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1.
PLoS Pathog ; 19(3): e1011174, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36877739

RESUMO

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the oocyst. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We confirm expression in male gametocytes and zygotes and show that actin II is associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. The function of actin II was investigated through mutational analysis, suggesting that long and stable filaments are necessary for male gametogenesis, while a second function in the oocyst stage also requires fine-tuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 µM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium.


Assuntos
Culicidae , Parasitos , Plasmodium , Animais , Masculino , Actinas/metabolismo , Parasitos/metabolismo , Citoesqueleto de Actina/metabolismo , Culicidae/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium/metabolismo
2.
Physiol Plant ; 176(5): e14534, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39284733

RESUMO

Bilberry (Vaccinium myrtillus L.) is a wild berry species that is prevalent in northern Europe. It is renowned and well-documented for its nutritional and bioactive properties, especially due to its anthocyanin content. However, an overview of biological systems governing changes in other crucial quality traits, such as size, firmness, and flavours, has received less attention. In the present study, we investigated detailed metabolomic and proteomic profiles at four different ripening stages of bilberry to provide a comprehensive understanding of overall quality during fruit ripening. By integrating omics datasets, we revealed a novel global regulatory network of plant hormones and physiological processes occurring during bilberry ripening. Key physiological processes, such as energy and primary metabolism, strongly correlate with elevated levels of gibberellic acids, jasmonic acid, and salicylic acid in unripe fruits. In contrast, as the fruit ripened, processes including flavour formation, cell wall modification, seed storage, and secondary metabolism became more prominent, and these were associated with increased abscisic acid levels. An indication of the increase in ethylene biosynthesis was detected during bilberry development, raising questions about the classification of non-climacteric and climacteric fruits. Our findings extend the current knowledge on the physiological and biochemical processes occurring during fruit ripening, which can serve as a baseline for studies on both wild and commercially grown berry species. Furthermore, our data may facilitate the optimization of storage conditions and breeding programs, as well as the future exploration of beneficial compounds in berries for new applications in food, cosmetics, and medicines.


Assuntos
Frutas , Metabolômica , Reguladores de Crescimento de Plantas , Proteômica , Vaccinium myrtillus , Frutas/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Proteômica/métodos , Reguladores de Crescimento de Plantas/metabolismo , Vaccinium myrtillus/metabolismo , Vaccinium myrtillus/genética , Vaccinium myrtillus/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Oxilipinas/metabolismo , Etilenos/metabolismo , Ácido Abscísico/metabolismo , Ciclopentanos/metabolismo , Giberelinas/metabolismo , Ácido Salicílico/metabolismo
3.
J Biol Chem ; 298(12): 102634, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36273584

RESUMO

Myosin B (MyoB) is a class 14 myosin expressed in all invasive stages of the malaria parasite, Plasmodium falciparum. It is not associated with the glideosome complex that drives motility and invasion of host cells. During red blood cell invasion, MyoB remains at the apical tip of the merozoite but is no longer observed once invasion is completed. MyoB is not essential for parasite survival, but when it is knocked out, merozoites are delayed in the initial stages of red blood cell invasion, giving rise to a growth defect that correlates with reduced invasion success. Therefore, further characterization is needed to understand how MyoB contributes to parasite invasion. Here, we have expressed and purified functional MyoB with the help of parasite-specific chaperones Hsp90 and Unc45, characterized its binding to actin and its known light chain MLC-B using biochemical and biophysical methods and determined its low-resolution structure in solution using small angle X-ray scattering. In addition to MLC-B, we found that four other putative regulatory light chains bind to the MyoB IQ2 motif in vitro. The purified recombinant MyoB adopted the overall shape of a myosin, exhibited actin-activated ATPase activity, and moved actin filaments in vitro. Additionally, we determined that the ADP release rate was faster than the ATP turnover number, and thus, does not appear to be rate limiting. This, together with the observed high affinity to actin and the specific localization of MyoB, may point toward a role in tethering and/or force sensing during early stages of invasion.


