RESUMO
DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.
Assuntos
Microscopia Crioeletrônica , DNA Helicases , DNA Polimerase teta , DNA Polimerase Dirigida por DNA , Humanos , DNA Helicases/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/antagonistas & inibidores , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/química , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/química , Piperazinas/farmacologia , Piperazinas/química , Linhagem Celular Tumoral , Ftalazinas/farmacologia , Ftalazinas/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Modelos Moleculares , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Ligação ProteicaRESUMO
DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Polθ) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Polθ is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Polθ-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Polθ helicase domain (Polθ-hel) in complex with DNA containing 3'-overhang. The structures reveal the sequential conformations adopted by Polθ-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Polθ-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Polθ-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Polθ-hel.
RESUMO
Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-ß (Aß) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aß42 (a-Aß42) and solid insoluble form s-Aß42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aß42 for AD treatment.
Assuntos
Peptídeos beta-Amiloides , Proteólise , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Humanos , Proteólise/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Linhagem Celular TumoralRESUMO
APOBEC3G (A3G) belongs to the AID/APOBEC cytidine deaminase family and is essential for antiviral immunity. It contains two zinc-coordinated cytidine-deaminase (CD) domains. The N-terminal CD1 domain is non-catalytic but has a strong affinity for nucleic acids, whereas the C-terminal CD2 domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains in DNA binding and editing is not fully understood. Here, our studies on rhesus macaque A3G (rA3G) show that the DNA editing function in linear and hairpin loop DNA is greatly enhanced by AA or GA dinucleotide motifs present downstream (in the 3'-direction) but not upstream (in the 5'-direction) of the target-C editing sites. The effective distance between AA/GA and the target-C sites depends on the local DNA secondary structure. We present two co-crystal structures of rA3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic CD1 domain in capturing AA/GA DNA and explaining our biochemical observations. Our structural and biochemical findings elucidate the molecular mechanism underlying the cooperative function between the non-catalytic and the catalytic domains of A3G, which is critical for its antiviral role and its contribution to genome mutations in cancer.
RESUMO
Metastasis, which is the spread of cancer cells into distant organs, is a critical determinant of prognosis in patients with cancer, and blood vessels are the major route for cancer cells to spread systemically. Extravasation is a critical process for the hematogenous metastasis; however, its underlying molecular mechanisms remain poorly understood. Here, we identified that senescent ECs highly express C-type lectin domain family 1 member B (CLEC-1b), and that endothelial CLEC-1b inhibits the hematogenous metastasis of a certain type of cancer. CLEC-1b expression was enhanced in ECs isolated from aged mice, senescent cultured human ECs, and ECs of aged human. CLEC-1b overexpression in ECs prevented the disruption of endothelial integrity, and inhibited the transendothelial migration of cancer cells expressing podoplanin (PDPN), a ligand for CLEC-1b. Notably, target activation of CLEC-1b in ECs decreased the hematogenous metastasis in the lungs by cancer cells expressing PDPN in mice. Our data reveal the protective role of endothelial CLEC-1b against cancer hematogenous metastasis. Considering the high CLEC-1b expression in senescent ECs, EC senescence may play a beneficial role with respect to the cancer hematogenous metastasis.
Assuntos
Lectinas Tipo C , Neoplasias , Idoso , Animais , Humanos , Camundongos , Plaquetas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Migração Transendotelial e TransepitelialRESUMO
During clozapine initiation, titration speed and concomitant valproate administration have been reported as risk factors for clozapine-induced fever and myocarditis. We tested the risk of concomitant valproate administration by stratifying patients according to titration rate. Concomitant valproate use was only associated with increased inflammatory adverse events in the slower titration group. The frequency of inflammatory adverse events was approximately 30 % during faster titration, regardless of concomitant valproate administration. However, the faster titration group with valproate had a higher frequency of severe adverse effects such as myocarditis. Clinicians should avoid concomitant valproate administration during clozapine initiation, regardless of titration rate.
