RESUMO
RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.
Assuntos
Microscopia Crioeletrônica , Glicina Hidroximetiltransferase , Glicina Hidroximetiltransferase/metabolismo , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/química , Humanos , RNA/metabolismo , RNA/genética , Serina/metabolismo , Regulação Alostérica , Ligação Proteica , Filogenia , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Glicina/metabolismo , Glicina/química , Sítios de LigaçãoRESUMO
Persistent organic pollutants (POPs), which encompass pesticides and industrial chemicals widely utilized across the globe, pose a covert threat to human health. ß-hexachlorocyclohexane (ß-HCH) is an organochlorine pesticide with striking stability, still illegally dumped in many countries, and recognized as responsible for several pathogenetic mechanisms. This study represents a pioneering exploration into the neurotoxic effects induced by the exposure to ß-HCH specifically targeting neuronal cells (N2a), microglia (BV-2), and C57BL/6 mice. As shown by western blot and qPCR analyses, the administration of ß-HCH triggered a modulation of NF-κB, a key factor influencing both inflammation and pro-inflammatory cytokines expression. We demonstrated by proteomic and western blot techniques epigenetic modifications in H3 histone induced by ß-HCH. Histone acetylation of H3K9 and H3K27 increased in N2a, and in the prefrontal cortex of C57BL/6 mice administered with ß-HCH, whereas it decreased in BV-2 cells and in the hippocampus. We also observed a severe detrimental effect on recognition memory and spatial navigation by the Novel Object Recognition Test (NORT) and the Object Place Recognition Task (OPRT) behavioural tests. Cognitive impairment was linked to decreased expression of the genes BDNF and SNAP-25, which are mediators involved in synaptic function and activity. The obtained results expand our understanding of the harmful impact produced by ß-HCH exposure by highlighting its implication in the pathogenesis of neurological diseases. These findings will support intervention programs to limit the risk induced by exposure to POPs. Regulatory agencies should block further illicit use, causing environmental hazards and endangering human and animal health.
Assuntos
Disfunção Cognitiva , Epigênese Genética , Hexaclorocicloexano , Histonas , Camundongos Endogâmicos C57BL , Animais , Hexaclorocicloexano/toxicidade , Disfunção Cognitiva/induzido quimicamente , Camundongos , Histonas/metabolismo , Epigênese Genética/efeitos dos fármacos , Masculino , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Doenças Neuroinflamatórias/induzido quimicamente , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Poluentes Ambientais/toxicidadeRESUMO
Phosphodiesterases (PDEs) are a superfamily of evolutionarily conserved cyclic nucleotide (cAMP/cGMP)-hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Within this family, the cGMP-dependent PDE5 is the major hydrolyzing enzyme in many mammalian tissues, where it regulates a number of cellular and tissular processes. Using Kluyveromyces lactis as a model organism, the murine PDE5A1, A2 and A3 isoforms were successfully expressed and studied, evidencing, for the first time, a distinct role of each isoform in the control, modulation and maintenance of the cellular redox metabolism. Moreover, we demonstrated that the short N-terminal peptide is responsible for the tetrameric assembly of MmPDE5A1 and for the mitochondrial localization of MmPDE5A2. We also analyzed MmPDE5A1, A2 and A3 using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), structural mass spectrometry (MS) and polyacrylamide gel electrophoresis in their native conditions (native-PAGE) and in the presence of redox agents. These analyses pointed towards the role of a few specific cysteines in the isoforms' oligomeric assembly and the loss of enzymatic activity when modified.
Assuntos
GMP Cíclico , Cisteína , Camundongos , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Isoformas de Proteínas , GMP Cíclico/metabolismo , Mamíferos/metabolismoRESUMO
Rheumatoid arthritis (RA) is a chronic autoimmune disease and rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) are the most frequently detected autoantibodies (autoAbs). To date, more than 20% of RA cases are still defined as seronegative forms (seronegative RA, SN-RA). The aim of this study was to identify new antigenic targets of autoAbs in RA patients, which can also be recognized in SN-RA. Using a proteomic approach, we tested sera from SN-RA patients by analyzing synovial fluid (SF) proteins from these patients. Sera from SN-RA patients revealed a strong reactive spot, corresponding to alpha 1 antitrypsin (A1AT). Reverse-phase nanoliquid chromatography and tandem mass spectrometry (Matrix Assisted Laser Desorption/Ionization-Time Of Flight, MALDI-TOF/TOF) confirmed the presence of A1AT in SF and showed that homocysteinylation was one of the post-translational modifications of A1AT. Homocysteinylated (Hcy)-A1AT immunoprecipitated from SN-RA patients' SFs and in vitro modified Hcy-A1AT were used as antigens by Enzyme-Linked ImmunoSorbent Assay (ELISA) to test the presence of specific autoAbs in sera from 111 SN-RA patients, 132 seropositive (SP)-RA patients, and from 95 patients with psoriatic arthritis, 40 patients with osteoarthritis, and 41 healthy subjects as control populations. We observed that a large portion of SN-RA patients (75.7%), and also most of SP-RA patients' sera (87.1%) displayed anti-Hcy-A1AT autoAbs (anti-HATA). Native A1AT was targeted at a lower rate by SP-RA patients autoAbs, while virtually no SN-RA patients' sera showed the presence of anti-native A1AT autoAbs. In conclusion, anti-HATA can be considered potential biomarkers for RA, also in the SN forms. The discovery of novel autoAbs targeting specific autoantigens can represent higher clinic significance for all RA patients' population.
Assuntos
Artrite Reumatoide/diagnóstico , Autoanticorpos/sangue , Autoantígenos/imunologia , alfa 1-Antitripsina/imunologia , Idoso , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Autoantígenos/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Voluntários Saudáveis , Homocisteína/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional , Testes Sorológicos , alfa 1-Antitripsina/metabolismoRESUMO
Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization.
Assuntos
Proteínas de Transporte/metabolismo , Citocinese , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Divisão Celular , Linhagem Celular , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , TetraploidiaRESUMO
The ß-amyloid (Aß) peptide plays a key role in the pathogenesis of Alzheimer's disease. The methionine (Met) residue at position 35 in Aß C-terminal domain is critical for neurotoxicity, aggregation, and free radical formation initiated by the peptide. The role of Met in modulating toxicological properties of Aß most likely involves an oxidative event at the sulfur atom. We therefore investigated the one- or two-electron oxidation of the Met residue of Aß25-35 fragment and the effect of such oxidation on the behavior of the peptide. Bicarbonate promotes two-electron oxidations mediated by hydrogen peroxide after generation of peroxymonocarbonate (HCO4-, PMC). The bicarbonate/carbon dioxide pair stimulates one-electron oxidations mediated by carbonate radical anion (CO3â¢-). PMC efficiently oxidizes thioether sulfur of the Met residue to sulfoxide. Interestingly, such oxidation hampers the tendency of Aß to aggregate. Conversely, CO3â¢- causes the one-electron oxidation of methionine residue to sulfur radical cation (MetSâ¢+). The formation of this transient reactive intermediate during Aß oxidation may play an important role in the process underlying amyloid neurotoxicity and free radical generation.
Assuntos
Peptídeos beta-Amiloides/química , Carbonatos/química , Radicais Livres/química , Fragmentos de Peptídeos/química , Agregados Proteicos , Humanos , OxirreduçãoRESUMO
Among natural products under investigation for their additive potential in cancer prevention and treatment, the flavonoid quercetin has received attention for its effects on the cell cycle arrest and apoptosis. In the past, we addressed this issue in K562 cells, a cellular model of the human chronic myeloid leukemia. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) proteomics with the aim to increase knowledge on the regulative and metabolic pathways modulated by quercetin in these cells. After 24 h of quercetin treatment, we observed that apoptosis was not completely established, thus we selected this time range to capture quantitative data. As a result, we were able to achieve a robust identification of 1703 proteins, and to measure fold changes between quercetin-treated and untreated cells for 1206 proteins. Through a bioinformatics functional analysis on a subset of 112 proteins, we propose that the apoptotic phenotype of K562 cells entails a significant modulation of the translational machinery, RNA metabolism, antioxidant defense systems, and enzymes involved in lipid metabolism. Finally, we selected eight differentially expressed proteins, validated their modulated expression in quercetin-treated K562 cells, and discussed their possible role in flavonoid cytotoxicity. This quantitative profiling, performed for the first time on this type of tumor cells upon treatment with a flavonoid, will contribute to revealing the molecular basis of the multiplicity of the effects selectively exerted by quercetin on K562 cells.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteoma/efeitos dos fármacos , Proteômica/métodos , Quercetina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Marcação por Isótopo , Células K562 , Metabolismo dos Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fatores de TempoRESUMO
STAT3 is an oncoprotein overexpressed in different types of tumors, including prostate cancer (PCa), and its activity is modulated by a variety of post-translational modifications (PTMs). Prostate cancer represents the most common cancer diagnosed in men, and each phase of tumor progression displays specific cellular conditions: inflammation is predominant in tumor's early stage, whereas oxidative stress is typical of clinically advanced PCa. The aim of this research is to assess the correspondence between the stimulus-specificity of STAT3 PTMs and definite STAT3-mediated transcriptional programs, in order to identify new suitable pharmacological targets for PCa treatment. Experiments were performed on less-aggressive LNCaP and more aggressive DU-145 cell lines, simulating inflammatory and oxidative-stress conditions. Cellular studies confirmed pY705-STAT3 as common denominator of all STAT3-mediated signaling. In addition, acK685-STAT3 was found in response to IL-6, whereas glutC328/542-STAT3 and pS727-STAT3 occurred upon tert-butyl hydroperoxyde (tBHP) treatment. Obtained results also provided evidence of an interplay between STAT3 PTMs and specific protein interactors such as P300 and APE1/Ref-1. In accordance with these outcomes, mRNA levels of STAT3-target genes seemed to follow the differing STAT3 PTMs. These results highlighted the role of STAT3 and its PTMs as drivers in the progression of PCa.
Assuntos
Neoplasias da Próstata/metabolismo , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Biomarcadores , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias da Próstata/genética , Transcrição GênicaRESUMO
In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Microglia/metabolismo , Proteômica/métodos , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Marcação por Isótopo , Camundongos , Microglia/efeitos dos fármacos , RatosRESUMO
The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP.
Assuntos
Antineoplásicos/química , Cisteína/química , Glicina Hidroximetiltransferase/química , Piruvatos/química , Serina/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cisteína/metabolismo , Citosol/química , Citosol/enzimologia , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glicina Hidroximetiltransferase/antagonistas & inibidores , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Cinética , Mitocôndrias/química , Mitocôndrias/enzimologia , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Relação Estrutura-AtividadeRESUMO
A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic because they sequester RNA-binding proteins, thus affecting various steps of post-transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of amyotrophic lateral sclerosis (ALS), is still poorly defined. In this work, we set out to characterise these mechanisms by identifying proteins that bind to C9orf72 RNA. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur-alpha and its binding partner fragile X mental retardation protein 1 (FMRP, also known as FMR1), which accumulate in intra-cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated with a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Núcleo Celular/metabolismo , Modelos Biológicos , Biossíntese de Proteínas , Proteínas/metabolismo , Expansão das Repetições de Trinucleotídeos , Esclerose Lateral Amiotrófica/patologia , Animais , Proteína C9orf72 , Proteínas de Ligação a DNA , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Camundongos , Neurônios Motores/metabolismo , Fosforilação , Proteínas de Ligação a Poli(A)/metabolismo , Ligação Proteica , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TranscriçãoRESUMO
We report that 3',5'-cyclic CMP undergoes nonenzymatic di- and trimerization at 20 °C under dry conditions upon proton or UV irradiation. The reaction involves stacking of the cyclic monomers and subsequent polymerization through serial transphosphorylations between the stacked monomers. Proton- and UV-induced oligomerization of 3',5'-cyclic CMP demonstrates that pyrimidines-similar to purines-might also have taken part in the spontaneous generation of RNA under plausible prebiotic conditions as well as in an extraterrestrial context. The observed polymerization of naturally occurring 3',5'-cyclic nucleotides supports the possibility that the extant genetic nucleic acids might have originated by way of a straight Occamian path, starting from simple reactions between plausibly preactivated monomers.
Assuntos
CMP Cíclico/química , CMP Cíclico/efeitos da radiação , Oligorribonucleotídeos/síntese química , RNA/síntese química , Dicroísmo Circular , Evolução Química , Modelos Químicos , Polimerização , Prótons , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Raios UltravioletaRESUMO
Thiotaurine, a thiosulfonate related to taurine and hypotaurine, is formed by a metabolic process from cystine and generated by a transulfuration reaction between hypotaurine and thiocysteine. Thiotaurine can produce hydrogen sulfide (H2S) from its sulfane sulfur moiety. H2S is a gaseous signaling molecule which can have regulatory roles in inflammatory process. In addition, sulfane sulfur displays the capacity to reversibly bind to other sulfur atoms. Thiotaurine inhibits PMA-induced activation of human neutrophils, and hinders neutrophil spontaneous apoptosis. Here, we present the results of a proteomic approach to study the possible effects of thiotaurine at protein expression level. Proteome analysis of human neutrophils has been performed comparing protein extracts of resting or PMA-activated neutrophils in presence or in absence of thiotaurine. In particular, PMA-stimulated neutrophils showed high level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression compared to the level of the same glycolytic enzyme in the resting neutrophils. Conversely, decreased expression of GAPDH has been observed when human neutrophils were incubated with 1 mM thiotaurine before activation with PMA. This result, confirmed by Western blot analysis, suggests again that thiotaurine shows a bioactive role in the mechanisms underlying the inflammatory process, influencing the energy metabolism of activated leukocytes and raises the possibility that thiotaurine, acting as a sulfur donor, could modulate neutrophil activation via persulfidation of target proteins, such as GAPDH.
Assuntos
Ativação de Neutrófilo/efeitos dos fármacos , Proteômica/métodos , Taurina/análogos & derivados , Humanos , Taurina/farmacologiaRESUMO
Genome polymorphisms are responsible for phenotypic differences between humans and for individual susceptibility to genetic diseases and therapeutic responses. Non-synonymous single-nucleotide polymorphisms (nsSNPs) lead to protein variants with a change in the amino acid sequence that may affect the structure and/or function of the protein and may be utilized as efficient structural and functional markers of association to complex diseases. This study is focused on nsSNP variants of the ligand binding domain of PPARγ a nuclear receptor in the superfamily of ligand inducible transcription factors that play an important role in regulating lipid metabolism and in several processes ranging from cellular differentiation and development to carcinogenesis. Here we selected nine nsSNPs variants of the PPARγ ligand binding domain, V290M, R357A, R397C, F360L, P467L, Q286P, R288H, E324K, and E460K, expressed in cancer tissues and/or associated with partial lipodystrophy and insulin resistance. The effects of a single amino acid change on the thermodynamic stability of PPARγ, its spectral properties, and molecular dynamics have been investigated. The nsSNPs PPARγ variants show alteration of dynamics and tertiary contacts that impair the correct reciprocal positioning of helices 3 and 12, crucially important for PPARγ functioning.
Assuntos
PPAR gama/química , PPAR gama/genética , Polimorfismo de Nucleotídeo Único , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Humanos , Ligantes , Simulação de Dinâmica Molecular , PPAR gama/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Desdobramento de Proteína/efeitos dos fármacos , Relação Estrutura-Atividade , Termodinâmica , Transcrição Gênica , Ureia/farmacologiaRESUMO
DS is the most frequent genetic cause of intellectual disability characterized by the anomalous presence of three copies of chromosome 21. One of the peculiar features of DS is the onset of Alzheimer's disease neuropathology after the age of 40years characterized by deposition of senile plaques and neurofibrillary tangles. Growing studies demonstrated that increased oxidative damage, accumulation of unfolded/damaged protein aggregates and dysfunction of intracellular degradative system are key players in neurodegenerative processes. In this study, redox proteomics approach was used to analyze the frontal cortex from DS subjects under the age of 40 compared with age-matched controls, and proteins found to be increasingly carbonylated were identified. Interestingly, our results showed that oxidative damage targets specifically different components of the intracellular quality control system such as GRP78, UCH-L1, V0-ATPase, cathepsin D and GFAP that couples with decreased activity of the proteasome and autophagosome formation observed. We also reported a slight but consistent increase of Aß 1-42 SDS- and PBS-soluble form and tau phosphorylation in DS versus CTR. We suggest that disturbance in the proteostasis network could contribute to the accumulation of protein aggregates, such as amyloid deposits and NFTs, which occur very early in DS. It is likely that a sub-optimal functioning of degradative systems occur in DS neurons, which in turn provide the basis for further accumulation of toxic protein aggregates. The results of this study suggest that oxidation of protein members of the proteostatis network is an early event in DS and might contribute to neurodegenerative phenomena.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Lobo Frontal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Deficiências na Proteostase/metabolismo , Adolescente , Adulto , Peptídeos beta-Amiloides/metabolismo , Estudos de Casos e Controles , Catepsina D/metabolismo , Criança , Pré-Escolar , Chaperona BiP do Retículo Endoplasmático , Feminino , Lobo Frontal/patologia , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Oxirredução , Estresse Oxidativo/fisiologia , Fagossomos/metabolismo , Fosforilação/fisiologia , Carbonilação Proteica/fisiologia , Proteômica/métodos , Deficiências na Proteostase/patologia , Ubiquitina Tiolesterase/metabolismo , Adulto Jovem , Proteínas tau/metabolismoRESUMO
The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged. In particular, the F360L mutant of PPARγ (PPARγ2 residue 388), which is associated with familial partial lipodystrophy, significantly decreases basal transcriptional activity and impairs stimulation by synthetic ligands. To date, the structural reason for this defective behaviour is unclear. Therefore, the crystal structure of PPARγ F360L together with the partial agonist LT175 has been solved and the mutant has been characterized by circular-dichroism spectroscopy (CD) in order to compare its thermal stability with that of the wild-type receptor. The X-ray analysis showed that the mutation induces dramatic conformational changes in the C-terminal part of the receptor ligand-binding domain (LBD) owing to the loss of van der Waals interactions made by the Phe360 residue in the wild type and an important salt bridge made by Arg357, with consequent rearrangement of loop 11/12 and the activation function helix 12 (H12). The increased mobility of H12 makes the binding of co-activators in the hydrophobic cleft less efficient, thereby markedly lowering the transactivation activity. The spectroscopic analysis in solution and molecular-dynamics (MD) simulations provided results which were in agreement and consistent with the mutant conformational changes observed by X-ray analysis. Moreover, to evaluate the importance of the salt bridge made by Arg357, the crystal structure of the PPARγ R357A mutant in complex with the agonist rosiglitazone has been solved.
Assuntos
Lipodistrofia Parcial Familiar/genética , Mutação , PPAR gama/química , Ativação Transcricional , Cristalização , Humanos , Mutagênese Sítio-Dirigida , PPAR gama/genéticaRESUMO
Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia-associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9-targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9-LDL receptor interaction, thereby attenuating the PCSK9-mediated impairment of LDL cholesterol clearance. The N-terminal sequence of human H ferritin was engineered to incorporate a 13-amino acid linear peptide (Pep2-8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9-binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin's ability to disrupt PCSK9-LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin-based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin-based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases.
Assuntos
LDL-Colesterol , Nanopartículas , Pró-Proteína Convertase 9 , Receptores de LDL , Humanos , Pró-Proteína Convertase 9/metabolismo , Pró-Proteína Convertase 9/química , Pró-Proteína Convertase 9/genética , Receptores de LDL/metabolismo , Receptores de LDL/química , Nanopartículas/química , LDL-Colesterol/metabolismo , Inibidores de PCSK9/farmacologia , Inibidores de PCSK9/química , Ferritinas/química , Ferritinas/metabolismo , Ligação ProteicaRESUMO
Chronic lymphocytic leukemia (CLL) is a widespread type of leukemia that predominantly targets B lymphocytes, undermining the balance between cell proliferation and apoptosis. In healthy B cells, miR-15/16, a tandem of microRNAs, functions as a tumor suppressor, curbing the expression of the antiapoptotic B cell lymphoma 2 protein (Bcl-2). Conversely, in CLL patients, a recurring deletion on chromosome 13q14, home to the miR15-a and miR16-1 genes, results in Bcl-2 overexpression, thereby fostering the onset of the pathology. In the present research, a novel approach utilizing humanized ferritin-based nanoparticles was employed to successfully deliver miR15-a and miR-16-1 into MEG01 cells, a model characterized by the classic CLL deletion and overexpression of the human ferritin receptor (TfR1). The loaded miR15-a and miR16-1, housed within modified HumAfFt, were efficiently internalized via the MEG01 cells and properly directed into the cytoplasm. Impressively, the concurrent application of miR15-a and miR16-1 demonstrated a robust capacity to induce apoptosis through the reduction in Bcl-2 expression levels. This technology, employing RNA-loaded ferritin nanoparticles, hints at promising directions in the battle against CLL, bridging the substantial gap left by traditional transfection agents and indicating a pathway that may offer hope for more effective treatments.
RESUMO
Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6 , and its proper distribution in the body are essential for human health. The PLP recycling pathway plays a crucial role in these processes and its defects cause severe neurological diseases. The enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), whose catalytic action yields PLP, is one of the key players in this pathway. Mutations in the gene encoding PNPO are responsible for a severe form of neonatal epilepsy. Recently, PNPO has also been described as a potential target for chemotherapeutic agents. Our laboratory has highlighted the crucial role of PNPO in the regulation of PLP levels in the cell, which occurs via a feedback inhibition mechanism of the enzyme, exerted by binding of PLP at an allosteric site. Through docking analyses and site-directed mutagenesis experiments, here we identified the allosteric PLP binding site of human PNPO. This site is located in the same protein region as the allosteric site we previously identified in the Escherichia coli enzyme homologue. However, the identity and arrangement of the amino acid residues involved in PLP binding are completely different and resemble those of the active site of PLP-dependent enzymes. The identification of the PLP allosteric site of human PNPO paves the way for the rational design of enzyme inhibitors as potential anti-cancer compounds.
Assuntos
Oxirredutases , Piridoxaminafosfato Oxidase , Humanos , Sítio Alostérico , Oxirredutases/metabolismo , Fosfatos , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/genética , Piridoxaminafosfato Oxidase/metabolismoRESUMO
Tauopathies, including Alzheimer's disease and Frontotemporal Dementia, are debilitating neurodegenerative disorders marked by cognitive decline. Despite extensive research, achieving effective treatments and significant symptom management remains challenging. Accurate diagnosis is crucial for developing effective therapeutic strategies, with hyperphosphorylated protein units and tau oligomers serving as reliable biomarkers for these conditions. This study introduces a novel approach using nanotechnology to enhance the diagnostic process for tauopathies. We developed humanized ferritin nanocages, a novel nanoscale delivery system, designed to encapsulate and transport a tau-specific fluorophore, BT1, into human retinal cells for detecting neurofibrillary tangles in retinal tissue, a key marker of tauopathies. The delivery of BT1 into living cells was successfully achieved through these nanocages, demonstrating efficient encapsulation and delivery into retinal cells derived from human induced pluripotent stem cells. Our experiments confirmed the colocalization of BT1 with pathological forms of tau in living retinal cells, highlighting the method's potential in identifying tauopathies. Using ferritin nanocages for BT1 delivery represents a significant contribution to nanobiotechnology, particularly in neurodegenerative disease diagnostics. This method offers a promising tool for the early detection of tau tangles in retinal tissue, with significant implications for improving the diagnosis and management of tauopathies. This study exemplifies the integration of nanotechnology with biomedical science, expanding the frontiers of nanomedicine and diagnostic techniques.