RESUMO
Anion exchanger 3 (AE3) is pivotal in regulating intracellular pH across excitable tissues, yet its structural intricacies and functional dynamics remain underexplored compared to other anion exchangers. This study unveils the structural insights into human AE3, including the cryo-electron microscopy structures for AE3 transmembrane domains (TMD) and a chimera combining AE3 N-terminal domain (NTD) with AE2 TMD (hAE3NTD2TMD). Our analyzes reveal a substrate binding site, an NTD-TMD interlock mechanism, and a preference for an outward-facing conformation. Unlike AE2, which has more robust acid-loading capabilities, AE3's structure, including a less stable inward-facing conformation due to missing key NTD-TMD interactions, contributes to its moderated pH-modulating activity and increased sensitivity to the inhibitor DIDS. These structural differences underline AE3's distinct functional roles in specific tissues and underscore the complex interplay between structural dynamics and functional specificity within the anion exchanger family, enhancing our understanding of the physiological and pathological roles of the anion exchanger family.
Assuntos
Antiporters , Humanos , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Antiporters/química , Antiporters/ultraestruturaRESUMO
Sphingomyelin (SM) has key roles in modulating mammalian membrane properties and serves as an important pool for bioactive molecules. SM biosynthesis is mediated by the sphingomyelin synthase (SMS) family, comprising SMS1, SMS2 and SMS-related (SMSr) members. Although SMS1 and SMS2 exhibit SMS activity, SMSr possesses ceramide phosphoethanolamine synthase activity. Here we determined the cryo-electron microscopic structures of human SMSr in complexes with ceramide, diacylglycerol/phosphoethanolamine and ceramide/phosphoethanolamine (CPE). The structures revealed a hexameric arrangement with a reaction chamber located between the transmembrane helices. Within this structure, a catalytic pentad E-H/D-H-D was identified, situated at the interface between the lipophilic and hydrophilic segments of the reaction chamber. Additionally, the study unveiled the two-step synthesis process catalyzed by SMSr, involving PE-PLC (phosphatidylethanolamine-phospholipase C) hydrolysis and the subsequent transfer of the phosphoethanolamine moiety to ceramide. This research provides insights into the catalytic mechanism of SMSr and expands our understanding of sphingolipid metabolism.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Esfingomielinas , Transferases (Outros Grupos de Fosfato Substituídos) , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Esfingomielinas/metabolismo , Esfingomielinas/química , Esfingomielinas/biossíntese , Ceramidas/metabolismo , Ceramidas/química , Etanolaminas/metabolismo , Etanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/química , Diglicerídeos/metabolismo , Diglicerídeos/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas de MembranaRESUMO
Lipid droplets (LDs) are dynamic lipid storage organelles that can sense and respond to changes in systemic energy balance. The size and number of LDs are controlled by complex and delicate mechanisms, among which, whether and which SNARE proteins mediate LD fusion, and the mechanisms governing this process remain poorly understood. Here we identified a SNARE complex, syntaxin 18 (STX18)-SNAP23-SEC22B, that is recruited to LDs to mediate LD fusion. STX18 targets LDs with its transmembrane domain spanning the phospholipid monolayer twice. STX18-SNAP23-SEC22B complex drives LD fusion in adiposome lipid mixing and content mixing in vitro assays. CIDEC/FSP27 directly binds STX18, SEC22B, and SNAP23, and promotes the lipid mixing of SNAREs-reconstituted adiposomes by promoting LD clustering. Knockdown of STX18 in mouse liver via AAV resulted in smaller liver and reduced LD size under high-fat diet conditions. All these results demonstrate a critical role of the SNARE complex STX18-SNAP23-SEC22B in LD fusion.
