RESUMEN
Polo-like kinase 1 (PLK1), which is crucial in cell cycle regulation, is considered a promising anticancer drug target. Herein, we present the N-degron pathway-based proteolysis targeting chimera (PROTAC) for PLK1 degradation, targeting the Polo-box domain (PBD). We identified DD-2 as the most potent PROTAC that selectively induces PLK1 degradation in cancer cells, including HeLa and nonsmall cell lung cancer (NSCLC), through the N-degron pathway. DD-2 exhibited significant in vitro anticancer effects, inducing G2/M arrest and apoptosis in HeLa and NSCLC cell lines. DD-2 showed significant tumor growth inhibition in a xenograft mouse model using HeLa and NSCLC cell lines, highlighting its potential in cancer treatment. Furthermore, the combination of DD-2 with tyrosine kinase inhibitor (TKI), osimertinib, effectively suppressed tumor growth in double-mutated H1975 cell lines, emphasizing DD-2's potential in combination cancer therapies. Collectively, this study demonstrates the potential of the N-degron pathway, especially using DD-2, for targeted cancer therapies.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Ciclo Celular , Quimera Dirigida a la Proteólisis , Proteínas Serina-Treonina Quinasas , Quinasa Tipo Polo 1 , Apoptosis , Degrones , Línea Celular Tumoral , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Puntos de Control de la Fase G2 del Ciclo Celular , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
OBJECTIVES: We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. METHODS: Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. RESULTS: ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. CONCLUSION: Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.
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Antiinfecciosos , Staphylococcus aureus Resistente a Meticilina , Humanos , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Lipopolisacáridos , Endopeptidasa K/farmacología , Pepsina A/farmacología , Tripsina/farmacología , Antiinfecciosos/farmacología , Antiinfecciosos/química , Antibacterianos/farmacología , Antibacterianos/química , Antiinflamatorios/farmacología , Bacterias , Biopelículas , Timidina/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
AQEE-30 is one of the VGF peptides, which are derived from the VGF polypeptide precursor, and related to various physiological phenomena including neuroprotective effects in Huntington's disease and amyotrophic lateral sclerosis (ALS). Although various functions of AQEE-30 have been reported so far, the structure of this peptide has not been reported yet. In this study, the structure of human AQEE-30 was investigated in hexafluoroisopropanol (HFIP) and dodecyl phosphocholine (DPC) micelle solutions, using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. CD results showed that AQEE-30 had a partial helical structure in aqueous buffer, and the helical structure was stabilized in the HFIP and DPC micelle solutions. The 3D structures determined by NMR spectroscopy showed that AQEE-30 adopted mainly α-helical structure in both the HFIP and DPC micelle solutions. The surface of AQEE-30 showed that it was predominantly negatively charged. The residues from 601 to 611 in both the HFIP and DPC micelle solutions showed amphiphilicity with four negatively charged residues, glutamate. The C-terminal consecutive arginine residues formed a partial positively charged surface. These results suggest an α-helical active structure of AQEE-30 in the cell-membrane environment.
Asunto(s)
Micelas , Neuropéptidos , Humanos , Dicroismo Circular , Membranas , Péptidos/química , Factores de Crecimiento NerviosoRESUMEN
Allergy-causing inflammatory diseases have attracted significant attention because they have emerged as global health problems linked to urbanization. Interleukin-33 (IL-33) plays an important role in producing inflammatory cytokines, and it has been suggested as a target for the diagnosis and treatment of allergy-causing inflammatory diseases. In this work, specific peptides that bind to IL-33 were identified by a phage display technique and their molecular interactions were described. The peptide-displaying phages were selected on the basis of relative binding affinity by using enzyme-linked immunosorbent assay (ELISA) and square wave voltammetry (SWV). The selected IL-33 specific peptide was identified as FGLEPRANLHFT. To investigate the molecular interactions between IL-33 and the affinity peptide, the peptide was separated from the phage particles, chemically synthesized and characterized by SWV, isothermal titration calorimetry (ITC), and microscale thermophoresis (MST). The binding constant (Kd) value with SWV, MST, and ITC was found to be 1.68 ± 0.37 µM, 5.98 ± 1.30 µM, and 2.68 ± 1.37 µM, respectively. Two-dimensional (2D) NMR spectral analysis was performed to elucidate the primary peptide binding site of IL-33, which was near the ST2-D3 and IL1RAcP-D3 binding interfaces. Based on these observations using two different approaches, we conclude that this approach could be applied not only for the design of new peptides or peptide biomimetics for drug development, but also for the creation of unique molecular recognition elements useful for allergy diagnosis.
