1): 3444, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103528

RESUMO

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.


Assuntos
Arginina/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Metilação/efeitos dos fármacos , Camundongos Nus , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína-Arginina N-Metiltransferases/deficiência , Proteínas Proto-Oncogênicas c-akt/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Hematol Oncol ; 39 Suppl 1: 83-87, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34105816

RESUMO

Follicular lymphoma (FL) is a paradigm of tumors that require the interaction between tumor and microenvironment cells to foster their development from initial steps to progression. Recent large-scale genome studies have uncovered multiple genetic alterations of FL that influence the microenvironment in two main directions, promoting tumor cell survival and proliferation and facilitating their evasion from immune antitumor signals. Understanding the crosstalk between tumor B-cells and the microenvironment will facilitate the identification of vulnerabilities that may offer novel targets for treatment of the patients. This review highlights recent findings showing the effect of common genetic mutations modulating the cell composition of the tumor microenvironment and the novel therapeutic perspectives to target these interactions.


Assuntos
Linfócitos B , Proliferação de Células , Linfoma Folicular , Mutação , Microambiente Tumoral , Linfócitos B/imunologia , Linfócitos B/patologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Humanos , Linfoma Folicular/genética , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Linfoma Folicular/terapia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
8.
MAbs ; 13(1): 1919285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34074219

RESUMO

The newly emerging variants of SARS-CoV-2 from South Africa (B.1.351/501Y.V2) and Brazil (P.1/501Y.V3) have led to a higher infection rate and reinfection of COVID-19 patients. We found that the mutations K417N, E484K, and N501Y within the receptor-binding domains (RBDs) of the virus could confer ~2-fold higher binding affinity to the human receptor, angiotensin converting enzyme 2 (ACE2), compared to the wildtype RBD. The mutated version of RBD also completely abolishes the binding of bamlanivimab, a therapeutic antibody, in vitro. Detailed analysis shows that the ~10-fold gain of binding affinity between ACE2 and Y501-RBD, which also exits in the high contagious variant B.1.1.7/501Y.V1 from the United Kingdom, is compromised by additional introduction of the K417/N/T mutation. Mutation of E484K leads to the loss of bamlanivimab binding to RBD, although this mutation does not affect the binding between RBD and ACE2.


Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Antivirais/metabolismo , COVID-19/virologia , Mutação , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/diagnóstico , COVID-19/tratamento farmacológico , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética
9.
Nat Commun ; 12(1): 3258, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059679

RESUMO

Autophagy can selectively target protein aggregates, pathogens, and dysfunctional organelles for the lysosomal degradation. Aberrant regulation of autophagy promotes tumorigenesis, while it is far less clear whether and how tumor-specific alterations result in autophagic aberrance. To form a link between aberrant autophagy selectivity and human cancer, we establish a computational pipeline and prioritize 222 potential LIR (LC3-interacting region) motif-associated mutations (LAMs) in 148 proteins. We validate LAMs in multiple proteins including ATG4B, STBD1, EHMT2 and BRAF that impair their interactions with LC3 and autophagy activities. Using a combination of transcriptomic, metabolomic and additional experimental assays, we show that STBD1, a poorly-characterized protein, inhibits tumor growth via modulating glycogen autophagy, while a patient-derived W203C mutation on LIR abolishes its cancer inhibitory function. This work suggests that altered autophagy selectivity is a frequently-used mechanism by cancer cells to survive during various stresses, and provides a framework to discover additional autophagy-related pathways that influence carcinogenesis.


Assuntos
Carcinogênese/genética , Macroautofagia/genética , Proteínas de Membrana/genética , Modelos Genéticos , Proteínas Musculares/genética , Neoplasias/genética , Algoritmos , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Simulação por Computador , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Técnicas de Silenciamento de Genes , Glicogênio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Musculares/metabolismo , Mutação , Neoplasias/mortalidade , Neoplasias/patologia , Via de Pentose Fosfato/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteoma/genética , RNA-Seq , Análise Serial de Tecidos , Efeito Warburg em Oncologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072457

RESUMO

Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are substantially reduced. To better understand the structural basis for these functional differences, the 2.15 Å crystal structure of CYP3A7 has been solved. Comparative analysis of CYP3A enzymes shows that decreased structural plasticity rather than the active site microenvironment defines the ligand binding ability of CYP3A7. In particular, a rotameric switch in the gatekeeping amino acid F304 triggers local and long-range rearrangements that transmit to the F-G fragment and alter its interactions with the I-E-D-helical core, resulting in a more rigid structure. Elongation of the ß3-ß4 strands, H-bond linkage in the substrate channel, and steric constraints in the C-terminal loop further increase the active site rigidity and limit conformational ensemble. Collectively, these structural distinctions lower protein plasticity and change the heme environment, which, in turn, could impede the spin-state transition essential for optimal reactivity and oxidation of substrates.


