RESUMO
In vitro, ß-amyloid (Aß) peptides form polymorphic fibrils, with molecular structures that depend on growth conditions, plus various oligomeric and protofibrillar aggregates. Here, we investigate structures of human brain-derived Aß fibrils, using seeded fibril growth from brain extract and data from solid-state nuclear magnetic resonance and electron microscopy. Experiments on tissue from two Alzheimer's disease (AD) patients with distinct clinical histories showed a single predominant 40 residue Aß (Aß40) fibril structure in each patient; however, the structures were different from one another. A molecular structural model developed for Aß40 fibrils from one patient reveals features that distinguish in-vivo- from in-vitro-produced fibrils. The data suggest that fibrils in the brain may spread from a single nucleation site, that structural variations may correlate with variations in AD, and that structure-specific amyloid imaging agents may be an important future goal.
Assuntos
Doença de Alzheimer/patologia , Amiloide/química , Encéfalo/patologia , Idoso , Amiloide/metabolismo , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Feminino , Humanos , Modelos BiológicosRESUMO
The penetrating growth of Li into the inorganic solid-state electrolyte (SSE) is one key factor limiting its practical application. Research to understand the underlying mechanism of Li penetration has been ongoing for years and is continuing. Here, we report an in situ scanning electron microscopy methodology to investigate the dynamic behaviors of isolated Li filaments in the garnet SSE under practical cycling conditions. We find that the filaments tend to grow in the SSE, while surprisingly, those filaments can self-dissolve with a decrease in the current density without a reversal of the current direction. We further build a coupled electro-chemo-mechanical model to assess the interplay between electrochemistry and mechanics during the dynamic evolution of filaments. We reveal that filament growth is strongly regulated by the competition between the electrochemical driving force and mechanical resistive force. The numerical results provide rational guidance for the design of solid-state batteries with excellent properties.
RESUMO
Self-assembling peptides represent a captivating area of study in nanotechnology and biomaterials. This interest is largely driven by their unique properties and the vast application potential across various fields such as catalytic functions. However, design complexities, including high-dimensional sequence space and structural diversity, pose significant challenges in the study of such systems. In this work, we explored the possibility of self-assembled peptides to catalyze the hydrolysis of hydrosilane for hydrogen production using ab initio calculations and carried out wet-lab experiments to confirm the feasibility of these catalytic reactions under ambient conditions. Further, we delved into the nuanced interplay between sequence, structural conformation, and catalytic activity by combining modeling with experimental techniques such as transmission electron microscopy and nuclear magnetic resonance and proposed a dual mode of the microstructure of the catalytic center. Our results reveal that although research in this area is still at an early stage, the development of self-assembled peptide catalysts for hydrogen production has the potential to provide a more sustainable and efficient alternative to conventional hydrogen production methods. In addition, this work also demonstrates that a computation-driven rational design supplemented by experimental validation is an effective protocol for conducting research on functional self-assembled peptides.
Assuntos
Hidrogênio , Peptídeos , Hidrogênio/química , Catálise , Peptídeos/química , Modelos Moleculares , HidróliseRESUMO
Recent studies have shown that long noncoding RNAs (lncRNAs) are critical regulators in the central nervous system (CNS). However, their roles in the cerebellum are currently unclear. In this work, we identified the isoform 204 of lncRNA Gm2694 (designated as lncRNA-Promoting Methylation (lncRNA-PM)) is highly expressed in the cerebellum and derived from the antisense strand of the upstream region of Cerebellin-1 (Cbln1), a well-known critical cerebellar synaptic organizer. LncRNA-PM exhibits similar spatiotemporal expression pattern as Cbln1 in the postnatal mouse cerebellum and activates the transcription of Cbln1 through Pax6/Mll1-mediated H3K4me3. In mouse cerebellum, lncRNA-PM, Pax6/Mll1, and H3K4me3 are all associated with the regulatory regions of Cbln1. Knockdown of lncRNA-PM in cerebellum causes deficiencies in Cbln1 expression, cerebellar synaptic integrity, and motor function. Together, our work reveals an lncRNA-mediated transcriptional activation of Cbln1 through Pax6-Mll1-H3K4me3 and provides novel insights of the essential roles of lncRNA in the cerebellum.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição PAX6/metabolismo , Precursores de Proteínas/metabolismo , RNA Longo não Codificante/metabolismo , Sinapses/metabolismo , Processamento Alternativo/genética , Cerebelo/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Humanos , Atividade Motora , Proteína de Leucina Linfoide-Mieloide/genética , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Longo não Codificante/genética , Ativação Transcricional/genéticaRESUMO
Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three ß-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation.