RESUMO
Endoplasmic reticulum-associated degradation (ERAD) maintains protein homeostasis by retrieving misfolded proteins from the endoplasmic reticulum (ER) lumen into the cytosol for degradation. The retrotranslocation of misfolded proteins across the ER membrane is an energy-consuming process, with the detailed transportation mechanism still needing clarification. We determined the cryo-EM structures of the hetero-decameric complex formed by the Derlin-1 tetramer and the p97 hexamer. It showed an intriguing asymmetric complex and a putative coordinated squeezing movement in Derlin-1 and p97 parts. With the conformational changes of p97 induced by its ATP hydrolysis activities, the Derlin-1 channel could be torn into a "U" shape with a large opening to the lipidic environment, thereby forming an entry for the substrates in the ER membrane. The EM analysis showed that p97 formed a functional protein complex with Derlin-1, revealing the coupling mechanism between the ERAD retrotranslocation and the ATP hydrolysis activities.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Complexo de Endopeptidases do Proteassoma , Humanos , Microscopia Crioeletrônica , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Hypophosphatasia (HPP) is a metabolic bone disease that manifests as developmental abnormalities in bone and dental tissues. HPP patients exhibit hypo-mineralization and osteopenia due to the deficiency or malfunction of tissue non-specific alkaline phosphatase (TNAP), which catalyzes the hydrolysis of phosphate-containing molecules outside the cells, promoting the deposition of hydroxyapatite in the extracellular matrix. Despite the identification of hundreds of pathogenic TNAP mutations, the detailed molecular pathology of HPP remains unclear. Here, to address this issue, we determine the crystal structures of human TNAP at near-atomic resolution and map the major pathogenic mutations onto the structure. Our study reveals an unexpected octameric architecture for TNAP, which is generated by the tetramerization of dimeric TNAPs, potentially stabilizing the TNAPs in the extracellular environments. Moreover, we use cryo-electron microscopy to demonstrate that the TNAP agonist antibody (JTALP001) forms a stable complex with TNAP by binding to the octameric interface. The administration of JTALP001 enhances osteoblast mineralization and promoted recombinant TNAP-rescued mineralization in TNAP knockout osteoblasts. Our findings elucidate the structural pathology of HPP and highlight the therapeutic potential of the TNAP agonist antibody for osteoblast-associated bone disorders.
Assuntos
Fosfatase Alcalina , Hipofosfatasia , Humanos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Hipofosfatasia/genética , Hipofosfatasia/metabolismo , Hipofosfatasia/patologia , Microscopia Crioeletrônica , Osso e Ossos/metabolismo , Osteoblastos/metabolismoRESUMO
The cell maintains its intracellular pH in a narrow physiological range and disrupting the pH-homeostasis could cause dysfunctional metabolic states. Anion exchanger 2 (AE2) works at high cellular pH to catalyze the exchange between the intracellular HCO3- and extracellular Cl-, thereby maintaining the pH-homeostasis. Here, we determine the cryo-EM structures of human AE2 in five major operating states and one transitional hybrid state. Among those states, the AE2 shows the inward-facing, outward-facing, and intermediate conformations, as well as the substrate-binding pockets at two sides of the cell membrane. Furthermore, critical structural features were identified showing an interlock mechanism for interactions among the cytoplasmic N-terminal domain and the transmembrane domain and the self-inhibitory effect of the C-terminal loop. The structural and cell-based functional assay collectively demonstrate the dynamic process of the anion exchange across membranes and provide the structural basis for the pH-sensitive pH-rebalancing activity of AE2.
Assuntos
Proteínas de Transporte de Ânions , Antiporters , Humanos , Antiportadores de Cloreto-Bicarbonato , Concentração de Íons de Hidrogênio , Membrana Celular/metabolismo , Homeostase , Antiporters/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Cloretos/metabolismoRESUMO
Pyruvate carboxylase (PC) catalyzes the two-step carboxylation of pyruvate to produce oxaloacetate, playing a key role in the maintenance of metabolic homeostasis in cells. Given its involvement in multiple diseases, PC has been regarded as a potential therapeutic target for obesity, diabetes, and cancer. Albeit acetyl-CoA has been recognized as the allosteric regulator of PC for over 60 years, the underlying mechanism of how acetyl-CoA induces PC activation remains enigmatic. Herein, by using time-resolved cryo-electron microscopy, we have captured the snapshots of PC transitional states during its catalytic cycle. These structures and the biochemical studies reveal that acetyl-CoA stabilizes PC in a catalytically competent conformation, which triggers a cascade of events, including ATP hydrolysis and the long-distance communication between the two reactive centers. These findings provide an integrated picture for PC catalysis and unveil the unique allosteric mechanism of acetyl-CoA in an essential biochemical reaction in all kingdoms of life.
