RESUMO
The adsorption of proteins onto surfaces significantly impacts biomaterials, medical devices, and biological processes. This study aims to provide insights into the irreversible adsorption process of multiprotein complexes, particularly focusing on the interaction between anti-His6 IgG antibodies and the His6-tagged P2X2 receptor. Traditional approaches to understanding protein adsorption have centered around kinetic and thermodynamic models, often examining individual proteins and surface coverage, typically through Molecular Dynamics (MD) simulations. In this research, we introduce a computational approach employing Autodesk Maya 3D software for the investigation of multiprotein complexes' adsorption behavior. Utilizing Atomic Force Microscopy (AFM) imaging and Maya 3D-based mechanical simulations, our study yields real-time structural and kinetic observations. Our combined experimental and computational findings reveal that the P2X2 receptor-IgG antibody complex likely undergoes absorption in an 'extended' configuration. Whereas the P2X2 receptor is less adsorbed once is complexed to the IgG antibody compared to its individual state, the opposite is observed for the antibody. This insight enhances our understanding of the role of protein-protein interactions in the process of protein adsorption.
Assuntos
Imunoglobulina G , Simulação de Dinâmica Molecular , Adsorção , Receptores Purinérgicos P2X2 , Microscopia de Força Atômica , Complexos MultiproteicosRESUMO
BACKGROUND & AIMS: Epigenetic regulation of gene expression plays a critical role in the development of liver cancer; however, the molecular mechanisms of epigenetic-driven liver cancers are not well understood. The aims of this study were to examine molecular mechanisms that cause the dedifferentiation of hepatocytes into cancer cells in aggressive hepatoblastoma and test if the inhibition of these mechanisms inhibits tumor growth. METHODS: We have analyzed CCAAT/Enhancer Binding Protein alpha (C/EBPα), Transcription factor Sp5, and histone deacetylase (HDAC)1 pathways from a large biobank of fresh hepatoblastoma (HBL) samples using high-pressure liquid chromatography-based examination of protein-protein complexes and have examined chromatin remodeling on the promoters of markers of hepatocytes and p21. The HDAC1 activity was inhibited in patient-derived xenograft models of HBL and in cultured hepatoblastoma cells and expression of HDAC1-dependent markers of hepatocytes was examined. RESULTS: Analyses of a biobank showed that a significant portion of HBL patients have increased levels of an oncogenic de-phosphorylated-S190-C/EBPα, Sp5, and HDAC1 compared with amounts of these proteins in adjacent regions. We found that the oncogenic de-phosphorylated-S190-C/EBPα is created in aggressive HBL by protein phosphatase 2A, which is increased within the nucleus and dephosphorylates C/EBPα at Ser190. C/EBPα-HDAC1 and Sp5-HDAC1 complexes are abundant in hepatocytes, which dedifferentiate into cancer cells. Studies in HBL cells have shown that C/EBPα-HDAC1 and Sp5-HDAC1 complexes reduce markers of hepatocytes and p21 via repression of their promoters. Pharmacologic inhibition of C/EBPα-HDAC1 and Sp5-HDAC1 complexes by Suberoylanilide hydroxamic acid (SAHA) and small interfering RNA-mediated inhibition of HDAC1 increase expression of hepatocyte markers, p21, and inhibit proliferation of cancer cells. CONCLUSIONS: HDAC1-mediated repression of markers of hepatocytes is an essential step for the development of HBL, providing background for generation of therapies for aggressive HBL by targeting HDAC1 activities.
Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Hepatócitos/metabolismo , Histona Desacetilase 1/metabolismo , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Quinases Ativadas por p21/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Hepatócitos/patologia , Histona Desacetilase 1/genética , Humanos , Neoplasias Hepáticas/patologia , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Quinases Ativadas por p21/genéticaRESUMO
The Retinoblastoma protein (pRb) is a key cell cycle regulator conserved in a wide variety of organisms. Experimental analysis of pRb's functions in animals and plants has revealed that this protein participates in cell proliferation and differentiation processes. In addition, pRb in animals and its orthologs in plants (RBR), are part of highly conserved protein complexes which suggest the possibility that analogies exist not only between functions carried out by pRb orthologs themselves, but also in the structure and roles of the protein networks where these proteins are involved. Here, we present examples of pRb/RBR participation in cell cycle control, cell differentiation, and in the regulation of epigenetic changes and chromatin remodeling machinery, highlighting the similarities that exist between the composition of such networks in plants and animals.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Montagem e Desmontagem da Cromatina , Epigênese Genética , Proteínas de Plantas/fisiologia , Proteína do Retinoblastoma/fisiologia , Animais , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/fisiologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Diferenciação Celular/genética , Dano ao DNA , Genes de Plantas , Genes do Retinoblastoma , Homeostase , Mamíferos/genética , Mamíferos/metabolismo , Modelos Moleculares , Família Multigênica , Complexos Multiproteicos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Proteínas de Plantas/química , Plantas/genética , Plantas/metabolismo , Conformação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteína do Retinoblastoma/química , Especificidade da Espécie , Células-Tronco/metabolismoRESUMO
Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, which associate with KHARON. KHAP1 is located only in the subpellicular microtubules, whereas KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.