Assuntos
Amiloide/química , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Motivos de Aminoácidos , Amiloide/metabolismo , Microscopia Crioeletrônica , Humanos , Necroptose , Fosforilação , Domínios Proteicos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Tumour-derived exosomes (TDEs) play an important role in tumourigenesis and progression by regulating components in the tumour microenvironment (TME), however, the role of TDE-related immune genes in hepatocellular carcinoma is not fully known. We systematically analysed TDE genes from ExoCarta and immune genes from Immport,Machine learning ultimately identified eight TDE-related prognostic immune genes and used them as the basis for constructing a risk model, which was constructed to better predict patients with hepatocellular carcinoma (HCC) compared with published prognostic models. There were significant differences between the high and low risk groups in terms of biological functioning. Low-risk group were more sensitive to immunotherapy, the sensitivity to oxaliplatin and cisplatin differed between the high- and low-risk groups, and knockout of the core gene RAC1 limited the malignant biological behaviour of hepatocellular carcinoma cells. In conclusion, TIRGs are effective in predicting the prognosis of patients with hepatocellular carcinoma and provide a new perspective on immunotherapy and chemotherapy for patients.
Assuntos
Carcinoma Hepatocelular , Exossomos , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Exossomos/genética , Neoplasias Hepáticas/genética , Linhagem Celular , Transformação Celular Neoplásica , Microambiente Tumoral/genética , PrognósticoRESUMO
Aggregation of amyloid-ß peptides into fibrils or other self-assembled states is central to the pathogenesis of Alzheimer's disease. Fibrils formed in vitro by 40- and 42-residue amyloid-ß peptides (Aß40 and Aß42) are polymorphic, with variations in molecular structure that depend on fibril growth conditions. Recent experiments suggest that variations in amyloid-ß fibril structure in vivo may correlate with variations in Alzheimer's disease phenotype, in analogy to distinct prion strains that are associated with different clinical and pathological phenotypes. Here we investigate correlations between structural variation and Alzheimer's disease phenotype using solid-state nuclear magnetic resonance (ssNMR) measurements on Aß40 and Aß42 fibrils prepared by seeded growth from extracts of Alzheimer's disease brain cortex. We compared two atypical Alzheimer's disease clinical subtypes-the rapidly progressive form (r-AD) and the posterior cortical atrophy variant (PCA-AD)-with a typical prolonged-duration form (t-AD). On the basis of ssNMR data from 37 cortical tissue samples from 18 individuals, we find that a single Aß40 fibril structure is most abundant in samples from patients with t-AD and PCA-AD, whereas Aß40 fibrils from r-AD samples exhibit a significantly greater proportion of additional structures. Data for Aß42 fibrils indicate structural heterogeneity in most samples from all patient categories, with at least two prevalent structures. These results demonstrate the existence of a specific predominant Aß40 fibril structure in t-AD and PCA-AD, suggest that r-AD may relate to additional fibril structures and indicate that there is a qualitative difference between Aß40 and Aß42 aggregates in the brain tissue of patients with Alzheimer's disease.