Assuntos
Citocromo P-450 CYP3A/química , Ligantes , Sequência de Aminoácidos , Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Polimorfismo Genético , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072574

RESUMO

Neurofibromatosis (NF) is a neurocutaneous syndrome characterized by the development of tumors of the central or peripheral nervous system including the brain, spinal cord, organs, skin, and bones. There are three types of NF: NF1 accounting for 96% of all cases, NF2 in 3%, and schwannomatosis (SWN) in <1%. The NF1 gene is located on chromosome 17q11.2, which encodes for a tumor suppressor protein, neurofibromin, that functions as a negative regulator of Ras/MAPK and PI3K/mTOR signaling pathways. The NF2 gene is identified on chromosome 22q12, which encodes for merlin, a tumor suppressor protein related to ezrin-radixin-moesin that modulates the activity of PI3K/AKT, Raf/MEK/ERK, and mTOR signaling pathways. In contrast, molecular insights on the different forms of SWN remain unclear. Inactivating mutations in the tumor suppressor genes SMARCB1 and LZTR1 are considered responsible for a majority of cases. Recently, treatment strategies to target specific genetic or molecular events involved in their tumorigenesis are developed. This study discusses molecular pathways and related targeted therapies for NF1, NF2, and SWN and reviews recent clinical trials which involve NF patients.


Assuntos
Suscetibilidade a Doenças , Neurilemoma/etiologia , Neurofibromatoses/etiologia , Neurofibromatose 1/etiologia , Neurofibromatose 2/etiologia , Neoplasias Cutâneas/etiologia , Animais , Biomarcadores Tumorais , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Genes da Neurofibromatose 1 , Genes da Neurofibromatose 2 , Predisposição Genética para Doença , Humanos , Modelos Biológicos , Terapia de Alvo Molecular , Mutação , Neurilemoma/diagnóstico , Neurilemoma/terapia , Neurofibromatoses/diagnóstico , Neurofibromatoses/terapia , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/terapia , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/terapia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/terapia , Resultado do Tratamento
12.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072607

RESUMO

Glioblastoma (GBM) is an extremely aggressive tumor originating from neural stem cells of the central nervous system, which has high histopathological and genomic diversity. Mitochondria are cellular organelles associated with the regulation of cellular metabolism, redox signaling, energy generation, regulation of cell proliferation, and apoptosis. Accumulation of mutations in mitochondrial DNA (mtDNA) leads to mitochondrial dysfunction that plays an important role in GBM pathogenesis, favoring abnormal energy and reactive oxygen species production and resistance to apoptosis and to chemotherapeutic agents. The present review summarizes the known mitochondrial DNA alterations related to GBM, their cellular and metabolic consequences, and their association with diagnosis, prognosis, and treatment.


Assuntos
DNA Mitocondrial , Variação Genética , Glioblastoma/genética , Mitocôndrias/genética , Linhagem Celular Tumoral , Metabolismo Energético , Genoma Mitocondrial , Genômica/métodos , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Lipogênese/genética , Mitocôndrias/metabolismo , Mutação
13.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072612

RESUMO

The BAG proteins are a family of multi-functional co-chaperones. In plants, BAG proteins were found to play roles both in abiotic and biotic stress tolerance. However, the function of Arabidopsis BAG2 remains largely unknown, whereas BAG6 is required for plants' defense to pathogens, although it remains unknown whether BAG6 is involved in plants' tolerance to abiotic stresses. Here, we show that both BAG2 and BAG6 are expressed in various tissues and are upregulated by salt, mannitol, and heat treatments and by stress-related hormones including ABA, ethylene, and SA. Germination of bag2, bag6 and bag2 bag6 seeds is less sensitive to ABA compared to the wild type (WT), whereas BAG2 and BAG6 overexpression lines are hypersensitive to ABA. bag2, bag6, and bag2 bag6 plants show higher survival rates than WT in drought treatment but display lower survival rates in heat-stress treatment. Consistently, these mutants showed differential expression of several stress- and ABA-related genes such as RD29A, RD29B, NCED3 and ABI4 compared to the WT. Furthermore, these mutants exhibit lower levels of ROS after drought and ABA treatment but higher ROS accumulation after heat treatment than the WT. These results suggest that BAG2 and BAG6 are negatively involved in drought stress but play a positive role in heat stress in Arabidopsis.