Assuntos
Antipsicóticos , Clozapina , Miocardite , Esquizofrenia , Humanos , Clozapina/efeitos adversos , Esquizofrenia/tratamento farmacológico , Ácido Valproico/efeitos adversos , Antipsicóticos/efeitos adversos , Miocardite/induzido quimicamente , Japão , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológicoRESUMO
SARS-CoV-2 non-structural protein 15 (Nsp15) is critical for productive viral replication and evasion of host immunity. The uridine-specific endoribonuclease activity of Nsp15 mediates the cleavage of the polyuridine [poly(U)] tract of the negative-strand coronavirus genome to minimize the formation of dsRNA that activates the host antiviral interferon signaling. However, the molecular basis for the recognition and cleavage of the poly(U) tract by Nsp15 is incompletely understood. Here, we present cryogenic electron microscopy (cryoEM) structures of SARS-CoV-2 Nsp15 bound to viral replication intermediate dsRNA containing poly(U) tract at 2.7-3.3 Å resolution. The structures reveal one copy of dsRNA binds to the sidewall of an Nsp15 homohexamer, spanning three subunits in two distinct binding states. The target uracil is dislodged from the base-pairing of the dsRNA by amino acid residues W332 and M330 of Nsp15, and the dislodged base is entrapped at the endonuclease active site center. Up to 20 A/U base pairs are anchored on the Nsp15 hexamer, which explains the basis for a substantially shortened poly(U) sequence in the negative strand coronavirus genome compared to the long poly(A) tail in its positive strand. Our results provide mechanistic insights into the unique immune evasion strategy employed by coronavirus Nsp15.
RESUMO
Clozapine-induced fever marks the beginning of its inflammatory and potentially life-threatening adverse effects, such as myocarditis. We retrospectively analyzed the correlation between clozapine titration rate and fever onset date in 254 Japanese patients, including 55 with treatment-resistant schizophrenia who developed clozapine-induced fever. Pearson's product-moment correlation indicated a significant delay in the fever onset date with slower titration. Most fever onset cases occurred within 4 weeks, even with slow titration. Therefore, clinicians should remain vigilant in monitoring clozapine-induced fever within 4 weeks of clozapine initiation, regardless of the titration rate.
RESUMO
Human APOBEC3 (A3) cytidine deaminases are antiviral factors that are particularly potent against retroviruses. As a countermeasure, HIV-1 uses a viral infectivity factor (Vif) to target specific human A3s for proteasomal degradation. Vif recruits cellular transcription cofactor CBF-ß and Cullin-5 (CUL5) RING E3 ubiquitin ligase to bind different A3s distinctively, but how this is accomplished remains unclear in the absence of the atomic structure of the complex. Here, we present the cryo-EM structures of HIV-1 Vif in complex with human A3H, CBF-ß and components of CUL5 ubiquitin ligase (CUL5, ELOB, and ELOC). Vif nucleates the entire complex by directly binding four human proteins, A3H, CBF-ß, CUL5, and ELOC. The structures reveal a large interface area between A3H and Vif, primarily mediated by an α-helical side of A3H and a five-stranded ß-sheet of Vif. This A3H-Vif interface unveils the basis for sensitivity-modulating polymorphism of both proteins, including a previously reported gain-of-function mutation in Vif isolated from HIV/AIDS patients. Our structural and functional results provide insights into the remarkable interplay between HIV and humans and would inform development efforts for anti-HIV therapeutics.
Assuntos
Síndrome da Imunodeficiência Adquirida , HIV-1 , Humanos , Ubiquitina-Proteína Ligases/genética , Antivirais , Citidina Desaminase , Proteínas Culina/genética , AminoidrolasesRESUMO
BACKGROUND: Higher frequencies of inflammatory adverse effects of clozapine have been reported in Japan. As the international titration protocol for Asians has set slower dose titration than the Japanese package insert, we hypothesized that a dose titration speed slower than the recommendation of the guideline would be associated with fewer inflammatory-related adverse events. METHODS: The medical records of all 272 patients who were first started on clozapine at seven hospitals between 2009 and 2023 were studied retrospectively. Of those, 241 were included in the analysis. The patients were divided into two groups regarding whether the titration speed was faster or slower than the guideline for Asians. The incidence of inflammatory adverse events with clozapine was compared between the groups. RESULTS: The frequency of inflammatory adverse events was 34 % (37/110) in the faster titration group and 13 % (17/131) in the slower titration group, and a significant difference was observed by Fisher exact test (odds ratio 3.38; 95 % confidence interval 1.71-6.91; p < 0.001). Serious adverse effects, fever for more than five days, and clozapine discontinuation were significantly more frequent in the faster titration group. Logistic regression analysis indicated significantly more inflammatory adverse events in the faster titration group (adjusted odds ratio 4.01; 95 % confidence interval 2.02-7.87; p < 0.001) considering age, sex, body mass index, concomitant valproic acid, and smoking as confounding factors. CONCLUSION: Clozapine-induced inflammatory adverse events were less frequent in Japanese individuals when a titration rate was more gradual than the protocol recommended in the Japanese package insert.
