RESUMO
CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.
Assuntos
Actinomycetales/enzimologia , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/química , DNA Viral/metabolismo , Multimerização Proteica , Imagem Individual de MoléculaRESUMO
Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.
Assuntos
Actinobacteria/genética , Actinobacteria/ultraestrutura , Sistemas CRISPR-Cas , Hibridização de Ácido Nucleico , Actinobacteria/química , Actinobacteria/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/ultraestrutura , Microscopia Crioeletrônica , Modelos Moleculares , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismoRESUMO
CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and â¼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA. PAPERCLIP.
Assuntos
Proteínas de Ligação a DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligação Proteica , Análise de Sequência de DNA/métodos , Sistemas CRISPR-Cas , Ensaio de Desvio de Mobilidade Eletroforética , Microscopia de Fluorescência , Motivos de NucleotídeosRESUMO
piRNA guides the action of PIWI proteins to silence deleterious transposons in animal reproductive tissues. Biogenesis of piRNA-induced silencing complex (piRISC) involves a multi-step process. In this issue, Matsumoto et al. report the first crystal structure of a PIWI-clade protein displaying a guide RNA, ready for action.
Assuntos
Proteínas Argonautas/genética , Proteínas de Drosophila/genética , Animais , Elementos de DNA Transponíveis , Drosophila melanogaster/genética , RNA Interferente Pequeno/genéticaRESUMO
Leading CRISPR-Cas technologies employ Cas9 and Cas12 enzymes that generate RNA-guided dsDNA breaks. Yet, the most abundant microbial adaptive immune systems, Type I CRISPRs, are under-exploited for eukaryotic applications. Here, we report the adoption of a minimal CRISPR-Cas3 from Neisseria lactamica (Nla) type I-C system to create targeted large deletions in the human genome. RNP delivery of its processive Cas3 nuclease and target recognition complex Cascade can confer â¼95% editing efficiency. Unexpectedly, NlaCascade assembly in bacteria requires internal translation of a hidden component Cas11 from within the cas8 gene. Furthermore, expressing a separately encoded NlaCas11 is the key to enable plasmid- and mRNA-based editing in human cells. Finally, we demonstrate that supplying cas11 is a universal strategy to systematically implement divergent I-C, I-D, and I-B CRISPR-Cas3 editors with compact sizes, distinct PAM preferences, and guide orthogonality. These findings greatly expand our ability to engineer long-range genome edits.
Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Deleção de Genes , Edição de Genes , Genoma Humano , Neisseria lactamica/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Células HEK293 , Células HeLa , Humanos , Neisseria lactamica/enzimologia , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.
Assuntos
Sistemas CRISPR-Cas/genética , Células-Tronco Embrionárias Humanas , RNA Guia de Cinetoplastídeos/genética , Deleção de Sequência/genética , Endonucleases/química , Endonucleases/genética , Escherichia coli/genética , Edição de Genes/métodos , Genoma Humano/genética , Genômica , Humanos , Ribonucleoproteínas/genéticaRESUMO
Short prokaryotic Ago accounts for most prokaryotic Argonaute proteins (pAgos) and is involved in defending bacteria against invading nucleic acids. Short pAgo associated with TIR-APAZ (SPARTA) has been shown to oligomerize and deplete NAD+ upon guide-mediated target DNA recognition. However, the molecular basis of SPARTA inhibition and activation remains unknown. In this study, we determined the cryogenic electron microscopy structures of Crenotalea thermophila SPARTA in its inhibited, transient and activated states. The SPARTA monomer is auto-inhibited by its acidic tail, which occupies the guide-target binding channel. Guide-mediated target binding expels this acidic tail and triggers substantial conformational changes to expose the Ago-Ago dimerization interface. As a result, SPARTA assembles into an active tetramer, where the four TIR domains are rearranged and packed to form NADase active sites. Together with biochemical evidence, our results provide a panoramic vision explaining SPARTA auto-inhibition and activation and expand understanding of pAgo-mediated bacterial defense systems.
Assuntos
Proteínas Argonautas , Bactérias , Proteínas Argonautas/genética , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Bactérias/genética , Células Procarióticas/metabolismo , DNA/genética , Ligação ProteicaRESUMO
Cephalotaxines harbor great medical potential, but their natural source, the endemic conifer Cephalotaxus is highly endangered, creating a conflict between biotechnological valorization and preservation of biodiversity. Here, we construct the whole biosynthetic pathway to the 1-phenethylisoquinoline scaffold, as first committed compound for phenylethylisoquinoline alkaloids (PIAs), combining metabolic modeling, and transcriptome mining of Cephalotaxus hainanensis to infer the biosynthesis for PIA precursor. We identify a novel protein, ChPSS, driving the Pictet-Spengler condensation and show that this enzyme represents the branching point where PIA biosynthesis diverges from the concurrent benzylisoquinoline-alkaloids pathway. We also pinpoint ChDBR as crucial step to form 4-hydroxydihydrocinnamaldehyde diverging from lignin biosynthesis. The elucidation of the early PIA pathway represents an important step toward microbe-based production of these pharmaceutically important alkaloids resolving the conflict between biotechnology and preservation of biodiversity.