Assuntos
Acetil-CoA Carboxilase , Piruvato Carboxilase , Humanos , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo , Acetilcoenzima A/metabolismo , Regulação Alostérica , Microscopia Crioeletrônica , Conformação Molecular , Acetil-CoA Carboxilase/metabolismoRESUMO
Mutations of PSEN1 have been reported in dilated cardiomyopathy pedigrees. Understanding the effects and mechanisms of PSEN1 in cardiomyocytes might have important implications for treatment of heart diseases. Here, we showed that PSEN1 was downregulated in ischemia-induced failing hearts. Functionally, cardiovascular specific PSEN1 deletion led to spontaneous death of the mice due to cardiomyopathy. At the age of 11 months, the ratio of the heart weight/body weight was slightly lower in the Sm22a-PSEN1-KO mice compared with that of the WT mice. Echocardiography showed that the percentage of ejection fraction and fractional shortening was significantly reduced in the Sm22a-PSEN1-KO group compared with the percent of these measures in the WT group, indicating that PSEN1-KO resulted in heart failure. The abnormally regulated genes resulted from PSEN1-KO were detected to be enriched in muscle development and dilated cardiomyopathy. Among them, several genes encode Ca2+ ion channels, promoting us to investigate the effects of PSEN1 KO on regulation of Ca2+ in isolated adult cardiomyocytes. Consistently, in isolated adult cardiomyocytes, PSEN1-KO increased the concentration of cytosolic Ca2+ and reduced Ca2+ concentration inside the sarcoplasmic reticulum (SR) lumen at the resting stage. Additionally, SR Ca2+ was decreased in the failing hearts of WT mice, but with the lowest levels observed in the failing hearts of PSEN1 knockout mice. These results indicate that the process of Ca2+ release from SR into cytoplasm was affected by PSEN1 KO. Therefore, the abnormalities in Ca2+ homeostasis resulted from downregulation of PSEN1 in failing hearts might contribute to aging-related cardiomyopathy, which might had important implications for the treatment of aging-related heart diseases.
Assuntos
Cálcio , Cardiomiopatia Dilatada , Animais , Cardiomiopatia Dilatada/genética , Homeostase , Camundongos , Camundongos Knockout , Miócitos Cardíacos/fisiologia , Retículo SarcoplasmáticoRESUMO
As the major component of cell membranes, phosphatidylcholine (PC) is synthesized de novo in the Kennedy pathway and then undergoes extensive deacylation-reacylation remodeling via Lands' cycle. The re-acylation is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT) and among the four LPCAT members in human, the LPCAT3 preferentially introduces polyunsaturated acyl onto the sn-2 position of lysophosphatidylcholine, thereby modulating the membrane fluidity and membrane protein functions therein. Combining the x-ray crystallography and the cryo-electron microscopy, we determined the structures of LPCAT3 in apo-, acyl donor-bound, and acyl receptor-bound states. A reaction chamber was revealed in the LPCAT3 structure where the lysophosphatidylcholine and arachidonoyl-CoA were positioned in two tunnels connected near to the catalytic center. A side pocket was found expanding the tunnel for the arachidonoyl CoA and holding the main body of arachidonoyl. The structural and functional analysis provides the basis for the re-acylation of lysophosphatidylcholine and the substrate preference during the reactions.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/química , Fosfolipídeos/química , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Acilação , Animais , Domínio Catalítico , Galinhas , Microscopia Crioeletrônica , Cristalografia por Raios X , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Modelos Moleculares , Fosfolipídeos/metabolismo , Multimerização Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Endoplasmic reticulum-associated degradation (ERAD) is a process directing misfolded proteins from the ER lumen and membrane to the degradation machinery in the cytosol. A key step in ERAD is the translocation of ER proteins to the cytosol. Derlins are essential for protein translocation in ERAD, but the mechanism remains unclear. Here, we solved the structure of human Derlin-1 by cryo-electron microscopy. The structure shows that Derlin-1 forms a homotetramer that encircles a large tunnel traversing the ER membrane. The tunnel has a diameter of about 12 to 15 angstroms, large enough to allow an α helix to pass through. The structure also shows a lateral gate within the membrane, providing access of transmembrane proteins to the tunnel, and thus, human Derlin-1 forms a protein channel for translocation of misfolded proteins. Our structure is different from the monomeric yeast Derlin structure previously reported, which forms a semichannel with another protein.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Microscopia Crioeletrônica , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Genetically engineered T cells expressing a chimeric antigen receptor (CAR) are rapidly emerging a promising new treatment for haematological and non-haematological malignancies. CAR-T therapy can induce rapid and durable clinical responses but is associated with unique acute toxicities. Moreover, CAR-T cells are vulnerable to immunosuppressive mechanisms. Here, we report that CAR-T cells release extracellular vesicles, mostly in the form of exosomes that carry CAR on their surface. The CAR-containing exosomes express a high level of cytotoxic molecules and inhibit tumour growth. Compared with CAR-T cells, CAR exosomes do not express Programmed cell Death protein 1 (PD1), and their antitumour effect cannot be weakened by recombinant PD-L1 treatment. In a preclinical in vivo model of cytokine release syndrome, the administration of CAR exosomes is relatively safe compared with CAR-T therapy. This study supports the use of exosomes as biomimetic nanovesicles that may be useful in future therapeutic approaches against tumours.