RESUMO
Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral infectivity factor (Vif) breaches this host A3G immunity by hijacking a cellular E3 ubiquitin ligase complex to target A3G for ubiquitination and degradation. The molecular mechanism of A3G targeting by Vif-E3 ligase is unknown, limiting the antiviral efforts targeting this host-pathogen interaction crucial for HIV-1 infection. Here, we report the cryo-electron microscopy structures of A3G bound to HIV-1 Vif in complex with T cell transcription cofactor CBF-ß and multiple components of the Cullin-5 RING E3 ubiquitin ligase. The structures reveal unexpected RNA-mediated interactions of Vif with A3G primarily through A3G's noncatalytic domain, while A3G's catalytic domain is poised for ubiquitin transfer. These structures elucidate the molecular mechanism by which HIV-1 Vif hijacks the host ubiquitin ligase to specifically target A3G to establish infection and offer structural information for the rational development of antiretroviral therapeutics.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Microscopia Crioeletrônica , Ubiquitina/metabolismo , Ligação Proteica , Desaminase APOBEC-3G/genética , Desaminase APOBEC-3G/metabolismoRESUMO
BACKGROUND: Although drug-coated balloon (DCB)-based stent-less percutaneous coronary intervention (PCI) for de-novo lesions has attracted more attention, outcomes of the DCB procedure for hemodialysis (HD) patients are reported to be inferior to those for non-HD patients, similarly to drug-eluting stent (DES). Recent several reports have shown that rotational atherectomy (RA) followed by DCB treatment (RA/DCB) could be an option of revascularization strategy particularly for calcified de-novo lesions even in the new-generation DES era; however, efficacy of the RA/DCB procedure for HD patients remains unclear. METHODS: A total of 47 consecutive cases (53 lesions) undergoing RA/DCB for de-novo lesions were enrolled. According to the presence/absence of HD at baseline, the 47 cases were divided into the HD cases (N.=16) and the non-HD cases (N.=31), and the 53 lesions were divided into the HD lesions (N.=20) and the non-HD lesions (N.=33). RESULTS: The HD cases had a significantly lower prevalence of dyslipidemia and smoking than the non-HD cases. Final RA burr size, DCB diameter used, and angiographic success rate of PCI did not significantly differ between the 2 groups. Preprocedural, post-procedural, and follow-up QCA parameters were also similar between the 2 groups. Twelve-month clinical outcomes were comparable between the 2 groups. CONCLUSIONS: Mid-term outcomes of stent-less PCI using RA/DCB for de-novo lesions in HD patients might be comparable to those in non-HD patients, suggesting efficacy of pretreatment of RA prior to DCB treatment in HD patients.
Assuntos
Angioplastia Coronária com Balão , Aterectomia Coronária , Doença da Artéria Coronariana , Stents Farmacológicos , Intervenção Coronária Percutânea , Humanos , Aterectomia Coronária/efeitos adversos , Aterectomia Coronária/métodos , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/cirurgia , Intervenção Coronária Percutânea/efeitos adversos , Angioplastia Coronária com Balão/efeitos adversos , Angioplastia Coronária com Balão/métodos , Stents Farmacológicos/efeitos adversos , Resultado do Tratamento , Angiografia Coronária/métodos , Stents , Diálise RenalRESUMO
BACKGROUND: Several recent reports have shown that a stentless interventional procedure using rotational atherectomy followed by drug-coated balloon (DCB) treatment (RA/DCB) is a potent revascularization therapy for calcified de novo lesions even in the new-generation drug-eluting stent era; however, the role of the RA/DCB procedure for noncalcified de novo lesions remains unclear. METHODS: A total of 47 consecutive patients (53 lesions) who underwent RA/DCB for coronary de novo lesions were enrolled. According to the presence or absence of severe calcification at target lesions on fluoroscopy, the 47 patients were divided into the noncalcified cases (n = 12) and the calcified cases (n = 35), and the 53 lesions were divided into the noncalcified lesions (n = 14) and the calcified lesions (n = 39). RESULTS: The noncalcified cases tended to have a higher frequency of bleeding risk and had a significantly lower prevalence of dual antiplatelet therapy compared with the calcified cases. The main lesion-specific factors for the RA/DCB procedure among the noncalcified lesions were presence of left circumflex coronary artery ostial lesion. The final burr size, DCB diameter used, and angiographic success rate did not significantly differ between the 2 groups. The noncalcified lesions had a larger reference diameter and a shorter lesion length than the calcified lesions, whereas acute gain and late lumen loss did not differ between the 2 groups. Nine-month clinical outcomes were comparable between the 2 groups. CONCLUSIONS: Under drug-eluting stent-unsuitable clinical or lesion conditions, acute and midterm outcomes of RA/DCB for noncalcified de novo lesions might be comparable with those for calcified de novo lesions.