Asunto(s)
Bacteriófagos , Técnicas de Visualización de Superficie Celular , Ensayo de Inmunoadsorción Enzimática , Interleucina-33 , Biblioteca de Péptidos , Péptidos , Unión ProteicaRESUMEN
Cancer has been identified as a leading cause of death worldwide, and the increasing number of cancer cases threatens to shorten the average life expectancy of people. Recently, we reported a 3-azido-3-deoxythymidine (AZT)-based amphipathic small molecule, ADG-2e that revealed a notable potency against tumor metastasis. To evaluate the anticancer potential of ADG-2e, we assessed its anticancer potency in vitro and in vivo. Anticancer screening of ADG-2e against cervical cancer cells, HeLa CCL2, and BT549 mammary gland ductal carcinoma showed significant inhibition of cancer cell proliferation. Furthermore, mechanistic investigations revealed that cancer cell death presumably proceeded through an oncosis mechanistic pathway because ADG-2e treated cells showed severe damage on the plasma membrane, a loss of membrane integrity, and leakage of α-tubulin and ß-actin. Finally, evaluation of the antitumorigenic potential of ADG-2e in mouse xenograft models revealed that this compound potentially inhibits cancer cell proliferation. Collectively, these findings suggest that ADG-2e can evolve as an anticancer agent, which may represent a model for nucleoside-based small molecule anticancer drug discovery.
RESUMEN
In this study, we used the zebrafish animal model to establish a bioassay by which physiological efficacy differential of alpha-melanocyte-stimulating hormone (α-MSH) analogues could be measured by melanosome dispersion in zebrafish larvae. Brain-skin connection research has purported the interconnectedness between the nervous system and skin physiology. Accordingly, the neuropeptide α-MSH is a key regulator in several physiological processes, such as skin pigmentation in fish. In mammals, α-MSH has been found to regulate motivated behavior, appetite, and emotion, including stimulation of satiety and anxiety. Several clinical and animal model studies of autism spectrum disorder (ASD) have already demonstrated the effectiveness of α-MSH in restoring the social deficits of autism. Therefore, we sought to analyze the effect of synthetic and naturally-occurring α-MSH variants amongst different species. Our results showed that unique α-MSH derivatives from several fish species produced differential effects on the degree of melanophore dispersion. Using α-MSH human form as a standard, we could identify derivatives that induced greater physiological effects; particularly, the synthetic analogue melanotan-II (MT-II) exhibited a higher capacity for melanophore dispersion than human α-MSH. This was consistent with previous findings in an ASD mouse model demonstrating the effectiveness of MT-II in improving ASD behavioral symptoms. Thus, the melanophore assay may serve as a useful screening tool for therapeutic candidates for novel drug discovery.
Asunto(s)
Larva/efectos de los fármacos , Melanóforos/efectos de los fármacos , Péptidos Cíclicos/farmacología , Pigmentación de la Piel , alfa-MSH/análogos & derivados , alfa-MSH/farmacología , Secuencia de Aminoácidos , Animales , Bioensayo , Humanos , Larva/crecimiento & desarrollo , Melanóforos/citología , Homología de Secuencia , Pez Cebra , alfa-MSH/químicaRESUMEN
Gold nanoparticles (AuNPs) have been widely used as nanocarriers in drug delivery to improve the efficiency of chemotherapy treatment and enhance early disease detection. The advantages of AuNPs include their excellent biocompatibility, easy modification and functionalization, facile synthesis, low toxicity, and controllable particle size. This study aimed to synthesize a conjugated citraconic anhydride link between morphologically homogeneous AuNPs and doxorubicin (DOX) (DOX-AuNP). The carrier was radiolabeled for tumor diagnosis using positron emission tomography (PET). The systemically designed DOX-AuNP was cleaved at the citraconic anhydride linker site under the mild acidic conditions of a cancer cell, thereby releasing DOX. Subsequently, the AuNPs aggregated via electrostatic attraction. HeLa cancer cells exhibited a high uptake of the radiolabeled DOX-AuNP. Moreover, PET tumor images were obtained using radiolabeled DOX-AuNP in cancer xenograft mouse models. Therefore, DOX-AuNP is expected to provide a valuable insight into the use of radioligands to detect tumors using PET.