Assuntos
Divisão Celular , Proteínas do Citoesqueleto/metabolismo , Flagelos/metabolismo , Leishmania mexicana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas do Citoesqueleto/genética , Flagelos/genética , Leishmania mexicana/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Complexos Multiproteicos/genética , Transporte Proteico , Proteínas de Protozoários/genéticaRESUMO
Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.
Assuntos
Complexos Multiproteicos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Códon de Iniciação , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Fatores de Iniciação de Peptídeos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Protein-protein interactions (PPIs) are fundamental in many biological processes and understanding these interactions is key for a myriad of applications including drug development, peptide design and identification of drug targets. The biological data deluge demands efficient and scalable methods to characterize and understand protein-protein interfaces. In this paper, we present ppiGReMLIN, a graph based strategy to infer interaction patterns in a set of protein-protein complexes. Our method combines an unsupervised learning strategy with frequent subgraph mining in order to detect conserved structural arrangements (patterns) based on the physicochemical properties of atoms on protein interfaces. To assess the ability of ppiGReMLIN to point out relevant conserved substructures on protein-protein interfaces, we compared our results to experimentally determined patterns that are key for protein-protein interactions in 2 datasets of complexes, Serine-protease and BCL-2. RESULTS: ppiGReMLIN was able to detect, in an automatic fashion, conserved structural arrangements that represent highly conserved interactions at the specificity binding pocket of trypsin and trypsin-like proteins from Serine-protease dataset. Also, for the BCL-2 dataset, our method pointed out conserved arrangements that include critical residue interactions within the conserved motif LXXXXD, pivotal to the binding specificity of BH3 domains of pro-apoptotic BCL-2 proteins towards apoptotic suppressors. Quantitatively, ppiGReMLIN was able to find all of the most relevant residues described in literature for our datasets, showing precision of at least 69% up to 100% and recall of 100%. CONCLUSIONS: ppiGReMLIN was able to find highly conserved structures on the interfaces of protein-protein complexes, with minimum support value of 60%, in datasets of similar proteins. We showed that the patterns automatically detected on protein interfaces by our method are in agreement with interaction patterns described in the literature.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Animais , Gráficos por Computador , Mineração de Dados , Complexos Multiproteicos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tripsina/química , Tripsina/metabolismoRESUMO
The ability to perceive the environment, an essential attribute in living organisms, is linked to the evolution of signaling proteins that recognize specific signals and execute predetermined responses. Such proteins constitute concerted systems that can be as simple as a unique protein, able to recognize a ligand and exert a phenotypic change, or extremely complex pathways engaging dozens of different proteins which act in coordination with feedback loops and signal modulation. To understand how cells sense their surroundings and mount specific adaptive responses, we need to decipher the molecular workings of signal recognition, internalization, transfer, and conversion into chemical changes inside the cell. Protein allostery and dynamics play a central role. Here, we review recent progress on the study of two-component systems, important signaling machineries of prokaryotes and lower eukaryotes. Such systems implicate a sensory histidine kinase and a separate response regulator protein. Both components exploit protein flexibility to effect specific conformational rearrangements, modulating protein-protein interactions, and ultimately transmitting information accurately. Recent work has revealed how histidine kinases switch between discrete functional states according to the presence or absence of the signal, shifting key amino acid positions that define their catalytic activity. In concert with the cognate response regulator's allosteric changes, the phosphoryl-transfer flow during the signaling process is exquisitely fine-tuned for proper specificity, efficiency and directionality.