Assuntos
Alcaloides , Benzilisoquinolinas , Cephalotaxus , Cephalotaxus/genética , BiotecnologiaRESUMO
Hematopoietic progenitor kinase 1 (HPK1), a member of mitogen-activated protein kinase kinase kinase kinase (MAP4K) family of Ste20 serine/threonine kinases, is a negative regulator of T-cell receptor (TCR) signaling. Inactivating HPK1 kinase has been reported to be sufficient to elicit antitumor immune response. Therefore, HPK1 has attracted much attention as a promising target for tumor immunotherapy. A few of HPK1 inhibitors have been reported, and none of them have been approved for clinical applications. Hence, more effective HPK1 inhibitors are needed. Herein, a series of structurally novel diaminotriazine carboxamides were rationally designed, synthesized and evaluated for their inhibitory activity against HPK1 kinase. Most of them exhibited potent inhibitory potency against HPK1 kinase. In particular, compound 15b showed more robust HPK1 inhibitory activity than that of 11d developed by Merck in kinase activity assay (IC50 = 3.1 and 8.2 nM, respectively). The significant inhibitory potency against SLP76 phosphorylation in Jurkat T cells further confirmed the efficacy of compound 15b. In human peripheral blood mononuclear cell (PBMC) functional assays, compound 15b more significantly induced the production of interleukin 2 (IL-2) and interferon γ (IFN-γ) relative to 11d. Furthermore, 15b alone or in combination with anti-PD-1 antibodies showed potent in vivo antitumor efficacy in MC38 tumor-bearing mice. Compound 15b represents a promising lead for the development of effective HPK1 small-molecule inhibitors.
Assuntos
Leucócitos Mononucleares , Transdução de Sinais , Animais , Humanos , Camundongos , Fosforilação , Células JurkatRESUMO
Endocrine therapy (ET) is a well-validated strategy for estrogen receptor α positive (ERα + ) breast cancer therapy. Despite the clinical success of current standard of care (SoC), endocrine-resistance inevitably emerges and remains a significant medical challenge. Herein, we describe the structural optimization and evaluation of a new series of selective estrogen receptor covalent antagonists (SERCAs) based on benzothiophene scaffold. Among them, compounds 15b and 39d were identified as two highly potent covalent antagonists, which exhibits superior antiproliferation activity than positive controls against MCF-7 cells and shows high selectivity over ERα negative (ERα-) cells. More importantly, their mode of covalent engagement at Cys530 residue was accurately illustrated by a cocrystal structure of 15b-bound ERαY537S (PDB ID: 7WNV) and intact mass spectrometry, respectively. Further in vivo studies demonstrated potent antitumor activity in MCF-7 xenograft mouse model and an improved safety profile. Collectively, these compounds could be promising candidates for future development of the next generation SERCAs for endocrine-resistant ERα + breast cancer.
Assuntos
Neoplasias da Mama , Antagonistas do Receptor de Estrogênio , Humanos , Camundongos , Animais , Feminino , Receptor alfa de Estrogênio , Receptores de Estrogênio , Cristalografia por Raios X , Neoplasias da Mama/tratamento farmacológico , Células MCF-7 , Antagonistas de EstrogêniosRESUMO
CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. The prespacer is bound by Cas1-Cas2 as a dual-forked DNA, and the terminal 3'-OH of each 3' overhang serves as an attacking nucleophile during integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. Here we present four structural snapshots from the type II-A system of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3' overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer integration process and the leader-proximal preference.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Enterococcus faecalis , Proteínas Associadas a CRISPR/química , DNA/química , DNA/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/imunologia , Escherichia coli/genética , Integrases/química , Integrases/metabolismo , Modelos MolecularesRESUMO
Casposase, a homolog of Cas1 integrase, is encoded by a superfamily of mobile genetic elements known as casposons. While family 2 casposase has been well documented in both function and structure, little is known about the other three casposase families. Here, we studied the family 1 casposase lacking the helix-turn-helix (HTH) domain from Candidatus Nitrosopumilus koreensis AR1 (Ca. N. koreensis). The determinants for integration by Ca. N. koreensis casposase were extensively investigated, and it was found that a 13-bp target site duplication (TSD) sequence, a minimal 3-bp leader and three different nucleotides of the TSD sequences are indispensable for target specific integration. Significantly, the casposase can site-specifically integrate a broad range of terminal inverted repeat (TIR)-derived oligonucleotides ranging from 7-nt to â¼4000-bp, and various oligonucleotides lacking the 5'-TTCTA-3' motif at the 3' end of TIR sequence can be integrated efficiently. Furthermore, similar to some Cas1 homologs, the casposase utilizes a 5'-ATAA-3' motif in the TSD as a molecular ruler to dictate nucleophilic attack at 9-bp downstream of the end of the ruler during the spacer-side integration. By characterizing the family 1 Ca. N. koreensis casposase, we have extended our understanding on mechanistic similarities and evolutionary connections between casposons and the adaptation elements of CRISPR-Cas immunity.