Assuntos
Doença de Alzheimer/classificação , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Amiloide/química , Fragmentos de Peptídeos/química , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Amiloide/metabolismo , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Córtex Cerebral/química , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Fenótipo , Análise de Componente PrincipalRESUMO
Human cystic echinococcosis (CE) is a zoonotic disorder triggered by the larval stage of Echinococcus granulosus (E. granulosus) and predominantly occurred in the liver and lungs. The M2 macrophage level is considerably elevated among the liver of patients with hepatic CE and performs an integral function in liver fibrosis. However, the mechanism of CE inducing polarisation of macrophage to an M2 phenotype is unknown. In this study, macrophage was treated with E. granulosus cyst fluid (EgCF) to explore the mechanism of macrophage polarisation. Consequently, the expression of the M2 macrophage and production of anti-inflammatory cytokines increased after 48 h treatment by EgCF. In addition, EgCF promoted polarisation of macrophage to an M2 phenotype by inhibiting the expression of transcriptional factor hypoxia-inducible factor 1-alpha (HIF-1α), which increased the expression of glycolysis-associated genes, including hexokinase 2 (HK2) and pyruvate kinase 2 (PKM2). The HIF-1α agonist ML228 also inhibited the induction of macrophage to an M2 phenotype by EgCF in vitro. Our findings indicate that E. granulosus inhibits glycolysis by suppressing the expression of HIF-1α.
Assuntos
Equinococose , Echinococcus granulosus , Humanos , Animais , Líquido Cístico , Echinococcus granulosus/genética , Macrófagos , PulmãoRESUMO
The liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix-helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix-helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix-helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Amiloide , Proteínas Amiloidogênicas , Esclerose Lateral Amiotrófica/metabolismo , Agregados ProteicosRESUMO
T-follicular helper (TFH) cells are a subset of CD4+ helper T cells that help germinal center (GC) B-cell differentiation and high-affinity antibody production during germinal center reactions. Whether important extracellular molecules control TFH differentiation is not fully understood. Here, we demonstrate that a secreted protein extracellular matrix protein 1 (ECM1) is critical for TFH differentiation and antibody response. A lack of ECM1 inhibited TFH cell development and impaired GC B-cell reactions and antigen-specific antibody production in an antigen-immunized mouse model. ECM1 was induced by IL-6 and IL-21 in TFH cells, promoting TFH differentiation by down-regulating the level of STAT5 phosphorylation and up-regulating Bcl6 expression. Furthermore, injection of recombinant ECM1 protein into mice infected with PR8 influenza virus promoted protective immune responses effectively, by enhancing TFH differentiation and neutralizing antibody production. Collectively, our data identify ECM1 as a soluble protein to promote TFH cell differentiation and antibody production.
Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Proteínas da Matriz Extracelular/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Proteínas da Matriz Extracelular/genética , Vírus da Influenza A/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucinas/genética , Interleucinas/imunologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Linfócitos T Auxiliares-Indutores/citologiaRESUMO
TDP-43 is a primary pathological hallmark protein of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, which may exist in the form of amyloid inclusions in the cells of patients. In addition to serving as a biomarker for these diseases, TDP-43 can also directly trigger neurodegeneration. We previously determined the amyloidogenic core region of TDP-43 (residues 311-360) and showed by solution NMR that this region includes two α-helices [(321-330) and (335-343)] in solution. We suggested that the helix-to-sheet structural transformation initiates TDP-43 aggregation. In the present study, X-ray diffraction shows that TDP-43 (311-360) aggregates adopt a cross-ß structure. Thioredoxin (Trx)-fused TDP-43 (311-360) can undergo liquid-liquid phase separation (LLPS) before fibrillation, suggesting that phase separation is an intermediate step before amyloid formation. Solid-state NMR (SSNMR), carried out to elucidate the structural changes of TDP-43 (311-360) at the atomic level, indicates five ß-strands of the amyloids formed, with the major two ß-strands contributed by the first helical region in the solution structure. The NMR evidence is also in support of the fibril having a parallel in-register conformation, implying a mechanism in which the helix-helix interactions in LLPS are converted into ß-strand parallel lateral association upon fibrillation. Our studies have assigned many key interresidue interactions that contribute to the stability of the fibril, including F316 with I318 and Q327 and W334 with A325, A326, A329, and S332. SSNMR with 1H detection reveals a unique close interaction between the indole Nε1-Hε1 of W334 and the side-chain carbonyl of Q343. This interaction could be a very important factor in initiating TDP-43 (311-360) folding/misfolding in LLPS.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas Amiloidogênicas/química , Proteínas de Ligação a DNA/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Transição de Fase , Conformação Proteica , Multimerização ProteicaRESUMO
Tooth enamel is formed in an extracellular environment. Amelogenin, the major component in the protein matrix of tooth enamel during the developing stage, could assemble into high molecular weight structures, regulating enamel formation. However, the molecular structure of amelogenin protein assembly at the functional state is still elusive. In this work, we found that amelogenin is able to induce calcium phosphate minerals into hydroxyapatite (HAP) structure in vitro at pH 6.0. Assessed using X-ray diffraction (XRD) and 31P solid-state NMR (SSNMR) evidence, the formed HAP mimics natural enamel closely. The structure of amelogenin protein assembly coexisting with the HAP was also studied using atomic force microscopy (AFM), transmission electron microscopy (TEM) and XRD, indicating the ß-amyloid structure of the protein. SSNMR was proven to be an important tool in detecting both the rigid and dynamic components of the protein assembly in the sample, and the core sequence 18EVLTPLKWYQSI29 was identified as the major segment contributing to the ß-sheet secondary structure. Our research suggests an amyloid structure may be an important factor in controlling HAP formation at the right pH conditions with the help of other structural components in the protein assembly.
Assuntos
Amelogenina/metabolismo , Proteínas Amiloidogênicas/metabolismo , Durapatita/metabolismo , Amelogenina/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/ultraestrutura , Proteínas do Esmalte Dentário/química , Proteínas do Esmalte Dentário/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Proteínas Recombinantes , Análise Espectral , Relação Estrutura-AtividadeRESUMO
High-order assemblies of amelogenin, the major protein in enamel protein matrix, are believed to act as the template for enamel mineral formation. The Leucine-rich amelogenin (LRAP) is a natural splice-variant of amelogenin, a functional protein in vivo, containing conserved domains of amelogenin. In this work, we showed LRAP aggregates hierarchically into assemblies with various sizes including scattered beads, beads-on-a-string and gel-like precipitations in the presence of both calcium and phosphate ions. Solid-state NMR combined with X-ray diffraction and microscopic techniques, was applied to give a picture of LRAP self-assemblies at the atomic level. Our results, for the first time, confirmed LRAP assemblies with different sizes all contained a consistent rigid segment with ß-sheet secondary structure (residues 12-27) and the ß-sheet segment would further assemble into amyloid-like structures.
Assuntos
Amelogenina/química , Proteínas Amiloidogênicas/química , Leucina/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas Recombinantes/química , Amelogenina/genética , Amelogenina/metabolismo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Cálcio/química , Cálcio/metabolismo , Concentração de Íons de Hidrogênio , Leucina/metabolismo , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Fosfatos/química , Fosfatos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Difração de Raios X/métodosRESUMO
Molecular self-assembly provides a chemical strategy for the synthesis of nanostructures by using the principles of nature, and peptides serve as the promising building blocks to construct adaptable molecular architectures. Recently, a series of heptapeptides with alternative hydrophobic and hydrophilic residues were reported to form amyloid-like structures, which are capable of catalyzing acyl ester hydrolysis with remarkable efficiency. However, information remains elusive about the atomic structures of the fibrils. What is the origin of the sequence-dependent catalytic activity? How is the ester hydrolysis catalyzed by the fibrils? In this work, the atomic structures of the aggregates were determined by using molecular modeling and further validated by solid-state NMR experiments, where the fibril with high activity adopts twisted parallel configuration within each layer, and the one with low activity is in flat antiparallel configuration. The polymorphism originates from the interactions between different regions of the building block peptides, where the delicate balance between rigidity and flexibility plays an important role. We further show that the p-nitrophenylacetate ( pNPA) hydrolysis reactions catalyzed by two different fibrils follow a similar mechanism, and the difference in microenvironment at the active site between the natural enzyme and the present self-assembled fibrils should account for the discrepancy in catalytic activities. The present work provides understanding of the structure and function of self-assembled fibrils formed with short peptides at an atomic level and thus sheds new insight on designing aggregates with better functions.
Assuntos
Biocatálise , Peptídeos/química , Peptídeos/metabolismo , Agregados Proteicos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fenilacetatos/metabolismo , Conformação Proteica , Zinco/metabolismoRESUMO
Biomineralization processes govern the formation of hierarchical hard tissues such as bone and teeth in living organisms, and mimicking these processes could lead to the design of new materials with specialized properties. However, such advances require structural characterization of the proteins guiding biomineral formation to understand and mimic their impact. In their "active" form, biomineralization proteins are bound to a solid surface, severely limiting our ability to use many conventional structure characterization techniques. Here, solid-state NMR spectroscopy was applied to study the intermolecular interactions of amelogenin, the most abundant protein present during the early stages of enamel formation, in self-assembled oligomers bound to hydroxyapatite. Intermolecular dipolar couplings were identified that support amelogenin dimer formation stabilized by residues toward the C-termini. These dipolar interactions were corroborated by molecular dynamics simulations. A ß-sheet structure was identified in multiple regions of the protein, which is otherwise intrinsically disordered in the absence of hydroxyapatite. To our knowledge, this is the first intermolecular protein-protein interaction reported for a biomineralization protein, representing an advancement in understanding enamel development and a new general strategy toward investigating biomineralization proteins.
Assuntos
Amelogenina/química , Amelogenina/metabolismo , Durapatita/metabolismo , Sequência de Aminoácidos , Animais , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação ProteicaRESUMO
We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg(18)-Leu mutant (R18L-CA), and R18L-CA spheres with 20-100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of (15)N,(13)C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in (15)N and (13)C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.
Assuntos
Capsídeo/química , HIV-1/química , Proteínas do Capsídeo , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
OBJECTIVES: To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin. RESULTS: Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 °C, naringinase showed enhanced α-L-rhamnosidase activity while the ß-D-glucosidase activity was abrogated. The addition of Mg(2+), Fe(2+) and Co(2+) was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 °C), linarin could be nearly completely transformed to tilianin with excellent selectivity (>98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and (1)H-NMR. CONCLUSION: A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the α-L-rhamnosidase activity of naringinase and blocked its ß-D-glucosidase activity.
Assuntos
Flavonoides/metabolismo , Glicosídeos/metabolismo , Complexos Multienzimáticos/metabolismo , beta-Glucosidase/metabolismo , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
OBJECTIVE: Sorafenib is a chemotherapeutic agent used to treat hepatocellular carcinoma (HCC). However, its clinical response rates are often low. Tumour-associated macrophages (TAMs) have been implicated in tumour resistance. The relationship between TAMs-derived exosomes and primary resistance to sorafenib in hepatocellular carcinoma is unclear. METHODS: The study analysed RNA-SEQ data from TCGA-LIHC to explore the relationship between TAMs and sorafenib IC50. THP-1-induced M2 macrophages were used as a model to investigate the relationship between M2 macrophage exosomes and primary resistance to sorafenib in hepatocellular carcinoma cells using apoptosis, colony generation, cell viability and dual luciferase. RESULTS: M2 macrophage score and sorafenib IC50 were positively correlated in hepatocellular carcinoma patients, M2 macrophage exosomes promoted sorafenib resistance in hepatocellular carcinoma cells, and M2-exo-miR-200c-3p facilitated the development of sorafenib resistance in hepatocellular carcinoma cells by mediating the activation of PI3K/AKT. CONCLUSION: We propose and demonstrate for the first time that M2 macrophage exosomes promote sorafenib resistance in hepatocellular carcinoma, providing a new perspective for the clinical treatment of hepatocellular carcinoma patients.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Resistencia a Medicamentos Antineoplásicos , Exossomos , Neoplasias Hepáticas , MicroRNAs , Sorafenibe , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Exossomos/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/efeitos dos fármacos , Macrófagos Associados a Tumor/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Células THP-1RESUMO
Receptor-interacting protein kinase 1 (RIPK1) is a therapeutic target in treating neurodegenerative diseases and cancers. RIPK1 has three distinct functional domains, with the center domain containing a receptor-interacting protein homotypic interaction motif (RHIM), which mediates amyloid formation. The functional amyloid formed by RIPK1 and/or RIPK3 is a crucial intermediate in regulating cell necroptosis. In this study, the amyloid structure of mouse RIPK1, formed by an 82-residue sequence centered at RHIM, is presented. It reveals the "N"-shaped folding of the protein subunit in the fibril with four ß-strands. The folding pattern is shared by several amyloid structures formed by proteins with RHIM, with the central ß-strand formed by the most conserved tetrad sequence I/VQI/VG. However, the solid-state NMR results indicate a structural difference between mouse RIPK1 and mouse RIPK3. A change in the structural rigidity is also suggested by the observation of weakened signals for mouse RIPK3 upon mixing with RIPK1 to form the RIPK1/RIPK3 complex fibrils. Our results provide vital information to understand the interactions between different proteins with RHIM, which will help us further comprehend the regulation mechanism in cell necroptosis.
Assuntos
Amiloide , Proteína Serina-Treonina Quinases de Interação com Receptores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Camundongos , Amiloide/metabolismo , Amiloide/química , Humanos , Necroptose , Sequência de Aminoácidos , Domínios Proteicos , Ligação Proteica , Modelos MolecularesRESUMO
The conditions present during enamel crystallite development change dramatically as a function of time, including the pH, protein concentration, surface type, and ionic strength. In this work, we investigate the role that two of these changing conditions, pH and ionic strength, have in modulating the interaction of the amelogenin, LRAP, with hydroxyapatite (HAP). Using solid-state NMR dipolar recoupling and chemical shift data, we investigate the structure, orientation, and dynamics of three regions in the N-terminus of the protein: L(15) to V(19), V(19) to L(23), and K(24) to S(28). These regions are also near the only phosphorylated residue in the protein pS(16); therefore, changes in the LRAP-HAP interaction as a function of phosphorylation (LRAP(-P) vs LRAP(+P)) were also investigated. All of the regions and conditions studied for the surface immobilized proteins showed restricted motion, with indications of slightly more mobility under all conditions for L(15)(+P) and K(24)(-P). The structure and orientation of the LRAP-HAP interaction in the N-terminus of the phosphorylated protein is very stable to changing solution conditions. From REDOR dipolar recoupling data, the structure and orientation in the region L(15)V(19)(+P) did not change significantly as a function of pH or ionic strength. The structure and orientation of the region V(19)L(23)(+P) were also stable to changes in pH, with the only significant change observed at high ionic strength, where the region becomes extended, suggesting this may be an important region in regulating mineral development. Chemical shift studies also suggest minimal changes in all three regions studied for both LRAP(-P) and LRAP(+P) as a function of pH or ionic strength, and also reveal that K(24) has multiple resolvable resonances, suggestive of two coexisting structures. Phosphorylation also alters the LRAP-HAP interface. All of the three residues investigated (L(15), V(19), and K(24)) are closer to the surface in LRAP(+P), but only K(24)S(28) changes structure as a result of phosphorylation, from a random coil to a largely helical structure, and V(19)L(23) becomes more extended at high ionic strength when phosphorylated. These observations suggest that ionic strength and dephosphorylation may provide switching mechanisms to trigger a change in the function of the N-terminus during enamel development.