Assuntos
Proteínas/metabolismo , Transdução de Sinais , Regulação Alostérica , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Células Eucarióticas/metabolismo , Histidina Quinase/química , Histidina Quinase/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fosforilação , Células Procarióticas/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Human syncytiotrophoblast mitochondria require the activity of the isocitrate dehydrogenase type 2 (IDH2) to obtain reduced coenzymes for progesterone (P4) synthesis. Data from the literature indicate that mitochondrial steroidogenic contact sites transform efficiently cholesterol into P4. In this research, we identified the IDH2 as a member of the steroidogenic contact site and analyzed the steroidogenic role of its activity. METHOD: Human syncytiotrophoblast mitochondria were isolated by differential centrifugation, and steroidogenic contact sites were obtained by osmotic shock and sucrose gradient ultracentrifugation. In-gel native activity assay, mass spectroscopy, and western blot were used to identify the association of proteins and their activities. P4 was determined by immunofluorescence. RESULTS: The IDH2 was mainly identified in steroidogenic contact sites, and its activity was associated with a complex of proteins with an apparent molecular mass of ~590â¯kDa. Mass spectroscopy showed many groups of proteins with several metabolic functions, including steroidogenesis and ATP synthesis. The IDH2 activity was coupled to P4 synthesis since in the presence of Ca2+ or Na2SeO3, inhibitors of the IDH2, the P4 production decreased. CONCLUSIONS: The human syncytiotrophoblast mitochondria build contact sites for steroidogenesis. The IDH2, a non-membrane protein, supplies the NADPH required for the synthesis of P4 in a complex (steroidosome) that associate the proteins required to transform efficiently cholesterol into P4, which is necessary in pregnancy to maintain the relationship between mother and fetus. GENERAL SIGNIFICANCE: The IDH2 is proposed as a check point in the regulation of placental steroidogenesis.
Assuntos
Isocitrato Desidrogenase/metabolismo , Complexos Multiproteicos/metabolismo , Placenta/metabolismo , Progesterona/metabolismo , Esteroides/biossíntese , Adolescente , Adulto , Feminino , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , Gravidez , Progesterona/análise , Ligação Proteica , Esteroides/análise , Trofoblastos/química , Trofoblastos/metabolismo , Trofoblastos/ultraestrutura , Adulto JovemRESUMO
Van der Waals forces are determinants of the formation of protein-ligand complexes. Physical models based on the Lennard-Jones potential can estimate van der Waals interactions with considerable accuracy and with a computational complexity that allows its application to molecular docking simulations and virtual screening of large databases of small organic molecules. Several empirical scoring functions used to evaluate protein-ligand interactions approximate van der Waals interactions with the Lennard-Jones potential. In this chapter, we present the main concepts necessary to understand van der Waals interactions relevant to molecular recognition of a ligand by the binding pocket of a protein target. We describe the Lennard-Jones potential and its application to calculate potential energy for an ensemble of structures to highlight the main features related to the importance of this interaction for binding affinity.
Assuntos
Desenho de Fármacos , Modelos Teóricos , Complexos Multiproteicos/química , Proteínas/química , Algoritmos , LigantesRESUMO
Ragulator is a pentamer composed of p18, MP1, p14, C7orf59, and hepatitis B virus X-interacting protein (HBXIP; LAMTOR 1-5) which acts as a lysosomal scaffold of the Rag GTPases in the amino acid sensitive branch of TORC1 signaling. Here, we present the crystal structure of human HBXIP-C7orf59 dimer (LAMTOR 4/5) at 2.9 Å and identify a phosphorylation site on C7orf59 which modulates its interaction with p18. Additionally, we demonstrate the requirement of HBXIP-C7orf59 to stabilize p18 and allow further binding of MP1-p14. The structure of the dimer revealed an unfolded N terminus in C7orf59 (residues 1-15) which was shown to be essential for p18 binding. Full-length p18 does not interact stably with MP1-p14 in the absence of HBXIP-C7orf59, but deletion of p18 residues 108-161 rescues MP1-p14 binding. C7orf59 was phosphorylated by protein kinase A (PKA) in vitro and mutation of the conserved Ser67 residue to aspartate prevented phosphorylation and negatively affected the C7orf59 interaction with p18 both in cell culture and in vitro. C7orf59 Ser67 was phosphorylated in human embryonic kidney 293T cells. PKA activation with forskolin induced dissociation of p18 from C7orf59, which was prevented by the PKA inhibitor H-89. Our results highlight the essential role of HBXIP-C7orf59 dimer as a nucleator of pentameric Ragulator and support a sequential model of Ragulator assembly in which HBXIP-C7orf59 binds and stabilizes p18 which allows subsequent binding of MP1-p14.