Assuntos
Exossomos/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Exossomos/metabolismo , Humanos , Ativação Linfocitária/imunologia , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Neoplasias/genética , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismoRESUMO
Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2-targeting phosphorylation site-specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2-specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain-only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)9-mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA-mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.-Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C., Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.
Assuntos
Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Animais Recém-Nascidos , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Insuficiência Cardíaca/metabolismo , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
PURPOSE: Both EGFR and PI3K-Akt signaling pathways have been used as therapeutically actionable targets, but resistance is frequently reported. In this report, we show that enrichment of the cancer stem cell (CSC) subsets and dysregulation of Notch signaling underlie the challenges to therapy and describe the development of bispecific antibodies targeting both HER and Notch signaling. EXPERIMENTAL DESIGN: We utilized cell-based models to study Notch signaling in drug-induced CSC expansion. Both cancer cell line models and patient-derived xenograft tumors were used to evaluate the antitumor effects of bispecific antibodies. Cell assays, flow cytometry, qPCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. RESULTS: We found that EGFR/Notch targeting bispecific antibodies exhibited a notable antistem cell effect in both in vitro and in vivo assays. Bispecific antibodies delayed the occurrence of acquired resistance to EGFR inhibitors in triple-negative breast cancer cell line-based models and showed efficacy in patient-derived xenografts. Moreover, the EGFR/Notch bispecific antibody PTG12 in combination with GDC-0941 exerted a stronger antitumor effect than the combined therapy of PI3K inhibitor with EGFR inhibitors or tarextumab in a broad spectrum of epithelial tumors. Mechanistically, bispecific antibody treatment inhibits the stem cell-like subpopulation, reduces tumor-initiating cell frequency, and downregulates the mesenchymal gene expression. CONCLUSIONS: These findings suggest that the coblockade of EGFR and Notch signaling has the potential to increase the response to PI3K inhibition, and PTG12 may gain clinical efficacy when combined with PI3K blockage in cancer treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptores Notch/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Anticorpos Biespecíficos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Receptores ErbB/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Humanos , Indazóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Sulfonamidas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Post-ischaemic heart failure is a major cause of death worldwide. Reperfusion of infarcted heart tissue after myocardial infarction has been an important medical intervention to improve outcomes. However, disturbances in Ca2+ and redox homeostasis at the cellular level caused by ischaemia/reperfusion remain major clinical challenges. In this study, we investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of a Type-2 ryanodine receptor (RyR2) degradation-associated gene, Presenilin 1 (PSEN1), to combat post-ischaemic heart failure. Adeno-associated viral PSEN1 gene delivery elevated PSEN1 protein expression in a post-infarction rat heart failure model, and this administration normalised the contractile dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca2+ handling and function. Moreover, PSEN1 gene transfer to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca2+ leak, thereby restoring the diminished intracellular Ca2+ transients and SR Ca2+ load. Moreover, PSEN1 gene transfer reversed the phosphorylation of RyR2 in failing cardiomyocytes. However, selective autophagy inhibition did not prevent the PSEN1-induced blockade of RyR2 degradation, making the participation of autophagy in PSEN1-associated RyR2 degradation unlikely. Our results established a role of the cardiac expression of PSEN1 with AAV9 vectors as a promising therapeutic approach for post-ischaemic heart failure.
