RESUMO
BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Alvo Mecanístico do Complexo 2 de Rapamicina , Melanoma , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Proteína Companheira de mTOR Insensível à Rapamicina , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Melanoma/genética , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica/métodos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Ensaios Antitumorais Modelo de Xenoenxerto , MAP Quinase Quinase Quinases/antagonistas & inibidoresRESUMO
CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients results in opposed rRNA amounts. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-CSB-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.
Assuntos
Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Ferroquelatase/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase I/genética , RNA Ribossômico/genética , Fatores de Transcrição/genética , Linhagem Celular Transformada , Sobrevivência Celular , Imunoprecipitação da Cromatina , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/metabolismo , Ferroquelatase/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase I/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Raios UltravioletaRESUMO
The genome sequencing of the tardigrade Ramazzottius varieornatus revealed a unique nucleosome-binding protein named damage suppressor (Dsup), which was discovered to be crucial for the extraordinary abilities of tardigrades in surviving extreme stresses, such as UV. Evidence in Dsup-transfected human cells suggests that Dsup mediates an overall response in DNA damage signaling, DNA repair, and cell cycle regulation, resulting in an acquired resistance to stress. Given these promising outcomes, our study attempts to provide a wider comprehension of the molecular mechanisms modulated by Dsup in human cells and to explore the Dsup-activated molecular pathways under stress. We performed a differential proteomic analysis of Dsup-transfected and control human cells under basal conditions and at 24 h recovery after exposure to UV-C. We demonstrate via enrichment and network analyses, for the first time, that even in the absence of external stimuli, and more significantly, after stress, Dsup activates mechanisms involved with the unfolded protein response, the mRNA processing and stability, cytoplasmic stress granules, the DNA damage response, and the telomere maintenance. In conclusion, our results shed new light on Dsup-mediated protective mechanisms and increases our knowledge of the molecular machineries of extraordinary protection against UV-C stress.
Assuntos
Proteômica , Tardígrados , Humanos , Animais , Tardígrados/genética , Tardígrados/metabolismo , Dano ao DNA , Reparo do DNA , Mapeamento CromossômicoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a form of chronic and irreversible fibrosing interstitial pneumonia of unknown etiology. Although antifibrotic treatments have shown a reduction of lung function decline and a slow disease progression, IPF is characterize by a very high mortality. Emerging evidence suggests that IPF increases the risk of lung carcinogenesis. Both diseases show similarities in terms of risk factors, such as history of smoking, concomitant emphysema, and viral infections, besides sharing similar pathogenic pathways. Lung cancer (LC) diagnosis is often difficult in IPF patients because of the diffuse lung injuries and abnormalities due to the underlying fibrosis. This is reflected in the lack of optimal therapeutic strategies for patients with both diseases. For this purpose, we performed a proteomic study on bronchoalveolar lavage fluid (BALF) samples from IPF, LC associated with IPF (LC-IPF) patients, and healthy controls (CTRL). Molecular pathways involved in inflammation, immune response, lipid metabolism, and cell adhesion were found for the dysregulated proteins in LC-IPF, such as TTHY, APOA1, S10A9, RET4, GDIR1, and PROF1. The correlation test revealed a relationship between inflammation- and lipid metabolism-related proteins. PROF1 and S10A9, related to inflammation, were up-regulated in LC-IPF BAL and serum, while APOA1 and APOE linked to lipid metabolism, were highly abundant in IPF BAL and low abundant in IPF serum. Given the properties of cytokine/adipokine of the nicotinamide phosphoribosyltransferase, we also evaluated its serum abundance, highlighting its down-regulation in LC-IPF. Our retrospective analyses of BAL samples extrapolated some potential biomarkers of LC-IPF useful to improve the management of these contemporary pathologies. Their differential abundance in serum samples permits the measurement of these potential biomarkers with a less invasive procedure.