Assuntos
Adaptação Fisiológica , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Estresse Fisiológico , Proteínas de Arabidopsis/metabolismo , Secas , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Desenvolvimento Vegetal/genética , Reguladores de Crescimento de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
14.
Viruses ; 13(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067878

RESUMO

COVID-19 is a highly infectious respiratory disease caused by the novel coronavirus SARS-CoV-2. It has become a global pandemic and its frequent mutations may pose new challenges for vaccine design. During viral infection, the Spike RBD of SARS-CoV-2 binds the human host cell receptor ACE2, enabling the virus to enter the host cell. Both the Spike and ACE2 are densely glycosylated, and it is unclear how distinctive glycan types may modulate the interaction of RBD and ACE2. Detailed understanding of these determinants is key for the development of novel therapeutic strategies. To this end, we perform extensive all-atom simulations of the (i) RBD-ACE2 complex without glycans, (ii) RBD-ACE2 with oligomannose MAN9 glycans in ACE2, and (iii) RBD-ACE2 with complex FA2 glycans in ACE2. These simulations identify the key residues at the RBD-ACE2 interface that form contacts with higher probabilities, thus providing a quantitative evaluation that complements recent structural studies. Notably, we find that this RBD-ACE2 contact signature is not altered by the presence of different glycoforms, suggesting that RBD-ACE2 interaction is robust. Applying our simulated results, we illustrate how the recently prevalent N501Y mutation may alter specific interactions with host ACE2 that facilitate the virus-host binding. Furthermore, our simulations reveal how the glycan on Asn90 of ACE2 can play a distinct role in the binding and unbinding of RBD. Finally, an energetics analysis shows that MAN9 glycans on ACE2 decrease RBD-ACE2 affinity, while FA2 glycans lead to enhanced binding of the complex. Together, our results provide a more comprehensive picture of the detailed interplay between virus and human receptor, which is much needed for the discovery of effective treatments that aim at modulating the physical-chemical properties of this virus.


Assuntos
Enzima de Conversão de Angiotensina 2/química , COVID-19/virologia , Polissacarídeos/química , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Sítios de Ligação , Glicosilação , Interações entre Hospedeiro e Microrganismos , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ligação Viral
15.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069029

RESUMO

The relationship between the two most prominent neuropathological hallmarks of Alzheimer's Disease (AD), extracellular amyloid-ß (Aß) deposits and intracellular accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFT), remains at present not fully understood. A large body of evidence places Aß upstream in the cascade of pathological events, triggering NFTs formation and the subsequent neuron loss. Extracellular Aß deposits were indeed causative of an increased tau phosphorylation and accumulation in several transgenic models but the contribution of soluble Aß peptides is still controversial. Among the different Aß variants, the N-terminally truncated peptide Aß4-42 is among the most abundant. To understand whether soluble Aß4-42 peptides impact the onset or extent of tau pathology, we have crossed the homozygous Tg4-42 mouse model of AD, exclusively expressing Aß4-42 peptides, with the PS19 (P301S) tau transgenic model. Behavioral assessment showed that the resulting double-transgenic line presented a partial worsening of motor performance and spatial memory deficits in the aged group. While an increased loss of distal CA1 pyramidal neurons was detected in young mice, no significant alterations in hippocampal tau phosphorylation were observed in immunohistochemical analyses.


Assuntos
Doença de Alzheimer/complicações , Peptídeos beta-Amiloides/metabolismo , Modelos Animais de Doenças , Transtornos da Memória/etiologia , Transtornos Motores/etiologia , Mutação , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Comportamento Animal , Feminino , Humanos , Masculino , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Motores/metabolismo , Transtornos Motores/patologia , Proteínas tau/metabolismo
16.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069057

RESUMO

Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.


Assuntos
Bacteriófago T7/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Mutação , Nucleotídeos/análise , Moldes Genéticos , Proteínas Virais/metabolismo , Sistema Livre de Células , RNA Polimerases Dirigidas por DNA/genética , Transcrição Genética , Proteínas Virais/genética
17.
Viruses ; 13(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071591

RESUMO

Several recently developed high-throughput techniques have changed the field of molecular virology. For example, proteomics studies reveal complete interactomes of a viral protein, genome-wide CRISPR knockout and activation screens probe the importance of every single human gene in aiding or fighting a virus, and ChIP-seq experiments reveal genome-wide epigenetic changes in response to infection. Deep mutational scanning is a relatively novel form of protein science which allows the in-depth functional analysis of every nucleotide within a viral gene or genome, revealing regions of importance, flexibility, and mutational potential. In this review, we discuss the application of this technique to RNA viruses including members of the Flaviviridae family, Influenza A Virus and Severe Acute Respiratory Syndrome Coronavirus 2. We also briefly discuss the reverse genetics systems which allow for analysis of viral replication cycles, next-generation sequencing technologies and the bioinformatics tools that facilitate this research.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mutação/genética , Vírus de RNA/genética , Análise de Sequência de RNA , Biologia Computacional , Biblioteca Gênica , Genoma Viral/genética , Vírus de RNA/classificação , Vírus de RNA/fisiologia , Genética Reversa , Proteínas Virais/genética
19.
BMC Pediatr ; 21(1): 259, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074250

RESUMO

BACKGROUND: Neonatal hyperbilirubinemia causing jaundice is common in East Asian population. Uridine diphosphate glucuronosyltransferase isoenzyme (UGT1A1) glucuronidates bilirubin and converts the toxic form of bilirubin to its nontoxic form. METHOD: A retrospective study was conducted to review clinical information of ABO hemolysis neonates (ABO HDN) admitted to the Department of Neonatology, referred for neonatal hyperbilirubinemia, in a large general hospital of southern China from 2011 to 2017. Variation status of UGT1A1 was determined by direct sequencing or genotype assays. RESULT: Sixty-nine ABO HDNs were included into the final analysis. UGT1A1 c.211 G > A mutation (UGT1A1*6, p.Arg71Gly, rs4148323) was significantly associated with the increased bilirubin level in ABO HDNs, after adjusted by age, sex and feeding method (P = 0.019 for TBIL, P = 0.02 for IBIL). Moreover, heterozygous and/or homozygous UGT1A1 mutations in the coding sequence region were significantly associated with the increased risk of developing hazardous hyperbilirubinemia (as defined by TSB > 427 umol/L) as compared those with a normal UGT1A1 genotype (ORadj = 9.16, 95%CI 1.99-42.08, P = 0.002) in the study cohort. CONCLUSION: UGT1A1 variant in coding region is actively involved in the pathogenesis of ABO hemolysis related neonatal hyperbilirubinemia. Genetic assessment of UGT1A1 may be useful for clinical diagnosis of neonatal unconjugated hyperbilirubinemia.


Assuntos
Hiperbilirrubinemia Neonatal , Bilirrubina , China , Glucuronosiltransferase/genética , Humanos , Hiperbilirrubinemia , Hiperbilirrubinemia Neonatal/genética , Recém-Nascido , Mutação , Estudos Retrospectivos
20.
BMC Pediatr ; 21(1): 256, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074259

RESUMO

BACKGROUND: Developmental and epileptic encephalopathies (DEEs) are a heterogeneous group of chronic encephalopathies characterized by epilepsy with comorbid intellectual disability that are frequently associated with de novo nonsynonymous coding variants in ion channels, cell-surface receptors, and other neuronally expressed genes. Mutations in TRPM3 were identified as the cause of DEE. We report a novel patient with DEE carrying a de novo missense mutation in TRPM3, p.(S1202T); this missense mutation has never been reported. CASE PRESENTATION: A 7-year and 2-month-old Chinese patient who had recurrent polymorphic seizures was clinically diagnosed with DEE. A de novo missense mutation in TRPM3, which has not yet been reported, was identified in this case. The patient had a clinical phenotype consistent with previous reports. CONCLUSIONS: These findings could expand the spectrum of TRPM3 mutations and might also support that de novo substitutions of TRPM3 are a cause of DEE.


Assuntos
Epilepsia , Deficiência Intelectual , Canais de Cátion TRPM , Criança , China , Epilepsia/genética , Humanos , Lactente , Deficiência Intelectual/genética , Mutação , Mutação de Sentido Incorreto , Fenótipo , Canais de Cátion TRPM/genética
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