Assuntos
Dependovirus/genética , Vetores Genéticos , Insuficiência Cardíaca/fisiopatologia , Isquemia Miocárdica/genética , Isquemia Miocárdica/fisiopatologia , Presenilina-1/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Homeostase , Ratos , Retículo Sarcoplasmático/metabolismo , Função Ventricular EsquerdaRESUMO
Different functionalized carbon materials were used as supports to prepare Pd/carbon catalysts (Pd/AC, Pd/AC-O, Pd/AC-Cl and Pd/AC-N). The results of various characterization techniques revealed that a substantial increase in the surface functional groups of the supports could influence the size of the Pd nanoparticles and the chemical states of the Pd species due to Pd-support interactions. During the hydrogenation reaction to synthesize dibenzylbiotinmethylester, the Pd/AC-N catalyst, based on a support that was treated with nitric acid (AC-N), provided a higher yield of dibenzylbiotinmethylester than the other catalysts. The increase in surface oxygen groups (mainly CO2-releasing groups) in the AC-N support was relevant to achieving selective hydrogenation. These groups can provide an efficient pathway for the reaction, which may be responsible for the high yield of dibenzylbiotinmethylester.
RESUMO
Recently, PSEN1 has been reported to have mutations in dilated cardiomyopathy pedigrees. However, the function and mechanism of PSEN1 in cardiomyopathy remains unresolved. Here, we established four types of genetically modified mice to determine the function of PSEN1 in cardiac development and pathology. PSEN1 null mutation resulted in perinatal death, retardation of heart growth, ventricular dilatation, septum defects, and valvular thickening. PSEN1 knockout in adults led to decreased muscle fibers, widened sarcomere Z lines and reduced lengths of sarcomeres in cardiomyocytes. Cardiovascular loss of function of PSEN1 induced by Sm22a-Cre or Myh6-Cre/ER/tamoxifen also resulted in severe ultrastructural abnormalities, such as relaxed gap junctions between neighboring cardiomyocytes. Functionally, cardiovascular deletion of PSEN1 caused spontaneous mortality from birth to adulthood and led to diastolic heart dysfunction, including decreased volume of the left ventricle at the end-systolic and end-diastolic stages. Additionally, in a myocardial ischemia model, deletion of PSEN1 in the cardiovascular system first protected mice by inducing adaptive hypertrophy but ultimately resulted in severe heart failure. Furthermore, a collection of genes was abnormally expressed in the hearts of cardiac-specific PSEN1 knockout mice. They were enriched in cell proliferation, calcium regulation, and so on. Taken together, dynamic regulation and abnormal function of PSEN1 underlie the pathogenesis of cardiovascular diseases due to ultrastructural abnormality of cardiomyocytes.
Assuntos
Deleção de Genes , Cardiopatias Congênitas/fisiopatologia , Presenilina-1/deficiência , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular Esquerda , Animais , Diástole , Regulação da Expressão Gênica , Predisposição Genética para Doença , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Camundongos Knockout , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Fenótipo , Presenilina-1/genética , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologiaRESUMO
HER2, a ligand-free tyrosine kinase receptor of the HER family, is frequently overexpressed in breast cancer. The anti-HER2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. Despite the effectiveness of trastuzumab, its efficacy remains variable and often modest. Thus, there is an urgent need to improve ErbB2-targeting therapy. Here, we describe a novel anti-HER2 antibody, 7C3, which was developed using hybridoma technique. Structural analysis confirms that the epitope of this antibody is in domain II/III of HER2. Moreover, a structural conformation change was observed in HER2 in complex with 7C3. Interestingly, this novel anti-HER2 antibody exhibits efficacy in blocking HER2/EGFR heterodimerization and signaling. The results highlight the different function role of HER2 domains and the unique potential of 7C3 to inhibit the HER2/EGFR heterodimer, which may complement current anti-HER2 treatments.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Receptores ErbB/imunologia , Receptor ErbB-2/imunologia , Animais , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Dimerização , Epitopos/imunologia , Feminino , Humanos , Camundongos Endogâmicos BALB C , Transdução de Sinais/imunologia , Trastuzumab/imunologiaRESUMO