Assuntos
Adenocarcinoma de Pulmão , Fibrose Pulmonar Idiopática , Neoplasias Pulmonares , Humanos , Estudos Retrospectivos , Proteômica/métodos , Fibrose Pulmonar Idiopática/metabolismo , Líquido da Lavagem Broncoalveolar , Fibrose , Inflamação , Adenocarcinoma de Pulmão/diagnóstico , Neoplasias Pulmonares/diagnóstico , BiomarcadoresRESUMO
Epiretinal membranes (ERMs) are sheets of tissue that pathologically develop in the vitreoretinal interface leading to progressive vision loss. They are formed by different cell types and by an exuberant deposition of extracellular matrix proteins. Recently, we reviewed ERMs' extracellular matrix components to better understand molecular dysfunctions that trigger and fuel the onset and development of this disease. The bioinformatics approach we applied delineated a comprehensive overview on this fibrocellular tissue and on critical proteins that could really impact ERM physiopathology. Our interactomic analysis proposed the hyaluronic-acid-receptor cluster of differentiation 44 (CD44) as a central regulator of ERM aberrant dynamics and progression. Interestingly, the interaction between CD44 and podoplanin (PDPN) was shown to promote directional migration in epithelial cells. PDPN is a glycoprotein overexpressed in various cancers and a growing body of evidence indicates its relevant function in several fibrotic and inflammatory pathologies. The binding of PDPN to partner proteins and/or its ligand results in the modulation of signaling pathways regulating proliferation, contractility, migration, epithelial-mesenchymal transition, and extracellular matrix remodeling, all processes that are vital in ERM formation. In this context, the understanding of the PDPN role can help to modulate signaling during fibrosis, hence opening a new line of therapy.
Assuntos
Membrana Epirretiniana , Vitreorretinopatia Proliferativa , Humanos , Membrana Epirretiniana/metabolismo , Membrana Epirretiniana/patologia , Proteínas da Matriz Extracelular , Fibrose , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Fatores de Transcrição , Vitreorretinopatia Proliferativa/metabolismoRESUMO
Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5' of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols ß and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.
Assuntos
RNA Helicases DEAD-box/metabolismo , Ribonucleotídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Linhagem Celular , RNA Helicases DEAD-box/química , DNA Polimerase beta/metabolismo , Humanos , Domínios Proteicos , Motivos de Ligação ao RNA , Ribonuclease H/química , Ribonuclease H/metabolismoRESUMO
Krabbe disease (KD) is a rare autosomal recessive disorder caused by mutations in the galactocerebrosidase gene (GALC). Defective GALC causes aberrant metabolism of galactolipids present almost exclusively in myelin, with consequent demyelinization and neurodegeneration of the central and peripheral nervous system (NS). KD shares some similar features with other neuropathies and heterozygous carriers of GALC mutations are emerging with an increased risk in developing NS disorders. In this work, we set out to identify possible variations in the proteomic profile of KD-carrier brain to identify altered pathways that may imbalance its homeostasis and that may be associated with neurological disorders. The differential analysis performed on whole brains from 33-day-old twitcher (galc -/-), heterozygous (galc +/-), and wild-type mice highlighted the dysregulation of several multifunctional factors in both heterozygous and twitcher mice. Notably, the KD-carrier mouse, despite its normal phenotype, presents the deregulation of vimentin, receptor of activated protein C kinase 1 (RACK1), myelin basic protein (MBP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP), transitional endoplasmic reticulum ATPase (VCP), and N-myc downstream regulated gene 1 protein (NDRG1) as well as changes in the ubiquitinated-protein pattern. Our findings suggest the carrier may be affected by dysfunctions classically associated with neurodegeneration: (i) alteration of (mechano) signaling and intracellular trafficking, (ii) a generalized affection of proteostasis and lipid metabolism, with possible defects in myelin composition and turnover, and (iii) mitochondrion and energy supply dysfunctions.
Assuntos
Leucodistrofia de Células Globoides , Doenças Neurodegenerativas , Animais , Camundongos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Proteômica , Modelos Animais de Doenças , Galactosilceramidase/genética , Galactosilceramidase/metabolismoRESUMO
DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.
