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1.
Nature ; 630(8016): 484-492, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38811729

RESUMO

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Modelos Moleculares , Domínios Proteicos , Loci Gênicos/genética , Clivagem do DNA , Bactérias/virologia , Bactérias/genética , Bactérias/imunologia , Bacteriófagos/genética , Edição de Genes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-38450723

RESUMO

BACKGROUND: The effect of a healthy lifestyle on dementia associated with multimorbidity is not well understood. Our objective is to examine whether the adoption of a healthy lifestyle could potentially reduce the elevated risk of dementia in individuals with and without multimorbidity. METHODS: We utilized data from the UK Biobank cohort. A comprehensive healthy lifestyle score, ranging from 0 to 6, was generated. Cox proportional hazards models were used to examine the associations between multimorbidity, the healthy lifestyle score, and the incidence risk of dementia. RESULTS: Over a median follow-up period of 12.5 years, 5 852 all-cause dementia were recorded. Multimorbidity including cardiovascular, metabolic, neuropsychiatric, and inflammation-related diseases was associated with a higher risk of subsequent dementia. Each additional chronic disease was associated with a hazard ratio (HR) of 1.38 (95% CI: 1.33, 1.44). Compared to individuals without multimorbidity and a healthy lifestyle score of 5-6, patients with multimorbidity and a lifestyle score of 0-1 had a significantly higher risk of dementia (HR: 3.13; 95% CI: 2.64, 3.72), but the risk was markedly attenuated among those with multimorbidity and a lifestyle score of 5-6. Among patients with 3 or more diseases, the HR for dementia was 0.53 (95%CI: 0.42, 0.68) when comparing a lifestyle score of 5-6 to 0-1. And we observed more pronounced association between them among people younger than 60 years old. CONCLUSIONS: Adherence to a combination of healthy lifestyle factors, especially at a young age, was associated with a significantly lower risk of dementia among participants with multimorbidity.


Assuntos
Demência , Multimorbidade , Humanos , Estudos Prospectivos , Fatores de Risco , Estilo de Vida , Estilo de Vida Saudável , Demência/epidemiologia , Demência/etiologia
3.
Science ; 383(6682): eadh4859, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38301022

RESUMO

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.


Assuntos
Clivagem do DNA , Endonucleases , RNA Catalítico , Animais , Microscopia Crioeletrônica , Endonucleases/química , Endonucleases/genética , Hidrólise , Íntrons , Conformação de Ácido Nucleico , Splicing de RNA , RNA Catalítico/química , RNA Catalítico/genética
4.
Cell Res ; 33(3): 229-244, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36650285

RESUMO

CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (~860 aa), from the environmental metagenome. Complexed with a large guide RNA (~170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5' C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a 'bracelet' architecture of Casπ effector encircling the DNA target at 3.4 Å resolution, substantially different from the canonical 'two-lobe' architectures of Cas12 and Cas9 nucleases. The large guide RNA serves as a 'two-arm' scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulation.


Assuntos
DNA , Edição de Genes , DNA/genética , Endonucleases/genética , Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas
5.
Mol Cell ; 82(6): 1199-1209.e6, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35219382

RESUMO

A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.


Assuntos
Edição de Genes , RNA Guia de Cinetoplastídeos , Animais , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Mamíferos/metabolismo , RNA/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
6.
J Biomol Struct Dyn ; 36(9): 2424-2435, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28714799

RESUMO

Myeloid cell leukemia-1 (Mcl-1) has been a validated and attractive target for cancer therapy. Over-expression of Mcl-1 in many cancers allows cancer cells to evade apoptosis and contributes to the resistance to current chemotherapeutics. Here, we identified new Mcl-1 inhibitors using a multi-step virtual screening approach. First, based on two different ligand-receptor complexes, 20 pharmacophore models were established by simultaneously using 'Receptor-Ligand Pharmacophore Generation' method and manual build feature method, and then carefully validated by a test database. Then, pharmacophore-based virtual screening (PB-VS) could be performed by using the 20 pharmacophore models. In addition, docking study was used to predict the possible binding poses of compounds, and the docking parameters were optimized before performing docking-based virtual screening (DB-VS). Moreover, a 3D QSAR model was established by applying the 55 aligned Mcl-1 inhibitors. The 55 inhibitors sharing the same scaffold were docked into the Mcl-1 active site before alignment, then the inhibitors with possible binding conformations were aligned. For the training set, the 3D QSAR model gave a correlation coefficient r2 of 0.996; for the test set, the correlation coefficient r2 was 0.812. Therefore, the developed 3D QSAR model was a good model, which could be applied for carrying out 3D QSAR-based virtual screening (QSARD-VS). After the above three virtual screening methods orderly filtering, 23 potential inhibitors with novel scaffolds were identified. Furthermore, we have discussed in detail the mapping results of two potent compounds onto pharmacophore models, 3D QSAR model, and the interactions between the compounds and active site residues.