Assuntos
Miosina não Muscular Tipo IIB , Plasmodium falciparum , Proteínas de Protozoários , Actinas/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Miosinas/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo
4.
J Biol Chem ; 298(12): 102614, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36265586

RESUMO

Collagen prolyl 4-hydroxylases (C-P4H) are α2ß2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the ß-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the ß/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact ß/PDI subunit, at 3.8 Å resolution. The CAT domain interacts with the a, b', and a' domains of the ß/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a ß-strand, which is the edge strand of its major antiparallel ß-sheet. This extra region of the CAT domain interacts tightly with the ß/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a' domain and tight hydrophobic interactions with the b' domain. Using this new information, the structure of the mature C-P4H-II α2ß2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer.


Assuntos
Prolil Hidroxilases , Isomerases de Dissulfetos de Proteínas , Humanos , Domínio Catalítico , Colágeno/metabolismo , Peptídeos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolil Hidroxilases/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Conformação Proteica
5.
Environ Res ; 228: 115930, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37076033

RESUMO

Per- and polyfluoroalkyl substances (PFAS) are stable organic chemicals, which have been used globally since the 1940s and have caused PFAS contamination around the world. This study explores perfluorooctanoic acid (PFOA) enrichment and destruction by a combined method of sorption/desorption and photocatalytic reduction. A novel biosorbent (PG-PB) was developed from raw pine bark by grafting amine groups and quaternary ammonium groups onto the surface of bark particles. The results of PFOA adsorption at low concentration suggest that PG-PB has excellent removal efficiency (94.8%-99.1%, PG-PB dosage: 0.4 g/L) to PFOA in the concentration range of 10 µg/L to 2 mg/L. The PG-PB exhibited high adsorption efficiency regarding PFOA, being 456.0 mg/g at pH 3.3 and 258.0 mg/g at pH 7 with an initial concentration of 200 mg/L. The groundwater treatment reduced the total concentration of 28 PFAS from 18 000 ng/L to 9900 ng/L with 0.8 g/L of PG-PB. Desorption experiments examined 18 types of desorption solutions, and the results showed that 0.05% NaOH and a mixture of 0.05% NaOH + 20% methanol were efficient for PFOA desorption from the spent PG-PB. More than 70% (>70 mg/L in 50 mL) and 85% (>85 mg/L in 50 mL) of PFOA were recovered from the first and second desorption processes, respectively. Since high pH promotes PFOA degradation, the desorption eluents with NaOH were directly treated with a UV/sulfite system without further adjustment. The final PFOA degradation and defluorination efficiency in the desorption eluents with 0.05% NaOH + 20% methanol reached 100% and 83.1% after 24 h reaction. This study proved that the combination of adsorption/desorption and a UV/sulfite system for PFAS removal is a feasible solution for environmental remediation.


Assuntos
Fluorocarbonos , Poluentes Químicos da Água , Água , Adsorção , Metanol , Hidróxido de Sódio , Fluorocarbonos/análise , Caprilatos , Poluentes Químicos da Água/análise
6.
BMC Med ; 20(1): 141, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35477570

RESUMO

BACKGROUND: Preterm birth is defined as live birth before 37 completed weeks of pregnancy, and it is a major problem worldwide. The molecular mechanisms that lead to onset of spontaneous preterm birth are incompletely understood. Prediction and evaluation of the risk of preterm birth is challenging as there is a lack of accurate biomarkers. In this study, our aim was to identify placental proteins that associate with spontaneous preterm birth. METHODS: We analyzed the proteomes from placentas to identify proteins that associate with both gestational age and spontaneous labor. Next, rare and potentially damaging gene variants of the identified protein candidates were sought for from our whole exome sequencing data. Further experiments we performed on placental samples and placenta-associated cells to explore the location and function of the spontaneous preterm labor-associated proteins in placentas. RESULTS: Exome sequencing data revealed rare damaging variants in SERPINA1 in families with recurrent spontaneous preterm deliveries. Protein and mRNA levels of alpha-1 antitrypsin/SERPINA1 from the maternal side of the placenta were downregulated in spontaneous preterm births. Alpha-1 antitrypsin was expressed by villous trophoblasts in the placenta, and immunoelectron microscopy showed localization in decidual fibrinoid deposits in association with specific extracellular proteins. siRNA knockdown in trophoblast-derived HTR8/SVneo cells revealed that SERPINA1 had a marked effect on regulation of the actin cytoskeleton pathway, Slit-Robo signaling, and extracellular matrix organization. CONCLUSIONS: Alpha-1 antitrypsin is a protease inhibitor. We propose that loss of the protease inhibition effects of alpha-1 antitrypsin renders structures critical to maintaining pregnancy susceptible to proteases and inflammatory activation. This may lead to spontaneous premature birth.