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Oro/química , Concentración de Iones de Hidrógeno , Nanopartículas del Metal/química , Neoplasias del Cuello Uterino/diagnóstico por imagen , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/uso terapéutico , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/uso terapéutico , Femenino , Oro/metabolismo , Células HeLa , Humanos , Ratones , Tomografía de Emisión de Positrones , Ensayo de Unión Radioligante , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC-MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer's disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.
Asunto(s)
Aminoaciltransferasas/metabolismo , Arginina/metabolismo , Retículo Endoplásmico/metabolismo , Vectores Genéticos/genética , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Arginina/química , Arginina/genética , Células HeLa , Humanos , Proteínas de la Membrana/genética , Especificidad por SustratoRESUMEN
Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. Studies have shown that Plk4 undergoes dynamic relocalization from a ring-like pattern around a centriole to a dot-like morphology at the procentriole assembly site and this event is central for inducing centriole biogenesis. However, the detailed mechanisms underlying Plk4's capacity to drive its symmetry-breaking ring-to-dot relocalization remain largely unknown. Here, we showed that Plk4 self-initiates this process in an autophosphorylation-dependent manner and that STIL, its downstream target, is not required for this event. Time-dependent analyses with mEOS-fused photoconvertible Plk4 revealed that a portion of ring-state Plk4 acquires a capacity, presumably through autophosphorylation, to linger around a centriole, ultimately generating a dot-state morphology. Interestingly, Plk4 WT, but not its catalytically inactive mutant, showed the ability to form a nanoscale spherical assembly in the cytosol of human cells or heterologous E. coli, demonstrating its autophosphorylation-dependent self-organizing capacity. At the biochemical level, Plk4 - unlike its N-terminal ßTrCP degron motif - robustly autophosphorylated the PC3 SSTT motif within its C-terminal cryptic polo-box, an event critical for inducing its physical clustering. Additional in vivo experiments showed that although STIL was not required for Plk4's initial ring-to-dot conversion, coexpressed STIL greatly enhanced Plk4's ability to generate a spherical condensate and recruit Sas6, a major component of the centriolar cartwheel structure. We propose that Plk4's autophosphorylation-induced clustering is sufficient to induce its ring-to-dot localization conversion and that subsequently recruited STIL potentiates this process to generate a procentriole assembly body critical for Plk4-dependent centriole biogenesis.
Asunto(s)
Centriolos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genética , Biocatálisis , Ciclo Celular/genética , Línea Celular Tumoral , Citosol/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Osteosarcoma/patología , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , Interferencia de ARN , TransfecciónRESUMEN
Polo-like kinase-1 (Plk1) plays a key role in mitosis and has been identified as an attractive anticancer drug target. Plk1 consists of two drug-targeting sites, namely, N-terminal kinase domain (KD) and C-terminal polo-box domain (PBD). As KD-targeting inhibitors are associated with severe side effects, here we report on the pyrazole-based Plk1 PBD inhibitor, KBJK557, which showed a remarkable in vitro anticancer effect by inducing Plk1 delocalization, mitotic arrest, and apoptosis in HeLa cells. Further, in vivo optical imaging analysis and antitumorigenic activities in mouse xenograft models demonstrate that KBJK557 preferentially accumulates in cancer cells and selectively inhibits cancer cell proliferation. Pharmacokinetic profiles and partition coefficients suggest that KBJK557 was exposed in the blood and circulated through the organs with an intermediate level of clearance (t1/2, 7.73 h). The present investigation offers a strategy for specifically targeting cancer using a newly identified small-molecule inhibitor that targets the Plk1 PBD.