Assuntos
Proteínas Associadas a CRISPR/genética , Integrases/genética , Integrases/metabolismo , Sequências Repetidas Terminais/genética , Archaea/genética , Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , Sequências Hélice-Volta-Hélice/genética , Sequenciamento de Nucleotídeos em Larga Escala , Oligonucleotídeos/genéticaRESUMO
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and seriously threatened public health and safety. Despite COVID-19 vaccines being readily popularized worldwide, targeted therapeutic agents for the treatment of this disease remain very limited. Here, we studied the inhibitory activity of the scutellarein and its methylated derivatives against SARS-CoV-2 main protease (Mpro) by the fluorescence resonance energy transfer (FRET) assay. Among all the methylated derivatives we studied, 4'-O-methylscutellarein exhibited the most promising enzyme inhibitory activity in vitro, with the half-maximal inhibitory concentration value (IC50) of 0.40 ± 0.03 µM. Additionally, the mechanism of action of the hits was further characterized through enzyme kinetic studies and molecular docking. Overall, our results implied that 4'-O-methylscutellarein could be a primary lead compound with clinical potential for the development of inhibitors against the SARS-CoV-2 Mpro.
Assuntos
Alcaloides , Proteases 3C de Coronavírus , Indóis , SARS-CoV-2 , Inibidores de Protease Viral , Alcaloides/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Humanos , Indóis/farmacologia , Cinética , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Inibidores de Protease Viral/farmacologiaRESUMO
A glycosyl transferase, isolated from Angelica decursiva a medical herb rich in coumarin, shows C-glycosyl transferase activity by in vitro activity assay using 5,7-dihydroxycoumarin as substrate, producing a C-glycosylated product at position C'8 along with the main product at C'6 position. Catalytic promiscuity assay shows that AdCGT also displays O- or C-glycosylation activity to other coumarins and flavonoids. When phloretin and 2,4,6-trihydroxyacetophenone were fed as substrates, AdCGT catalyzed the formation of di-C-glycosides. Therefore, AdCGT is a multifunctional glycosyltransferase with a broad substrate acceptability. This work highlights the potential of AdCGT as a catalyst for glycosylation of coumarin and reveals a new regio-selective C-glycosyltransferase, providing a basis for exploring the mechanism of coumarin glycosylation.
Assuntos
Angelica , Angelica/química , Angelica/metabolismo , Biocatálise , Cumarínicos/química , Glicosídeos , Glicosiltransferases/metabolismoRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.
Assuntos
Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/química , DNA/metabolismo , Escherichia coli/química , RNA Bacteriano/química , Pareamento de Bases , Sequência de Bases , Cristalografia por Raios X , DNA Helicases/química , DNA Helicases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Motivos de Nucleotídeos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismoRESUMO
AIMS: Despite considerable therapeutic advances, there is still a dearth of evidence on the molecular determinants of cardiac hypertrophy that culminate in heart failure. Neuraminidases are a family of enzymes that catalyze the cleavage of terminal sialic acids from glycoproteins or glycolipids. This study sought to characterize the role of neuraminidases in pathological cardiac hypertrophy and identify pharmacological inhibitors targeting mammalian neuraminidases. METHODS AND RESULTS: Neuraminidase 1 (NEU1) was highly expressed in hypertrophic hearts of mice and rats, and this elevation was confirmed in patients with hypertrophic cardiomyopathy (n = 7) compared with healthy controls (n = 7). The increased NEU1 was mainly localized in cardiomyocytes by co-localization with cardiac troponin T. Cardiomyocyte-specific NEU1 deficiency alleviated hypertrophic phenotypes in response to transverse aortic constriction or isoproterenol hydrochloride infusion, while NEU1 overexpression exacerbated the development of cardiac hypertrophy. Mechanistically, co-immunoprecipitation coupled with mass spectrometry, chromatin immunoprecipitation, and luciferase assays demonstrated that NEU1 translocated into the nucleus and interacted with GATA4, leading to Foetal gene (Nppa and Nppb) expression. Virtual screening and experimental validation identified a novel compound C-09 from millions of compounds that showed favourable binding affinity to human NEU1 (KD = 0.38 µM) and effectively prevented the development of cardiac remodelling in cellular and animal models. Interestingly, anti-influenza drugs zanamivir and oseltamivir effectively inhibited mammalian NEU1 and showed new indications of cardio-protection. CONCLUSIONS: This work identifies NEU1 as a critical driver of cardiac hypertrophy and inhibition of NEU1 opens up an entirely new field of treatment for cardiovascular diseases.