CONTEXTE: Plusieurs rapports récents ont montré qu'une revascularisation sans endoprothèse effectuée par athérectomie rotationnelle (AR) suivie d'un traitement par ballonnet médicamenté (BM) constitue une méthode efficace pour traiter les nouvelles lésions calcifiées, même à l'ère des endoprothèses médicamentées de nouvelle génération; on ne connaît toutefois pas bien l'utilité de l'intervention par AR et BM en cas de nouvelles lésions non calcifiées. MÉTHODOLOGIE: Au total, 47 patients consécutifs (53 lésions) ayant subi une intervention par AR et BM pour traiter de nouvelles lésions coronariennes ont été admis dans l'étude. Ces 47 patients ont été répartis en deux groupes, en fonction de l'absence (n = 12) ou de la présence (n = 35) de lésions . cibles sévèrement calcifiées observées à la fluoroscopie. Les 53 lésions ont aussi été réparties en deux groupes : lésions non calcifiées (n = 14) et lésions calcifiées (n = 39). RÉSULTATS: Les patients n'ayant pas de lésion calcifiée étaient généralement plus susceptibles de présenter des saignements et significativement moins nombreux à être sous bithérapie antiplaquettaire, comparativement aux patients ayant des lésions calcifiées. Dans le cas des lésions non calcifiées, la principale caractéristique justifiant une AR et un traitement par BM était la présence d'une lésion ostiale du rameau circonflexe de l'artère coronaire gauche. La taille de la dernière fraise utilisée, le diamètre du BM utilisé et le taux de réussite objectivée par angiographie étaient comparables dans les deux groupes. Les lésions non calcifiées avaient un diamètre de référence plus grand et étaient plus courtes que les lésions calcifiées, tandis que le gain aigu et la perte luminale tardive étaient similaires dans les deux groupes. Les résultats cliniques à neuf mois étaient aussi similaires dans les deux groupes. CONCLUSIONS: Lorsque les conditions cliniques ou les lésions ne se prêtent pas à l'utilisation d'une endoprothèse médicamentée, le traitement des nouvelles lésions non calcifiées par AR et BM pourrait donner des résultats immédiats et à moyen terme comparables à ceux du traitement des nouvelles lésions calcifiées.
RESUMO
Dopamine supersensitivity psychosis is a clinical concept characterized by an unstable psychotic state and tardive dyskinesia in schizophrenia patients at the chronic stage. This state is thought to be induced by compensatory upregulation of dopamine D2 receptors, which is provoked by long-term and/or high-dose medications. Recent clinical data suggest that patients who responded well to medication but later exhibit dopamine supersensitivity develop tolerance to antipsychotics' effects and eventually transit to treatment-resistant schizophrenia, indicating that dopamine supersensitivity could be an etiology contributing to treatment-resistant schizophrenia. However, clinicians and researchers consider dopamine supersensitivity psychosis a minor phenomenon during the clinical course and do not make much of it. This opinion is often based on numerous clinical data indicating that dopamine supersensitivity psychosis is a relatively rare event. This review examines the data dealing with dopamine supersensitivity with the five themes of frequency, severity, withdrawal studies, switching to aripiprazole, and tardive dyskinesia. These effects of these themes on discussions of the clinical meaning of dopamine supersensitivity psychosis are then reviewed. The present review will help clinicians speculate about the background of severe psychopathology in a given patient; to make diagnoses of treatment-resistant schizophrenia and dopamine supersensitivity psychosis; and plan antipsychotic medication regimens with the goal of achieving better long-term prognosis.
Assuntos
Antipsicóticos , Transtornos Psicóticos , Esquizofrenia , Antipsicóticos/uso terapêutico , Dopamina , Humanos , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/tratamento farmacológico , Esquizofrenia/diagnóstico , Esquizofrenia/tratamento farmacológico , Esquizofrenia Resistente ao TratamentoRESUMO
Arterial atherosclerosis is the main pathological cause of coronary artery disease and peripheral arterial disease. Atherosclerosis is a chronic condition characterized by the presence of cholesterol-rich macrophages in the arterial intima. Accumulation of cholesterol in these macrophages is due to increased oxidation of low-density lipoprotein (LDL) and its uptake via scavenger receptors on the macrophages. Cholesterol efflux from the cholesterol-laden macrophages into high-density lipoprotein (HDL) is also a key process in maintaining cholesterol homeostasis and preventing cholesterol accumulation. Four pathways for the efflux of cholesterol to HDL exist in macrophages, including passive and active pathways. Several HDL characteristics determine cholesterol efflux capacity, namely composition, oxidative status, and HDL size. Oxidation of LDL and HDL, as well as an imbalance in cholesterol uptake and efflux, could lead to the accumulation of cholesterol in macrophages and initiation of atherosclerogenesis. Epidemiological studies have demonstrated that polyphenol-rich foods reduce cardiovascular events in the general population and in patients at risk of cardiovascular diseases. Many studies have reported that polyphenols in polyphenol-rich foods have anti-atherosclerotic properties by preventing cholesterol accumulation in macrophages through the suppression of lipoproteins oxidation and regulation of cholesterol uptake and efflux.