Assuntos
Cromatina/metabolismo , Genoma Humano , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Reparo de DNA por Recombinação , Proteína de Replicação A/genética , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Instabilidade Genômica , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteína de Replicação A/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismoRESUMO
In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of IPF, BALF proteomics is providing interesting new insights into its pathogenesis. To the best of our knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as the EVs isolation technique, and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider system involvement in the disease pathogenesis and support the importance of pre-fractioning of complex samples, such as BALF, in order to let low-abundant proteins-mediated pathways emerge.
Assuntos
Biomarcadores , Líquido da Lavagem Broncoalveolar , Vesículas Extracelulares/metabolismo , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , Proteoma , Proteômica , Idoso , Cromatografia Líquida , Suscetibilidade a Doenças , Eletroforese em Gel Bidimensional , Vesículas Extracelulares/ultraestrutura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
Reactive astrocytes are a hallmark of neurodegenerative disease including multiple sclerosis. It is widely accepted that astrocytes may adopt alternative phenotypes depending on a combination of environmental cues and intrinsic features in a highly plastic and heterogeneous manner. However, we still lack a full understanding of signals and associated signaling pathways driving astrocyte reaction and of the mechanisms by which they drive disease. We have previously shown in the experimental autoimmune encephalomyelitis mouse model that deficiency of the molecular adaptor Rai reduces disease severity and demyelination. Moreover, using primary mouse astrocytes, we showed that Rai contributes to the generation of a pro-inflammatory central nervous system (CNS) microenvironment through the production of nitric oxide and IL-6 and by impairing CD39 activity in response to soluble factors released by encephalitogenic T cells. Here, we investigated the impact of Rai expression on astrocyte function both under basal conditions and in response to IL-17 treatment using a proteomic approach. We found that astrocytes and astrocyte-derived extracellular vesicles contain a set of proteins, to which Rai contributes, that are involved in the regulation of oligodendrocyte differentiation and myelination, nitrogen metabolism, and oxidative stress. The HIF-1α pathway and cellular energetic metabolism were the most statistically relevant molecular pathways and were related to ENOA and HSP70 dysregulation.
Assuntos
Astrócitos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Vesículas Extracelulares/metabolismo , Interleucina-17/farmacologia , Neuroproteção , Oligodendroglia/fisiologia , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Animais , Diferenciação Celular , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/fisiopatologia , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina , Proteômica , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismoRESUMO
Osteogenesis imperfecta (OI) is a heritable disorder that mainly affects the skeleton. The inheritance is mostly autosomal dominant and associated to mutations in one of the two genes, COL1A1 and COL1A2, encoding for the type I collagen α chains. According to more than 1500 described mutation sites and to outcome spanning from very mild cases to perinatal-lethality, OI is characterized by a wide genotype/phenotype heterogeneity. In order to identify common affected molecular-pathways and disease biomarkers in OI probands with different mutations and lethal or surviving phenotypes, primary fibroblasts from dominant OI patients, carrying COL1A1 or COL1A2 defects, were investigated by applying a Tandem Mass Tag labeling-Liquid Chromatography-Tandem Mass Spectrometry (TMT LC-MS/MS) proteomics approach and bioinformatic tools for comparative protein-abundance profiling. While no difference in α1 or α2 abundance was detected among lethal (type II) and not-lethal (type III) OI patients, 17 proteins, with key effects on matrix structure and organization, cell signaling, and cell and tissue development and differentiation, were significantly different between type II and type III OI patients. Among them, some non-collagenous extracellular matrix (ECM) proteins (e.g., decorin and fibrillin-1) and proteins modulating cytoskeleton (e.g., nestin and palladin) directly correlate to the severity of the disease. Their defective presence may define proband-failure in balancing aberrances related to mutant collagen.
Assuntos
Biomarcadores/metabolismo , Cromatografia Líquida/métodos , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/patologia , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Pré-Escolar , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Osteogênese Imperfeita/genética , Proteoma/metabolismoRESUMO
Spinal muscular atrophy (SMA) type 1 is a severe infantile autosomal-recessive neuromuscular disorder caused by a survival motor neuron 1 gene (SMN1) mutation and characterized by progressive muscle weakness. Without supportive care, SMA type 1 is rapidly fatal. The antisense oligonucleotide nusinersen has recently improved the natural course of this disease. Here, we investigated, with a functional proteomic approach, cerebrospinal fluid (CSF) protein profiles from SMA type 1 patients who underwent nusinersen administration to clarify the biochemical response to the treatment and to monitor disease progression based on therapy. Six months after starting treatment (12 mg/5 mL × four doses of loading regimen administered at days 0, 14, 28, and 63), we observed a generalized reversion trend of the CSF protein pattern from our patient cohort to that of control donors. Notably, a marked up-regulation of apolipoprotein A1 and apolipoprotein E and a consistent variation in transthyretin proteoform occurrence were detected. Since these multifunctional proteins are critically active in biomolecular processes aberrant in SMA, i.e., synaptogenesis and neurite growth, neuronal survival and plasticity, inflammation, and oxidative stress control, their nusinersen induced modulation may support SMN improved-expression effects. Hence, these lipoproteins and transthyretin could represent valuable biomarkers to assess patient responsiveness and disease progression.
Assuntos
Terapia Genética , Oligonucleotídeos/farmacologia , Proteoma/análise , Atrofias Musculares Espinais da Infância/terapia , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Atrofias Musculares Espinais da Infância/líquido cefalorraquidiano , Atrofias Musculares Espinais da Infância/tratamento farmacológico , Atrofias Musculares Espinais da Infância/genéticaRESUMO
In Krabbe disease, a mutation in GALC gene causes widespread demyelination determining cell death by apoptosis, mainly in oligodendrocytes and Schwann cells. Less is known on the molecular mechanisms induced by this deficiency. Here, we report an impairment in protein synthesis and degradation and in proteasomal clearance with a potential accumulation of the misfolded proteins and induction of the endoplasmic reticulum stress in the brain of 6-day-old twitcher mice (TM) (model of Krabbe disease). In particular, an imbalance of the immunoproteasome function was highlighted, useful for shaping adaptive immune response by neurological cells. Moreover, our data show an involvement of cytoskeleton remodeling in Krabbe pathogenesis, with a lamin meshwork disaggregation in twitcher oligodendrocytes in 6-day-old TM. This study provides interesting protein targets and mechanistic insight on the early onset of Krabbe disease that may be promising options to be tested in combination with currently available therapies to rescue Krabbe phenotype.
Assuntos
Leucodistrofia de Células Globoides/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Oligodendroglia/metabolismo , Proteostase , Animais , Modelos Animais de Doenças , Feminino , Laminas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligodendroglia/ultraestrutura , ProteômicaRESUMO
OBJECTIVE: To investigate whether a probiotic supplementation to cART patients modifies the cerebrospinal fluid (CSF) proteome and improves neurocognitive impairment. METHODS: 26 CSF samples from 13 HIV-positive patients [six patients living with HIV (PLHIV) and seven patients with a history of AIDS (PHAIDS)] were analyzed. All patients underwent to neurocognitive evaluation and blood sampling at baseline and after 6 months of oral bacteriotherapy. Immune phenotyping and activation markers (CD38 and HLA-DR) were evaluated on peripheral blood mononuclear cells (PBMC). Plasma levels of IL-6, sCD14, and MIP-1ß were detected, by enzyme-linked immunosorbent assay (ELISA). Functional proteomic analysis of CSF sample was conducted by two-dimensional electrophoresis; a multivariate analysis was performed by principal component analysis (PCA) and data were enriched by STRING software. RESULTS: Oral bacteriotherapy leads to an improvement on several cognitive test and neurocognitive performance in both groups of HIV-positive subjects. A reduction in the percentage of CD4+CD38+HLA-DR+ T cells was also observed at peripheral level after the probiotic intake (p = 0.008). In addition, the probiotic supplementation to cART significantly modifies protein species composition and abundance at the CSF level, especially those related to inflammation (ß2-microglobulin p = 0.03; haptoglobin p = 0.06; albumin p = 0.003; hemoglobin p = 0.003; immunoglobulin heavy chains constant region p = 0.02, transthyretin p = 0.02) in PLHIV and PHAIDS. CONCLUSIONS: Our results suggest that oral bacteriotherapy as a supplement to cART could exert a role in the amelioration of inflammation state at peripheral and CNS level.
Assuntos
Complexo AIDS Demência/microbiologia , Infecções por HIV/complicações , Infecções por HIV/microbiologia , Probióticos/farmacologia , Complexo AIDS Demência/imunologia , Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/microbiologia , Adulto , Fármacos Anti-HIV/uso terapêutico , Líquido Cefalorraquidiano/efeitos dos fármacos , Líquido Cefalorraquidiano/imunologia , Disfunção Cognitiva/etiologia , Feminino , Infecções por HIV/imunologia , Humanos , Masculino , Microbiota/efeitos dos fármacos , Pessoa de Meia-Idade , Boca/microbiologia , ProteomaRESUMO
Benralizumab and mepolizumab are new therapies for severe eosinophilic asthma. They are both humanized IgG antibodies, targeting the IL-5 receptor and IL-5, respectively, suppressing the corresponding pathways. No specific biomarkers have been proposed to evaluate treatment response to benralizumab or mepolizumab. The aim of this proteomic study was to compare serum protein profiles of patients with severe eosinophilic asthma before and after anti-IL5 or anti-IL5R therapies. Proteomic analysis highlighted 22 differently abundant spots. Among the proteins identified, CAYP1, A1AT and A2M expression was significantly modified in both groups of patients after therapies while ceruloplasmin showed a significant modification in the group of benralizumab treatment. These differentially expressed proteins could be potential biomarkers of response to mepolizumab and benralizumab treatments and need further evaluation.
Assuntos
Anticorpos Monoclonais Humanizados , Asma , Proteínas de Ligação ao Cálcio/sangue , Eosinofilia , Interleucina-5/antagonistas & inibidores , alfa 1-Antitripsina/sangue , alfa-Macroglobulinas/agonistas , Adulto , Antiasmáticos/administração & dosagem , Antiasmáticos/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/imunologia , Asma/sangue , Asma/diagnóstico , Asma/tratamento farmacológico , Asma/fisiopatologia , Biomarcadores Farmacológicos/sangue , Monitoramento de Medicamentos/métodos , Eosinofilia/sangue , Eosinofilia/diagnóstico , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Proteômica/métodos , Índice de Gravidade de Doença , alfa-Macroglobulinas/análiseRESUMO
Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative disorder limited to the lung. New findings, starting from our proteomics studies on IPF, suggest that systemic involvement with altered molecular mechanisms and metabolic disorder is an underlying cause of fibrosis. The role of metabolic dysregulation in the pathogenesis of IPF has not been extensively studied, despite a recent surge of interest. In particular, our studies on bronchoalveolar lavage fluid have shown that the renin-angiotensin-aldosterone system (RAAS), the hypoxia/oxidative stress response, and changes in iron and lipid metabolism are involved in onset of IPF. These processes appear to interact in an intricate manner and to be related to different fibrosing pathologies not directly linked to the lung environment. The disordered metabolism of carbohydrates, lipids, proteins and hormones has been documented in lung, liver, and kidney fibrosis. Correcting these metabolic alterations may offer a new strategy for treating fibrosis. This paper focuses on the role of metabolic dysregulation in the pathogenesis of IPF and is a continuation of our previous studies, investigating metabolic dysregulation as a new target for fibrosis therapy.
Assuntos
Fibrose Pulmonar Idiopática/metabolismo , Animais , Humanos , Fibrose Pulmonar Idiopática/patologia , Ferro/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/patologia , Estresse Oxidativo , ProteômicaRESUMO
Acute exacerbations (AEs) are among the main causes of death in idiopathic pulmonary fibrosis (IPF) patients. In this study proteomic comparative analysis of bronchoalveolar lavage (BAL) fluid samples was performed in stable IPF patients versus AEs IPF group to identify AE pathogenetic mechanisms and novel potential predictive biomarkers. A functional proteomic analysis of BAL fluid samples from stable and AE-IPF patients was conducted in a population of 27 IPF patients. Fifty-one differentially abundant spots were observed and identified by mass spectrometry. Enrichment analysis found proteins of interest involved in the regulation of macrophages and lipid metabolism receptors. In acute exacerbation IPF group, differentially abundant proteins were involved in propagation of the ß-catenin WNT transduction signal, and proteins up-regulated in lung carcinogenesis (IGKC, S100A9, PEDF, IGHG1, ALDOA, A1AT, HPT, CO3 and PIGR) and acute phase proteins involved in protease-antiprotease imbalance (such as A1AT fragments). Dot-blot analysis of A1AT C-36 peptide allowed validating our findings, confirming up-regulation in AE IPF patients and suggesting its potential pathogenetic role. A crucial role of protease/antiprotease imbalance, clathrin-mediated endocytosis signalling and carcinogenesis emerged in IPF patients developing acute exacerbations.
Assuntos
Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Fibrose Pulmonar Idiopática/metabolismo , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/metabolismo , Progressão da Doença , Endocitose/fisiologia , Feminino , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Pulmão/fisiopatologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Transdução de Sinais/genética , Regulação para Cima , beta Catenina/metabolismoRESUMO
Objective: SSc is a rare severe connective tissue disorder. Its prognosis is mainly related to the development of pulmonary fibrosis (PF)-SSc and pulmonary arterial hypertension. No known therapy for PF-SSc modifies progressive lung fibrotic involvement. Research is therefore aimed at a deeper understanding of complex pathogenetic mechanisms and the possibility of new prognostic biomarkers and therapeutic targets. Methods: Towards the first of these aims, we conducted functional proteomic analysis of bronchoalveolar lavage samples from PF-SSc patients and smoker and non-smoker controls. Results: The differential expression pattern revealed by principal component analysis highlighted a specific protein profile of PF-SSc with respect to control samples, and enrichment analysis shed light on process networks involved in pathogenesis. The proteins identified are known to be involved in lung inflammation of PF-SSc-induced IL6 signalling, the complement system, innate immunity, Jak-STAT, the kallikrein-kinin system, blood coagulation, the immune response mediated by phagocytosis and phagosomes in antigen presentation. In particular, our MetaCore network suggested C3a, APOAI, 14-3-3ε, SPFA2 and S100A6 as potential biomarkers; these are upstream molecules involved in lung fibrosis, innate immunity and vascular damage occurring in PF-SSc. Conclusion: This report provides a molecular overview of pathological processes in PF-SSc, pinpointing possible new disease biomarkers and therapeutic targets.
Assuntos
Proteínas 14-3-3/análise , Lavagem Broncoalveolar/métodos , Proteínas de Ciclo Celular/análise , Proteômica/métodos , Fibrose Pulmonar/genética , Proteína A6 Ligante de Cálcio S100/análise , Escleroderma Sistêmico/genética , Idoso , Biomarcadores/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/imunologia , Escleroderma Sistêmico/complicaçõesRESUMO
In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Escherichia coli/genética , Lipoproteínas/metabolismo , Neisseria meningitidis/metabolismo , Aggregatibacter actinomycetemcomitans/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Lipoproteínas/genética , Neisseria meningitidis/genética , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação BacterianaRESUMO
Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl(+/-) to investigate the molecular basis of OI phenotypic variability. Brtl(+/-) resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl(+/-) mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl(+/-) lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-ß signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment.