RESUMO
Deposition of amyloid plaques in the brains of Alzheimer's disease (AD) patients is a hallmark of the disease. AD plaques consist primarily of the beta-amyloid (Aß) peptide but can contain other factors such as lipids, proteoglycans, and chaperones. So far, it is unclear how the cellular environment modulates fibril polymorphism and how differences in fibril structure affect cell viability. The small heat-shock protein (sHSP) alpha-B-Crystallin (αBC) is abundant in brains of AD patients, and colocalizes with Aß amyloid plaques. Using solid-state NMR spectroscopy, we show that the Aß40 fibril seed structure is not replicated in the presence of the sHSP. αBC prevents the generation of a compact fibril structure and leads to the formation of a new polymorph with a dynamic N-terminus. We find that the N-terminal fuzzy coat and the stability of the C-terminal residues in the Aß40 fibril core affect the chemical and thermodynamic stability of the fibrils and influence their seeding capacity. We believe that our results yield a better understanding of how sHSP, such as αBC, that are part of the cellular environment, can affect fibril structures related to cell degeneration in amyloid diseases.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Cadeia B de alfa-Cristalina , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Humanos , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/genética , Amiloide/química , Amiloide/metabolismoRESUMO
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Choque Térmico Pequenas/química , Chaperonas Moleculares/química , Conformação Proteica , Sequência de Aminoácidos , Animais , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/classificação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico Pequenas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mutação , Filogenia , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
Small heat-shock proteins (sHsps) compose the most widespread family of molecular chaperones. The human genome encodes 10 different sHsps (HspB1-10). It has been shown that HspB1 (Hsp27), HspB5 (αB-crystallin), and HspB6 (Hsp20) can form hetero-oligomers in vivo However, the impact of hetero-oligomerization on their structure and chaperone mechanism remains enigmatic. Here, we analyzed hetero-oligomer formation in human cells and in vitro using purified proteins. Our results show that the effect of hetero-oligomer formation on the composition of the sHsp ensembles and their chaperone activities depends strongly on the respective sHsps involved. We observed that hetero-oligomer formation between HspB1 and HspB5 leads to an ensemble that is dominated by species larger than the individual homo-oligomers. In contrast, the interaction of dimeric HspB6 with either HspB1 or HspB5 oligomers shifted the ensemble toward smaller oligomers. We noted that the larger HspB1-HspB5 hetero-oligomers are less active and that HspB6 activates HspB5 by dissociation to smaller oligomer complexes. The chaperone activity of HspB1-HspB6 hetero-oligomers, however, was modulated in a substrate-specific manner, presumably due to the specific enrichment of an HspB1-HspB6 heterodimer. These heterodimeric species may allow the tuning of the chaperone properties toward specific substrates. We conclude that sHsp hetero-oligomerization exerts distinct regulatory effects depending on the sHsps involved.
Assuntos
Proteínas de Choque Térmico Pequenas/metabolismo , Multimerização Proteica , Células CACO-2 , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células MCF-7RESUMO
Analysis of membrane proteins is still inadequately represented in diagnostics despite their importance as drug targets and biomarkers. One main reason is the difficult handling caused by their insolubility in aqueous buffer solutions. The nanodisc technology was developed to circumvent this challenge and enables analysis of membrane proteins with standard research methods. However, existing nanodisc generation protocols rely on time-consuming membrane isolation and protein purification from overexpression systems. In the present study, we present a novel, simplified procedure for the rapid generation of nanodiscs directly from intact cells. Workflow and duration of the nanodisc preparation were shortened without reducing the reconstitution efficiency, and all the steps were modified for the use of only standard laboratory equipment. This protocol was successfully applied to various human cell types, such as cultivated human embryonic kidney 293 (HEK-293) cells, as well as freshly isolated human red blood cells and platelets. In addition, the reconstitution of membrane proteins from cell organelles was achieved. The use of endogenous lipids ensures a native-like environment, which promotes native protein (re)folding. Nanodisc generation was verified by size exclusion chromatography and EM, whereas incorporation of different membrane proteins was demonstrated by Western blot analysis. Our protocol enabled the rapid incorporation of endogenous membrane proteins from human cells into nanodiscs, which can be applied to analytical approaches.
Assuntos
Plaquetas/metabolismo , Eritrócitos/metabolismo , Rim/metabolismo , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Plaquetas/ultraestrutura , Cromatografia em Gel , Eritrócitos/ultraestrutura , Células HEK293 , Humanos , Rim/ultraestrutura , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Organelas/metabolismo , Organelas/ultraestrutura , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Dobramento de Proteína , Redobramento de ProteínaRESUMO
A concise asymmetric synthesis has been developed to prepare idasanutlin, a small molecule MDM2 antagonist. Idasanutlin is currently being investigated as a potential treatment for various solid tumors and hematologic malignancies. The highly congested pyrrolidine core, containing four contiguous stereocenters, was constructed via a Cu(I)/(R)-BINAP catalyzed [3+2]-cycloaddition reaction. This optimized copper(I)-catalyzed process has been used to produce more than 1500 kg of idasanutlin. The manufacturing process will be described, highlighting the exceptionally selective and consistent cycloaddition/isomerization/hydrolysis sequence. The excellent yields, short cycle times and reduction in waste streams result in a sustainable production process with low environmental impact.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirrolidinas/síntese química , para-Aminobenzoatos/síntese química , Catálise , Cobre , Reação de Cicloadição , Hidrólise , IsomerismoRESUMO
Members of the kinesin-8 motor class have the remarkable ability to both walk towards microtubule plus-ends and depolymerise these ends on arrival, thereby regulating microtubule length. To analyse how kinesin-8 multitasks, we studied the structure and function of the kinesin-8 motor domain. We determined the first crystal structure of a kinesin-8 and used cryo-electron microscopy to calculate the structure of the microtubule-bound motor. Microtubule-bound kinesin-8 reveals a new conformation compared with the crystal structure, including a bent conformation of the α4 relay helix and ordering of functionally important loops. The kinesin-8 motor domain does not depolymerise stabilised microtubules with ATP but does form tubulin rings in the presence of a non-hydrolysable ATP analogue. This shows that, by collaborating, kinesin-8 motor domain molecules can release tubulin from microtubules, and that they have a similar mechanical effect on microtubule ends as kinesin-13, which enables depolymerisation. Our data reveal aspects of the molecular mechanism of kinesin-8 motors that contribute to their unique dual motile and depolymerising functions, which are adapted to control microtubule length.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Cinesinas/genética , Microtúbulos/metabolismo , Modelos Moleculares , Ligação ProteicaRESUMO
The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.
Assuntos
Proteínas de Choque Térmico Pequenas , Proteínas de Choque Térmico Pequenas/metabolismo , ProteínasRESUMO
Coronavirus infections are a world-wide threat to human health. A promising strategy to develop a broadly active antiviral is the use of fusion proteins consisting of an antibody IgG Fc region and a human ACE2 domain to which the viral spike proteins bind. Here we create antiviral fusion proteins based on IgM scaffolds. The hexameric ACE2-IgM-Fc fusions can be efficiently produced in mammalian cells and they neutralize the infectious virus with picomolar affinity thus surpassing monomeric ACE2-IgM-Fc by up to 96-fold in potency. In addition, the ACE2-IgM fusion shows increased neutralization efficiency for the highly infectious SARS-CoV-2 omicron variant in comparison to prototypic SARS-CoV-2. Taken together, these multimeric IgM fusions proteins are a powerful weapon to fight coronavirus infections.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Animais , Humanos , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Antivirais/farmacologia , Peptidil Dipeptidase A , Ligação Proteica , Imunoglobulina M , MamíferosRESUMO
AA amyloidosis is one of the most prevalent forms of systemic amyloidosis and affects both humans and other vertebrates. In this study, we compare MAS solid-state NMR data with a recent cryo-EM study of fibrils involving full-length murine SAA1.1. We address the question whether the specific requirements for the reconstitution of an amyloid fibril structure by cryo-EM can potentially yield a bias towards a particular fibril polymorph. We employ fibril seeds extracted from in to vivo material to imprint the fibril structure onto the biochemically produced protein. Sequential assignments yield the secondary structure elements in the fibril state. Long-range DARR and PAR experiments confirm largely the topology observed in the ex-vivo cryo-EM study. We find that the ß-sheets identified in the NMR experiments are similar to the ß-sheets found in the cryo-EM study, with the exception of amino acids 33-42. These residues cannot be assigned by solid-state NMR, while they adopt a stable ß-sheet in the cryo-EM structure. We suggest that the differences between MAS solid-state NMR and cryo-EM data are a consequence of a second conformer involving residues 33-42. Moreover, we were able to characterize the dynamic C-terminal tail of SAA in the fibril state. The C-terminus is flexible, remains detached from the fibrils, and does not affect the SAA fibril structure as confirmed further by molecular dynamics simulations. As the C-terminus can potentially interact with other cellular components, binding to cellular targets can affect its accessibility for protease digestion.
RESUMO
Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. Its chaperone activity is activated by oligomer dissociation at heat shock temperatures. Hsp26 contains 9 phosphorylation sites in different structural elements. Our analysis of phospho-mimetic mutations shows that phosphorylation activates Hsp26 at permissive temperatures. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, several phosphorylation sites in the C-terminal extension, which link subunits within the oligomer, are sensitive to the introduction of negative charges. In all cases, the intrinsic inhibition of chaperone activity is relieved and the N-terminal domain becomes accessible for substrate protein binding. The weakening of domain interactions within and between subunits by phosphorylation to activate the chaperone activity in response to proteotoxic stresses independent of heat stress could be a general regulation principle of sHsps.
Assuntos
Proteínas de Choque Térmico/metabolismo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Dicroísmo Circular , Microscopia Crioeletrônica , Transferência Ressonante de Energia de Fluorescência , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Espectrometria de Massas em Tandem , TemperaturaRESUMO
The prevalent model for cataract formation in the eye lens posits that damaged crystallin proteins form light-scattering aggregates. The α-crystallins are thought to counteract this process as chaperones by sequestering misfolded crystallin proteins. In this scenario, chaperone pool depletion would result in lens opacification. Here we analyze lenses from different mouse strains that develop early-onset cataract due to point mutations in α-, ß-, or γ-crystallin proteins. We find that these mutant crystallins are unstable in vitro; in the lens, their levels are substantially reduced, and they do not accumulate in the water-insoluble fraction. Instead, all the other crystallin proteins, including the α-crystallins, are found to precipitate. The changes in protein composition and spatial organization of the crystallins observed in the mutant lenses suggest that the imbalance in the lenticular proteome and altered crystallin interactions are the bases for cataract formation, rather than the aggregation propensity of the mutant crystallins.
Assuntos
Catarata/metabolismo , Cristalinas/metabolismo , Cristalino , Agregação Patológica de Proteínas , Animais , Cristalino/metabolismo , Cristalino/patologia , Camundongos , Chaperonas Moleculares/metabolismo , Proteoma/metabolismoRESUMO
A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs.
Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanoestruturas/química , Canal de Ânion 1 Dependente de Voltagem/genética , Fenômenos Biofísicos , Membrana Celular/química , Membrana Celular/genética , Humanos , Proteínas de Membrana/genética , Fosfolipídeos/química , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Canal de Ânion 1 Dependente de Voltagem/química , Proteína bcl-X/química , Proteína bcl-X/genéticaRESUMO
Cytoplasmic dynein, a microtubule-based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 µm. We also identified the ratio of Mg2+ -free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.
Assuntos
Trifosfato de Adenosina/química , Citoplasma/química , Dineínas/química , Pinças Ópticas , Trifosfato de Adenosina/metabolismo , Animais , Citoplasma/metabolismo , Dineínas/metabolismo , SuínosRESUMO
The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.
Assuntos
Cadeia A de alfa-Cristalina/química , Microscopia Crioeletrônica , Humanos , Cristalino/química , Modelos Moleculares , Oxirredução , Conformação Proteica , Multimerização Proteica , Desdobramento de Proteína , Cadeia A de alfa-Cristalina/ultraestruturaRESUMO
PURPOSE: Multiple cell types secrete exosome-like extracellular vesicles (ELVs) to the extracellular environment. Pathological conditions can produce characteristic changes to the vesicle cargo. We investigated if ionizing radiation is capable of inducing changes in the protein and microRNA (miRNA) cargo of ELVs. MATERIALS AND METHODS: Whole blood samples from healthy donors were irradiated with 2 Gy gamma rays and then peripheral blood mononuclear cells and plasma were separated from residual blood and co-cultivated for 24 h. The released ELVs were collected by differential ultracentrifugation from irradiated and non-irradiated samples. microRNAs and proteins were quantified by qPCR and label-free proteomics. RESULTS: Here we report a first characterization of radiation-induced changes in the protein and miRNA cargo of ELVs isolated from plasma. Proteome analysis of ELVs identified 214 proteins, of which nine significantly changed their abundance after irradiation. The radiation-induced down-regulation of afamin and serpine peptidase F1 was confirmed by immunoblotting. miRNA expression profiling identified 58 different exosomal miRNAs, the expression of miR-204-5p, miR-92a-3p and miR-31-5p was significantly increased in ELVs from irradiated samples. CONCLUSIONS: This study provides evidence that radiation-induced changes occur in the protein and miRNA cargo of plasma ELVs. These data imply a novel systemic communication pathway between irradiated and non-irradiated cells and tissues.
Assuntos
Exossomos/metabolismo , Exossomos/efeitos da radiação , Leucócitos Mononucleares/metabolismo , Proteínas de Membrana/sangue , MicroRNAs/sangue , Radiação Ionizante , Adulto , Idoso , Células Cultivadas , Relação Dose-Resposta à Radiação , Feminino , Humanos , Leucócitos Mononucleares/efeitos da radiação , Pessoa de Meia-Idade , Proteoma/metabolismo , Doses de RadiaçãoRESUMO
BACKGROUND: A simplified conditioning protocol including single-dose preoperative thymic and low-dose whole body irradiation with or without subsequent donor bone marrow transplantation (BMTx) can be applied in porcine lung transplantation. We hypothesized that this protocol would prolong allograft survival. METHODS: Left-sided single lung transplantation from major histocompatibility complex (MHC)-mismatched donors was performed in 27 minipigs. Recipients received whole body (1.5 Gy) and thymic irradiation (7 Gy) before transplantation (IRR group, n=6), intravenous immunosuppression with methylprednisolone, cyclosporine, and azathioprine for 27 postoperative days (IS group, n=5) or both (IRR+IS group, n=10). BMTx group recipients were treated with irradiation, immunosuppression and a donor bone marrow infusion on postoperative day 1. Peripheral blood leukocyte phenotype and donor cell chimerism were monitored by flow cytometry. Purified CD25+ T cells from long-term survivors or rejecting animals were used for in vitro MLR suppression assays. RESULTS: Median graft survival was: IRR 12 days, IS 55 days, IRR+IS 239 days, and BMTx 80 days (P<0.0001). Early peripheral blood macrochimerism was substantial in both the IRR+IS and the BMTX group but was lost in all groups after day 80. The frequency and suppressive function of CD4+CD25+ T cells were enhanced in IRR+IS group long-term survivors. CONCLUSION: Although donor bone marrow infusion was not beneficial in our model, a substantial proportion of the animals treated with irradiation and a 28-day course of immunosuppression accepted their lung allografts long term. The mechanism involved in maintaining allograft tolerance may be based on peripheral T-cell regulation.
Assuntos
Sobrevivência de Enxerto/efeitos da radiação , Transplante de Pulmão/imunologia , Linfócitos T Reguladores/efeitos da radiação , Animais , Transplante de Medula Óssea , Antígenos CD4/análise , Sobrevivência de Enxerto/imunologia , Pulmão/imunologia , Pulmão/patologia , Teste de Cultura Mista de Linfócitos , Modelos Animais , Receptores de Interleucina-2/análise , Suínos , Linfócitos T Reguladores/imunologia , Timo/citologia , Timo/efeitos da radiação , Transplante Homólogo , Irradiação Corporal TotalRESUMO
Exosomes are nanometer-sized extracellular vesicles that are believed to function as intercellular communicators. Here, we report that exosomes are able to modify the radiation response of the head and neck cancer cell lines BHY and FaDu. Exosomes were isolated from the conditioned medium of irradiated as well as non-irradiated head and neck cancer cells by serial centrifugation. Quantification using NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response of other cells the exosomes were transferred to non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes isolated from both irradiated and non-irradiated cells by irradiated recipient cells compared to non-irradiated recipient cells. Functional analyses by exosome transfer indicated that all exosomes (from non-irradiated and irradiated donor cells) increase the proliferation of non-irradiated recipient cells and the survival of irradiated recipient cells. The survival-promoting effects are more pronounced when exosomes isolated from irradiated compared to non-irradiated donor cells are transferred. A possible mechanism for the increased survival after irradiation could be the increase in DNA double-strand break repair monitored at 6, 8 and 10 h after the transfer of exosomes isolated from irradiated cells. This is abrogated by the destabilization of the exosomes. Our results demonstrate that radiation influences both the abundance and action of exosomes on recipient cells. Exosomes transmit prosurvival effects by promoting the proliferation and radioresistance of head and neck cancer cells. Taken together, this study indicates a functional role of exosomes in the response of tumor cells to radiation exposure within a therapeutic dose range and encourages that exosomes are useful objects of study for a better understanding of tumor radiation response.
Assuntos
Carcinoma de Células Escamosas/radioterapia , Sobrevivência Celular/efeitos da radiação , Exossomos/fisiologia , Neoplasias de Cabeça e Pescoço/radioterapia , Linhagem Celular Tumoral/efeitos da radiação , Proliferação de Células/fisiologia , Proliferação de Células/efeitos da radiação , Exossomos/ultraestrutura , Humanos , Microscopia EletrônicaRESUMO
Septic cardiomyopathy affects up to 70% of patients with septic shock and the derangement of cardiac mitochondrial function contributes to the likelihood of death. However, at present, there is no specific therapeutic drug available. The peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α) and coactivator-1ß (PGC-1ß) modulate members of the PPARs, which regulate mitochondrial energy metabolism and the production of mitochondrial reactive oxygen species in the heart. This study investigated the potential of the newly developed synthetic antimicrobial peptide 19-2.5 (Pep2.5) to attenuate mitochondrial dysfunction in murine cardiomyocytes stimulated with serum from septic shock patients. Pep2.5 treatment attenuated the suppression of PPAR-α, PPAR-γ ( P = 0.0004 and P = 0.0001, respectively) and PGC-1α/ß ( P = 0.0008 and P = 0.0147, respectively) in cardiomyocytes stimulated with serum from septic shock patients compared with untreated cells. Pep2.5 treatment enhanced the mitochondrial maximum respiration ( P < 0.0001), increased cellular ATP levels ( P < 0.0001) and reduced the production of mitochondrial reactive oxygen species. Thus, the administration of Pep2.5 may have the potential as a promising therapeutic approach in septic cardiomyopathy by attenuating mitochondrial dysfunction in the septic heart.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Sepse/terapia , Idoso , Idoso de 80 Anos ou mais , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/fisiologia , Miócitos Cardíacos/fisiologia , Oxirredução/efeitos dos fármacos , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sepse/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The alkyl chain in the backbone of sphingosine derivatives can be exchanged with functionalised (labelled) side chains in a single step under cross-metathesis reaction conditions.
Assuntos
Ceramidas/química , Reagentes de Ligações Cruzadas/química , Metano/química , Esfingomielinas/química , Metilação , Estrutura Molecular , Esfingosina/químicaRESUMO
Small heat-shock proteins, including αB-crystallin (αB), play an important part in protein homeostasis, because their ATP-independent chaperone activity inhibits uncontrolled protein aggregation. Mechanistic details of human αB, particularly in its client-bound state, have been elusive so far, owing to the high molecular weight and the heterogeneity of these complexes. Here we provide structural insights into this highly dynamic assembly and show, by using state-of-the-art NMR spectroscopy, that the αB complex is assembled from asymmetric building blocks. Interaction studies demonstrated that the fibril-forming Alzheimer's disease Aß1-40 peptide preferentially binds to a hydrophobic edge of the central ß-sandwich of αB. In contrast, the amorphously aggregating client lysozyme is captured by the partially disordered N-terminal domain of αB. We suggest that αB uses its inherent structural plasticity to expose distinct binding interfaces and thus interact with a wide range of structurally variable clients.