Asunto(s)
Antineoplásicos/uso terapéutico , Barbitúricos/uso terapéutico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Barbitúricos/síntesis química , Barbitúricos/metabolismo , Barbitúricos/farmacocinética , Carbocianinas/química , Proteínas de Ciclo Celular/metabolismo , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Colorantes Fluorescentes/química , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células HeLa , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Estructura Molecular , Neoplasias/diagnóstico , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1RESUMEN
TZP4 is a triazine-based amphipathic polymer designed to mimic the amphipathic structure found in antimicrobial peptides. TZP4 showed potent antimicrobial activity comparable to melittin against antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. TZP4 showed high resistance to proteolytic degradation and low tendency to develop drug resistance. The results from membrane depolarization, SYTOX Green uptake, flow cytometry, and gel retardation revealed that the mechanism of antimicrobial action of TZP4 involved an intracellular target rather than the bacterial cell membrane. Furthermore, TZP4 suppressed the messenger RNA levels of inducible nitric oxide synthase and tumor necrosis factor-α (TNF-α) and inhibited the release of nitric oxide and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BODIPY-TR-cadaverine displacement and dissociation of fluorescein isothiocyanate (FITC)-labeled LPS assays revealed that TZP4 strongly bound to LPS and disaggregated the LPS oligomers. Flow cytometric analysis demonstrated that TZP4 inhibits the binding of FITC-conjugated LPS to RAW264.7 cells. These observations indicate that TZP4 may exert its antiendotoxic activity by directly binding with LPS and inhibiting the interaction between LPS and CD14+ cells. Collectively, TZP4 is a promising drug candidate for the treatment of endotoxic shock and sepsis caused by Gram-negative bacterial infections.
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Antiinfecciosos , Péptidos Antimicrobianos , Polímeros , Triazinas , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antiinfecciosos/toxicidad , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/toxicidad , Supervivencia Celular/efectos de los fármacos , Farmacorresistencia Bacteriana , Endotoxinas/antagonistas & inhibidores , Lipopolisacáridos/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Polímeros/química , Polímeros/farmacología , Polímeros/toxicidad , Células RAW 264.7 , Triazinas/química , Triazinas/farmacología , Triazinas/toxicidadRESUMEN
To combat the escalating rise of antibacterial resistance, the development of antimicrobial peptides (AMPs) with a unique mode of action is considered an attractive strategy. However, proteolytic degradation of AMPs remains the greatest challenge in their transformation into therapeutics. Herein, we synthesized Fmoc-triazine amino acids that differ from each other by anchoring either cationic or hydrophobic residues. These unnatural amino acids were adopted for solid-phase peptide synthesis (SPPS) to synthesize a series of amphipathic antimicrobial peptidomimetics. From the antimicrobial screening, we found that the trimer, BJK-4 is the most potent short antimicrobial peptidomimetic without showing hemolytic activity and it displayed enhanced proteolytic stability. Moreover, the mechanism of action to kill bacteria was found to be an intracellular targeting.
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Materiales Biomiméticos/síntesis química , Proteínas Citotóxicas Formadoras de Poros/síntesis química , Triazinas/química , Aminoácidos/química , Animales , Materiales Biomiméticos/farmacología , Células Cultivadas , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Hemólisis , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Proteínas Citotóxicas Formadoras de Poros/farmacología , Estabilidad Proteica , Pseudomonas aeruginosa/efectos de los fármacos , Células RAW 264.7 , Ovinos , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Antimicrobial peptides (AMP) with anticancer activity have drawn remarkable attention in modern treatments. However, long peptide length and protease instability are the most addressing factors, which hampers their further development as therapeutic agents. In view of this, herein, we designed and synthesized a series of AZT-based cationic small molecule incorporating a variety of hydrophobic groups and cationic charges, including amine and guanidine groups to mimic the amphipathic structure of AMPs. These compounds were evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria. Through an extensive structure activity relationship study (SAR), we identified ADG-2e as the most potent antibacterial agent, which exhibited remarkable potency against drug resistant bacterial strains such as MRSA and MDRPA. Further, ADG-2e was examined for their anti-metastatic ability by investigating the cancer cell migration and invasiveness through scratch wound-healing assay and transwell invasive assay, respectively. In addition, time-lapse cell tracking analysis also performed for analyzing the cell movement pattern. Treatment of ADG-2e against metastatic breast cancer cells (MDA-MB-231) suppressed tumor cell migration by multi-directional lamellipodium formation, indicating their anti-metastatic potential. Thus, our cationic AZT based small molecules may evolve as an appealing class of antibacterial agents with anti-metastasis potential.
Asunto(s)
Antibacterianos/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Zidovudina/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias de la Mama/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas , Cicatrización de Heridas/efectos de los fármacos , Zidovudina/síntesis química , Zidovudina/químicaRESUMEN
Cep57 has been characterized as a component of a pericentriolar complex containing Cep63 and Cep152. Interestingly, Cep63 and Cep152 self-assemble into a pericentriolar cylindrical architecture, and this event is critical for the orderly recruitment of Plk4, a key regulator of centriole duplication. However, the way in which Cep57 interacts with the Cep63-Cep152 complex and contributes to the structure and function of Cep63-Cep152 self-assembly remains unknown. We demonstrate that Cep57 interacts with Cep63 through N-terminal motifs and associates with Cep152 via Cep63. Three-dimensional structured illumination microscopy (3D-SIM) analyses suggested that the Cep57-Cep63-Cep152 complex is concentrically arranged around a centriole in a Cep57-in and Cep152-out manner. Cep57 mutant cells defective in Cep63 binding exhibited improper Cep63 and Cep152 localization and impaired Sas6 recruitment for procentriole assembly, proving the significance of the Cep57-Cep63 interaction. Intriguingly, Cep63 fused to a microtubule (MT)-binding domain of Cep57 functioned in concert with Cep152 to assemble around stabilized MTs in vitro Thus, Cep57 plays a key role in architecting the Cep63-Cep152 assembly around centriolar MTs and promoting centriole biogenesis. This study may offer a platform to investigate how the organization and function of the pericentriolar architecture are altered by disease-associated mutations found in the Cep57-Cep63-Cep152 complex.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/análisis , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Nucleares/análisis , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de ProteínasRESUMEN
Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. How Plk4 dynamically promotes its symmetry-breaking relocalization and achieves its procentriole-assembly state remains unknown. Here we show that Plk4 is a unique kinase that utilizes its autophosphorylated noncatalytic cryptic polo-box (CPB) to phase separate and generate a nanoscale spherical condensate. Analyses of the crystal structure of a phospho-mimicking, condensation-proficient CPB mutant reveal that a disordered loop at the CPB PB2-tip region is critically required for Plk4 to generate condensates and induce procentriole assembly. CPB phosphorylation also promotes Plk4's dissociation from the Cep152 tether while binding to downstream STIL, thus allowing Plk4 condensate to serve as an assembling body for centriole biogenesis. This study uncovers the mechanism underlying Plk4 activation and may offer strategies for anti-Plk4 intervention against genomic instability and cancer.
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Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Biogénesis de Organelos , Proteínas Serina-Treonina Quinasas/genética , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Imagenología Tridimensional , Microscopía Confocal , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Células Sf9 , SpodopteraRESUMEN
Polo-like kinase 1 (Plk1) regulates cell cycle and cell proliferation, and is currently considered a potential biomarker in clinical trials for many cancers. A characteristic feature of Plks is their C-terminal polo-box domain (PBD). Pro-Leu-His-Ser-pThr (PLHS[pT])-the phosphopeptide inhibitor of the PBD of Plk1-induces apoptosis in cancer cells. However, because of the low cell membrane-penetration ability of PLHS[pT], new approaches are required to overcome these drawbacks. We therefore developed a vitamin E (VE) conjugate that is biodegradable by intracellular redox enzymes as an anticancer drug-delivery system. To ensure high efficiency of membrane penetration, we synthesized VE-S-S-PLHS[pT]KY (1) by conjugating PLHS[pT] to VE via a disulfide bond. We found that 1 penetrated cancer cell membranes, blocked cancer cell proliferation, and induced apoptosis in cancer cells through cell cycle arrest in the G2/M phase. We synthesized a radiolabeled peptide (124I-1), and the radioligand was evaluated in in vivo tumor uptake using positron emission tomography. This study shows that combination conjugates are an excellent strategy for specifically targeting Plk PBD. These conjugates have a dual function, with possible uses in anticancer therapy and tumor diagnosis.