Epidermal growth factor receptor (EGFR) blockade and radiation are efficacious in the treatment of cancer, but resistance is commonly reported. Studies have suggested that dysregulation of Notch signaling and enrichment of the cancer stem cell population underlie these treatment challenges. Our data show that dual targeting of EGFR and Notch2/3 receptors with antibody CT16 not only inhibited signaling mediated by these receptors but also showed a strong anti-stem cell effect both in vitro and in vivo. Treatment with CT16 prevented acquired resistance to EGFR inhibitors and radiation in non-small cell lung cancer (NSCLC) cell line models and patient-derived xenograft tumors. CT16 also had a superior radiosensitizing impact compared with EGFR inhibitors. CT16 in combination with radiation had a larger antitumor effect than the combination of radiation with EGFR inhibitors or tarextumab. Mechanistically, CT16 treatment inhibits the stem cell-like subpopulation, which has a high mesenchymal gene expression and DNA repair activity, and reduces tumor-initiating cell frequency. This finding highlights the capacity of a combined blockade of EGFR and Notch signaling to augment the response to radiation and suggests that CT16 may achieve clinical efficacy when combined with radiation in NSCLC treatment.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Receptores Notch/antagonistas & inibidores , Aldeído Desidrogenase/metabolismo , Animais , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Antineoplásicos/farmacologia , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos SCID , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Recent studies have shown that some members of the tripartite motif-containing protein (TRIM) family serve as important regulators of tumorigenesis. However, the biological role of TRIM14 in osteosarcoma remains to be established. In this study, we showed that TRIM14 is upregulated in human osteosarcoma specimens and cell lines, and correlated with osteosarcoma progression and shorter patient survival times. Functional studies demonstrated that overexpression of TRIM14 enhances osteosarcoma cell proliferation, clone formation, cell cycle procession, migration and invasion in vitro and promotes tumor growth in vivo, and conversely, its silencing has the opposite effects. Furthermore, TRIM14 overexpression induced activation of the AKT pathway. Inhibition of AKT expression reversed the TRIM14-mediated promotory effects on cell growth and mobility, in addition to TRIM14-induced epithelial-to-mesenchymal transition (EMT) and cyclin D1 upregulation. Our findings collectively suggest that TRIM14 functions as an oncogene by upregulating the AKT signaling pathway in osteosarcoma cells, supporting its potential utility as a therapeutic target for this disease.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Proteínas de Transporte/genética , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Adulto , Animais , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Osteossarcoma/mortalidade , Osteossarcoma/patologia , Prognóstico , Proteínas com Motivo Tripartido , Carga Tumoral , Adulto JovemRESUMO
The human epidermal growth factor receptor (EGFR) targeting chimeric monoclonal antibody, cetuximab (Erbitux®), is a widely used drug in the treatment of metastatic colorectal cancer. However, the activation of the extensive crosstalk among the EGFR family receptors as well as other tyrosine kinase receptors (RTKs) impairs the efficacy of the drug by fueling acquired resistance. To identify the responsible potential activation pathway underlying cetuximab resistance and generate novel treatment strategies, cetuximab-resistant colorectal cancer cell lines were generated and validated and a functional RNAi screen targeting human RTKs was used to identify extensive receptor tyrosine kinase signaling networks established in resistant cancer cells. MET, Axl, and IGF-1R were identified as contributors to the acquired resistance to cetuximab. Targeting vectored immunoprophylaxis (VIPs) to different RTKs were generated and characterized. Different VIP approaches were evaluated in vivo with parental and cetuximab-resistance xenografts and the RTKs in resistant cancer xenografts were inhibited with VIPs via re-sensitization to cetuximab treatment. Combination of VIPs was more broadly efficacious, mechanistically, due to co-blocking the EGFR/Axl/MET signaling pathway, which was cross-activated in the resistant cell lines. Moreover, a VIP-based procedural treatment strategy not only eliminated the tumor but also afforded long-lasting protection from tumor recurrence and resistance. Overall, EGFR-related RTK pathway-network activation represents a novel mechanism underlying cetuximab resistance. A broad VIP combination strategy and VIP-based procedural treatment strategy may be a recommended addition to cetuximab-based targeted therapy. Our results establish a new principle to achieve combined RTK inhibition and reverse drug resistance using a VIP approach.