Assuntos
Cardiomiopatia Hipertrófica , Insuficiência Cardíaca , Animais , Cardiomegalia , Humanos , Camundongos , Miócitos Cardíacos , Neuraminidase , RatosRESUMO
The COVID-19 pandemic is still affecting many people worldwide and causing a heavy burden to global health. To eliminate the disease, SARS-CoV-2, the virus responsible for the pandemic, can be targeted in several ways. One of them is to inhibit the 2'-O-methyltransferase (nsp16) enzyme that is crucial for effective translation of viral RNA and virus replication. For methylation of substrates, nsp16 utilizes S-adenosyl methionine (SAM). Binding of a small molecule in the protein site where SAM binds can disrupt the synthesis of viral proteins and, as a result, the replication of the virus. Here, we performed high-throughput docking into the SAM-binding site of nsp16 for almost 40 thousand structures, prepared for compounds from three libraries: Enamine Coronavirus Library, Enamine Nucleoside Mimetics Library, and Chemdiv Nucleoside Analogue Library. For the top scoring ligands, semi-empirical quantum-chemical calculations were performed, to better estimate protein-ligand binding enthalpy. Relying upon the calculated binding energies and predicted docking poses, we selected 21 compounds for experimental testing.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Metiltransferases/química , Simulação de Acoplamento Molecular , Pandemias , RNA Viral/genética , S-Adenosilmetionina , Proteínas não Estruturais Virais/metabolismoRESUMO
Gene knock-in refers to the insertion of exogenous functional genes into a target genome to achieve continuous expression. Currently, most knock-in tools are based on site-directed nucleases, which can induce double-strand breaks (DSBs) at the target, following which the designed donors carrying functional genes can be inserted via the endogenous gene repair pathway. The size of donor genes is limited by the characteristics of gene repair, and the DSBs induce risks like genotoxicity. New generation tools, such as prime editing, transposase, and integrase, can insert larger gene fragments while minimizing or eliminating the risk of DSBs, opening new avenues in the development of animal models and gene therapy. However, the elimination of off-target events and the production of delivery carriers with precise requirements remain challenging, restricting the application of the current knock-in treatments to mainly in vitro settings. Here, a comprehensive review of the knock-in tools that do not/minimally rely on DSBs and use other mechanisms is provided. Moreover, the challenges and recent advances of in vivo knock-in treatments in terms of the therapeutic process is discussed. Collectively, the new generation of DSBs-minimizing and large-fragment knock-in tools has revolutionized the field of gene editing, from basic research to clinical treatment.
Assuntos
Quebras de DNA de Cadeia Dupla , Técnicas de Introdução de Genes , Terapia Genética , Técnicas de Introdução de Genes/métodos , Animais , Humanos , Terapia Genética/métodos , Edição de Genes/métodosRESUMO
Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a "negative cooperativity" mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes.
Assuntos
Multimerização Proteica , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Peptídeos/química , Peptídeos/metabolismo , Modelos Moleculares , Alanina/química , Alanina/metabolismo , Alanina/análogos & derivados , Domínios Proteicos , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/química , Processamento de Proteína Pós-Traducional , Ligação Proteica , Ligases/metabolismo , Ligases/química , SulfetosRESUMO
Hepatocellular carcinoma (HCC) is a prevalent and lethal malignancy worldwide. The MET gene, which encodes receptor tyrosine kinase c-Met, is aberrantly activated in various solid tumors, including non-small cell lung cancer and HCC. In this study, we identified a novel c-Met inhibitor 54 by virtual screening and structural optimization. Compound 54 showed potent c-Met inhibition with an IC50 value of 0.45 ± 0.06 nM. It also exhibited high selectivity among 370 kinases and potent anti-proliferative activity against MET-amplified HCC cells. Moreover, compound 54 displayed significant anti-tumor efficacy in vivo, making it a potential candidate for HCC treatment in future studies.