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Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas de Ciclo Celular/metabolismo , Fosfopéptidos/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Vitamina E/química , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Células HeLa , Humanos , Mitosis/efectos de los fármacos , Quinasa Tipo Polo 1RESUMEN
Considering the emergence of bacterial resistance and low proteolytic stability of antimicrobial peptides (AMPs), herein we developed a series of ultra-short triazine based amphipathic polymers (TZP) that are connected with ethylene diamine linkers instead of protease sensitive amide bond. The most potent oligomers, TZP3 and TZP5 not only displayed potent antibacterial action on various drug-resistant pathogens but also exhibited a strong synergic antibacterial activity in combination with chloramphenicol against multidrug-resistant Pseudomonas aeruginosa (MDRPA). Since most of atopic dermatitis (AD) infections are caused by bacterial colonization, we evaluated the potency of TZP3 and TZP5 on AD in vitro and in vivo. In vitro AD analysis of these two polymers showed significant inhibition against the release of ß-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells. In AD-like skin lesions in BALB/c mice model, these two polymers displayed significant potency in suppressing dermal and epidermal thickness, mast cell infiltration and pro-inflammatory cytokines expression. Moreover, these polymers exhibited remarkable efficacy over the allergies caused by the imbalance of Th1/Th2 by regulating total IgE and IgG2a. Finally, the impact of treatment effects of these polymers was examined through analyzing the weights and sizes of spleen and lymph node of AD-induced mice.
Asunto(s)
Antibacterianos/farmacología , Polímeros/farmacología , Tensoactivos/farmacología , Triazinas/farmacología , Animales , Antibacterianos/química , Bacterias/efectos de los fármacos , Citocinas/metabolismo , Dermatitis Atópica/sangre , Dermatitis Atópica/patología , Modelos Animales de Enfermedad , Farmacorresistencia Microbiana/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Hemólisis , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Mediadores de Inflamación/metabolismo , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Mastocitos/efectos de los fármacos , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Péptido Hidrolasas/metabolismo , Polímeros/química , Ovinos , Piel/efectos de los fármacos , Piel/patología , Bazo/efectos de los fármacos , Bazo/patología , Triazinas/químicaRESUMEN
Complex amyloid aggregation of amyloid-ß (1-40) (Aß1-40) in terms of monomer structures has not been fully understood. Herein, we report the microscopic mechanism and pathways of Aß1-40 aggregation with macroscopic viewpoints through tuning its initial structure and solubility. Partial helical structures of Aß1-40 induced by low solvent polarity accelerated cytotoxic Aß1-40 amyloid fibrillation, while predominantly helical folds did not aggregate. Changes in the solvent polarity caused a rapid formation of ß-structure-rich protofibrils or oligomers via aggregation-prone helical structures. Modulation of the pH and salt concentration transformed oligomers to protofibrils, which proceeded to amyloid formation. We reveal diverse molecular mechanisms underlying Aß1-40 aggregation with conceptual energy diagrams and propose that aggregation-prone partial helical structures are key to inducing amyloidogenesis. We demonstrate that context-dependent protein aggregation is comprehensively understood using the macroscopic phase diagram, which provides general insights into differentiation of amyloid formation and phase separation from unfolded and folded structures.
Asunto(s)
Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/ultraestructura , Fragmentos de Péptidos/ultraestructura , Agregación Patológica de Proteínas/genética , Conformación Proteica en Hélice alfa/genética , Enfermedad de Alzheimer/patología , Amiloide/química , Amiloide/genética , Péptidos beta-Amiloides/química , Humanos , Fragmentos de Péptidos/química , Conformación Proteica en Lámina beta/genética , Pliegue de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , SolubilidadRESUMEN
The cell is constructed by higher-order structures and organelles through complex interactions among distinct structural constituents. The centrosome is a membraneless organelle composed of two microtubule-derived structures called centrioles and an amorphous mass of pericentriolar material. Super-resolution microscopic analyses in various organisms revealed that diverse pericentriolar material proteins are concentrically localized around a centriole in a highly organized manner. However, the molecular nature underlying these organizations remains unknown. Here we show that two human pericentriolar material scaffolds, Cep63 and Cep152, cooperatively generate a heterotetrameric α-helical bundle that functions in conjunction with its neighboring hydrophobic motifs to self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4, a key regulator for centriole duplication. Mutations disrupting the self-assembly abrogate Plk4-mediated centriole duplication. Because pericentriolar material organization is evolutionarily conserved, this work may offer a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms.