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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.
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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
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.
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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.
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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
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.
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Ferritinas , Retina , Tauopatias , Proteínas tau , Humanos , Proteínas tau/metabolismo , Ferritinas/metabolismo , Retina/metabolismo , Retina/patologia , Tauopatias/metabolismo , Tauopatias/patologia , Tauopatias/diagnóstico , Células-Tronco Pluripotentes Induzidas/metabolismo , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologiaRESUMO
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.
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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.
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Oxirredutases , Piridoxaminafosfato Oxidase , Humanos , Sítio Alostérico , Oxirredutases/metabolismo , Fosfatos , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/genética , Piridoxaminafosfato Oxidase/metabolismoRESUMO
Introduction: Anisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized. Methods: Genetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis. Results and discussion: EVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans' hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts.
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Anisaquíase , Anisakis , Doenças dos Peixes , Parasitos , Animais , Humanos , Anisakis/genética , Larva , Proteômica , Anisaquíase/etiologia , Anisaquíase/parasitologia , Doenças dos Peixes/parasitologiaRESUMO
Microglia, the macrophage-like glial cells, behave as sentinels against exogenous pathogens invading the neural tissue. Their commitment is not only confined to the defensive function, but they also perform balancing trophic activities such as neuronal postnatal development, remodeling and pruning of synapses. Likewise, microglia-derived extracellular vesicles (EVs) can play strategic roles in maintaining a healthy brain by modulating neuronal activity and by controlling neurite outgrowth as well as innate immune response. Nevertheless, strong evidence also points to their role in the development of neurodegenerative pathologies such as Alzheimer's disease (AD). Here, we explored EV protein content released by BV2 microglial cells in a resting state and after stimulation with beta-amyloid peptides (Aß), mimicking conditions occurring in AD. In the resting BV2 cells, we extended the list of proteins present in mouse microglia EV cargo with respect to those reported in the Vesiclepedia exosome database while, in amyloid-triggered microglia, we highlighted a pronounced drop in EV protein content. Focusing on Rab11A, a key factor in the recycling routes of amyloid species, we observed a dramatic decrease of this protein in Aß-treated microglia EV cargo with respect to the EVs from the untreated sample. This decrease might affect the delivery of Rab11A to neurons thus increasing the harmful amyloid burden in neuronal cells that eventually may lead to their death. We tentatively proposed that alterations observed in EVs derived from Aß-treated microglia may represent molecular features that, among others, shape the disease-associated microglial phenotype, a recently proposed subset of microglial population, present in neurodegenerative pathologies.
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Doença de Alzheimer , Vesículas Extracelulares , Camundongos , Animais , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Microglia/metabolismo , Microglia/patologia , Proteoma/metabolismo , Doença de Alzheimer/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologiaRESUMO
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.
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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
Peptide nucleic acids (PNAs) play a key role in prebiotic chemistry as a chimera between RNA and proteins. We developed an alternative synthesis of bioactive PNA's diaminopurine and guanine analogues from prebiotic compounds, such as aminomalononitrile (AMN), urea, and guanidine, using a two-step multicomponent microwave-assisted and solvent-free approach in the presence of selected amino acids. The novel derivatives showed selective inhibitory activity against influenza virus A/Puerto Rico/8/34 H1N1 encompassing the range of nanomolar activity. Derivatives decorated with the tyrosine residue showed the highest inhibitory activity against the virus.
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Aspergillus fumigatus is a disease-causing, opportunistic fungus that can establish infection due to its capacity to respond to a wide range of environmental conditions. Secreted proteins and metabolites, which play a critical role in fungal-host interactions and pathogenesis, are modulated by epigenetic players, such as bromodomain and extraterminal domain (BET) proteins. In this study, we evaluated the in vitro and in vivo capability of the BET inhibitor JQ1 to modulate the extracellular proteins and virulence of A. fumigatus. The abundance of 25 of the 76 extracellular proteins identified through LC-MS/MS proteomic analysis changed following JQ1 treatment. Among them, a ribonuclease, a chitinase, and a superoxide dismutase were dramatically downregulated. Moreover, the proteomic analysis of A. fumigatus intracellular proteins indicated that Abr2, an intracellular laccase involved in the last step of melanin synthesis, was absent in the JQ1-treated group. To investigate at which level this downregulation occurred and considering the ability of JQ1 to modulate gene expression we checked the level of ABR2, Chitinase, and Superoxide dismutase mRNA expression by qRT-PCR. Finally, the capacity of JQ1 to reduce the virulence of A. fumigatus has been proved using Galleria mellonella larvae, which are an in vivo model to evaluate fungal virulence. Overall, the promising activity exhibited by JQ1 suggests that A. fumigatus is sensitive to BET inhibition and BET proteins may be a viable target for developing antifungal agents.
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The pyridoxal 5'-phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP-dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP-dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.
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Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Lisina/metabolismo , Fosfato de Piridoxal , Proteínas/química , Estabilidade Proteica , Homeostase , Fosfatos/metabolismo , Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/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.
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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
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.
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Peptídeos beta-Amiloides/química , Carbonatos/química , Radicais Livres/química , Fragmentos de Peptídeos/química , Agregados Proteicos , Humanos , OxirreduçãoRESUMO
Celiac disease (CD) is an autoimmune enteropathy caused by an intolerance to gluten proteins. It has been hypothesized that probiotic bacteria may exert beneficial effects by modulating inflammatory processes and by sustaining peptide hydrolysis at the intestinal level. This study aims at evaluating the capacity of a probiotic mixture (two different strains of lactobacilli and three of bifidobacteria) to hydrolyze gluten peptides following simulated gastrointestinal digestion of gliadin (PT-gliadin). The capacity of bacterial hydrolysates to counteract the toxic effects of gliadin-derived peptides in Caco-2 cells was also assessed. The protein and peptide mixtures, untreated or proteolyzed with the probiotic preparation, were analyzed before and after each proteolytic step with different techniques (SDS-PAGE, reverse phase HPLC, filtration on different molecular cut-off membranes). These experiments demonstrated that PT-gliadin can be further digested by bacteria into lower molecular weight peptides. PT-gliadin, untreated or digested with the probiotics, was then used to evaluate oxidative stress, IL-6 cytokine production and expression of tight junctions' proteins-such as occludin and zonulin-in Caco-2 cells. PT-gliadin induced IL-6 production and modulation and redistribution of zonulin and occludin, while digestion with the probiotic strains reversed these effects. Our data indicate that this probiotic mixture may exert a protective role in CD.
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Bifidobacterium , Gliadina/metabolismo , Gliadina/toxicidade , Lactobacillus , Probióticos/farmacologia , Hidrolisados de Proteína/farmacologia , Bifidobacterium/metabolismo , Células CACO-2 , Doença Celíaca/prevenção & controle , Doença Celíaca/terapia , Haptoglobinas/metabolismo , Humanos , Hidrólise , Interleucina-6/metabolismo , Lactobacillus/metabolismo , Peso Molecular , Ocludina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Probióticos/uso terapêutico , Precursores de Proteínas/metabolismo , ProteóliseRESUMO
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.
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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
Diabetes mellitus is one of the major risk factors for cognitive dysfunction. The pathogenesis of brain impairment caused by chronic hyperglycemia is complex and includes mitochondrial dysfunction, neuroinflammation, neurotransmitters' alteration, and vascular disease, which lead to cognitive impairment, neurodegeneration, loss of synaptic plasticity, brain aging, and dementia. Glucagon-like peptide-1 (GLP-1), a gut released hormone, is attracting attention as a possible link between metabolic and brain impairment. Several studies have shown the influence of GPL-1 on neuronal functions such as thermogenesis, blood pressure control, neurogenesis, neurodegeneration, retinal repair, and energy homeostasis. Moreover, modulation of GLP-1 activity can influence amyloid ß peptide aggregation in Alzheimer's disease (AD) and dopamine (DA) levels in Parkinson's disease (PD). GLP-1 receptor agonists (GLP-1RAs) showed beneficial actions on brain ischemia in animal models, such as the reduction of cerebral infarct area and the improvement of neurological deficit, acting mainly through inhibition of oxidative stress, inflammation, and apoptosis. They might also exert a beneficial effect on the cognitive impairment induced by diabetes or obesity improving learning and memory by modulating synaptic plasticity. Moreover, GLP-1RAs reduced hippocampal neurodegeneration. Besides this, there are growing evidences on neuroprotective effects of these agonists in animal models of neurodegenerative diseases, regardless of diabetes. In PD animal models, GPL-1RAs were able to protect motor activity and dopaminergic neurons whereas in AD models, they seemed to improve nearly all neuropathological features and cognitive functions. Although further clinical studies of GPL-1RAs in humans are needed, they seem to be a promising therapy for diabetes-associated cognitive decline.
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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.
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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
The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDAapproved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HHGLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HHregulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogenactivated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogenactivated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HHregulated transcription factor. Phosphorylation occurred at multiple residues in the Cterminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 1433. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy.
Assuntos
Proteínas Hedgehog/genética , MAP Quinase Quinase Quinase 1/genética , Meduloblastoma/genética , Proteína GLI1 em Dedos de Zinco/genética , Anilidas/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Piridinas/administração & dosagem , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Amyloid-treated microglia prime and sustain neuroinflammatory processes in the central nervous system activating different signalling pathways inside the cells. Since a key role for PARP-1 has been demonstrated in inflammation and in neurodegeneration, we investigated PARylated proteins in resting and in ß-amyloid peptide treated BV2 microglial cells. A total of 1158 proteins were identified by mass spectrometry with 117 specifically modified in the amyloid-treated cells. Intervention of PARylation on the proteome of microglia showed to be widespread in different cellular districts and to affect various cellular pathways, highlighting the role of this dynamic post-translational modification in cellular regulation. Ubiquitination is one of the more enriched pathways, encompassing PARylated proteins like NEDD4, an E3 ubiquitine ligase and USP10, a de-ubiquitinase, both associated with intracellular responses induced by ß-amyloid peptide challenge. PARylation of NEDD4 may be involved in the recruiting of this protein to the plasma membrane where it regulates the endocytosis of AMPA receptors, whereas USP10 may be responsible for the increase of p53 levels in amyloid stimulated microglia. Unfolded protein response and Endoplasmic Reticulum Stress pathways, strictly correlated with the Ubiquitination process, also showed enrichment in PARylated proteins. PARylation may thus represent one of the molecular switches responsible for the transition of microglia towards the inflammatory microglia phenotype, a pivotal player in brain diseases including neurodegenerative processes. The establishment of trials with PARP inhibitors to test their efficacy in the containment of neurodegenerative diseases may be envisaged.