Assuntos
Antineoplásicos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes , Fluxo de Trabalho
7.
Chem Biodivers ; 9(12): 2754-60, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23255445

RESUMO

Two new labdane diterpenoids, namely hedycoronals A and B (1 and 2, resp.), were isolated from the rhizomes of Hedychium coronarium, together with eight known diterpenoids, 4-11, and a known diarylheptanoid, 3. The structures of 1 and 2 were established by detailed interpretation of their 1D- and 2D-NMR spectra and HR-ESI-MS data. Inhibitory activities against human umbilical vein endothelial cells (HUMECs) proliferation and cytotoxic activities against four cancer cell lines were assessed for all the isolates. Most of these metabolites showed moderate or potent cytotoxic activities against four cancer cell lines. Moreover, compounds 3 and 8 exhibited promising inhibitory activities against HUMECs with the IC(50) values of 6.4 to 3.3 µM.


Assuntos
Diarileptanoides/química , Diterpenos/química , Zingiberaceae/química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Diarileptanoides/farmacologia , Diarileptanoides/toxicidade , Diterpenos/farmacologia , Diterpenos/toxicidade , Células Endoteliais da Veia Umbilical Humana , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Rizoma/química
8.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 19(2): 455-8, 2011 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-21518507

RESUMO

The aim of this study was to investigate the expression and immunologic regulation function of interleukin-23 and its related cytokines in chronic idiopathic thrombocytopenic purpura (ITP) patients. Levels of cytokines in peripheral blood mononuclear cells (PBMNC) were detected by reverse-transcription real-time polymerase chain reaction in 30 patients with chronic ITP and 15 healthy volunteers. The quantity of IL-23, IL-12, IL-17 in serum was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that low detectable mRNA levels of IL-23p19, IL-12p35, IL-27 and IL-12p40 were found in all patients and healthy persons. Trace of IL-17 mRNA were expressed in PBMNC of part of patients and normal controls. Levels of IL-12p35, IL-27, IL-17 mRNA between healthy persons and chronic ITP patients were not statistically different. Compared with normal controls, patients showed the lower expression levels of IL-23p19 and IL-12p40 mRNA (p < 0.01). The IL-12 levels of chronic ITP patients were significantly higher than that of normal controls (p < 0.01). The IL-23 and IL-17 levels of chronic ITP patients were same to that of normal controls. It is concluded that the imbalance of T cell subsets in ITP patients mainly associated with IL-12, but not with IL-23 and IL-17.


Assuntos
Interleucina-12/metabolismo , Interleucina-23/metabolismo , Púrpura Trombocitopênica Idiopática/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Doença Crônica , Feminino , Humanos , Subunidade p35 da Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-17/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto Jovem
9.
J Pharm Anal ; 1(3): 197-202, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29403699

RESUMO

Euphorbia lathyris (Caper spurge) is a toxic and potent Chinese materia medica (T/PCMM). This study sought a method for identifying five diterpenoids (Euphorbia factors L1-L3, L7a and L8) with the spectra of UV and mass, quantifying three diterpenoids L1, L2, and L8 in crude extracts of unprocessed and processed E. lathyris seeds by liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS). The analysis was achieved on an Agilent Eclipse XDB-C18 column (4.6 mm×150 mm i.d., 5 µm) with an isocratic elution with a mobile phase consisting of water and acetonitrile at a flow rate of 0.25 mL/min at column temperature of 30 °C and UV detection was set at 272 nm. An ESI source was used with a positive ionization mode. The calibration curve was linear in the ranges of 9.9-79 µg/mL for Euphorbia factor L1, 3.8-30.5 µg/mL for Euphorbia factor L2, and 1.0-20.6 µg/mL for Euphorbia factor L8. The average recoveries (n=6) of three diterpenoids were 98.39%, 91.10% and 96.94%, respectively, with RSD of 2.5%, 2.4% and 2.1%, respectively. The contents of the three diterpenoids in processed E. lathyris seeds were 3.435, 1.367 and 0.286 mg/g, respectively, which decreased more sharply than those in unprocessed E. lathyris seeds which were 4.915, 1.944 and 0.425 mg/g, respectively. The method is simple, accurate, reliable and reproducible, and it can be applied to control the quality of unprocessed and processed E. lathyris seeds.

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