Assuntos
Trabalho de Parto Prematuro , Nascimento Prematuro , Éxons , Feminino , Humanos , Recém-Nascido , Trabalho de Parto Prematuro/genética , Placenta/metabolismo , Gravidez , Nascimento Prematuro/genética , Proteômica , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
7.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066314

RESUMO

HDL particles can be structurally modified in atherosclerotic disorders associated with low HDL cholesterol level (HDL-C). We studied whether the lipidome of the main phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) species of HDL2 and HDL3 subfractions is associated with premature coronary heart disease (CHD) or metabolic syndrome (MetS) in families where common low HDL-C predisposes to premature CHD. The lipidome was analyzed by LC-MS. Lysophosphatidylcholines were depleted of linoleic acid relative to more saturated and shorter-chained acids containing species in MetS compared with non-affected subjects: the ratio of palmitic to linoleic acid was elevated by more than 30%. A minor PC (16:0/16:1) was elevated (28-40%) in MetS. The contents of oleic acid containing PCs were elevated relative to linoleic acid containing PCs in MetS; the ratio of PC (16:0/18:1) to PC (16:0/18:2) was elevated by 11-16%. Certain PC and SM ratios, e.g., PC (18:0/20:3) to PC (16:0/18:2) and a minor SM 36:2 to an abundant SM 34:1, were higher (11-36%) in MetS and CHD. The fatty acid composition of certain LPCs and PCs displayed a characteristic pattern in MetS, enriched with palmitic, palmitoleic or oleic acids relative to linoleic acid. Certain PC and SM ratios related consistently to CHD and MetS.


Assuntos
Doença da Artéria Coronariana/metabolismo , Ácidos Graxos/metabolismo , Lipoproteínas HDL/metabolismo , Síndrome Metabólica/metabolismo , Fosfolipídeos/metabolismo , Adulto , Família , Feminino , Humanos , Lipidômica , Masculino , Pessoa de Meia-Idade , Fatores de Risco
8.
BMC Cardiovasc Disord ; 19(1): 306, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856737

RESUMO

BACKGROUND: Calcific aortic valve disease (CAVD) is an atheroinflammatory process; finally it leads to progressive calcification of the valve. There is no effective pharmacological treatment for CAVD and many of the underlying molecular mechanisms remain unknown. We conducted a proteomic study to reveal novel factors associated with CAVD. METHODS: We compared aortic valves from patients undergoing valvular replacement surgery due to non-calcified aortic insufficiency (control group, n = 5) to a stenotic group (n = 7) using two-dimensional difference gel electrophoresis (2D-DIGE). Protein spots were identified with mass spectrometry. Western blot and immunohistochemistry were used to validate the results in a separate patient cohort and Ingenuity Pathway Analysis (IPA) was exploited to predict the regulatory network of CAVD. RESULTS: We detected an upregulation of complement 9 (C9), serum amyloid P-component (APCS) and transgelin as well as downregulation of heat shock protein (HSP90), protein disulfide isomerase A3 (PDIA3), annexin A2 (ANXA2) and galectin-1 in patients with aortic valve stenosis. The decreased protein expression of HSP90 was confirmed with Western blot. CONCLUSIONS: We describe here a novel data set of proteomic changes associated with CAVD, including downregulation of the pro-inflammatory cytosolic protein, HSP90.


Assuntos
Estenose da Valva Aórtica/metabolismo , Valva Aórtica/química , Valva Aórtica/patologia , Calcinose/metabolismo , Proteínas de Choque Térmico HSP90/análise , Adulto , Idoso , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Calcinose/patologia , Estudos de Casos e Controles , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteômica , Transdução de Sinais
9.
J Cell Mol Med ; 22(2): 968-981, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29193784

RESUMO

Understanding of timing of human parturition is incomplete. Therefore, we carried out proteomic analyses of full-term placentas from uncomplicated pregnancies to identify protein signatures associated with the onset of spontaneous delivery. We found quantitative associations of 10 proteins with spontaneous term birth, evident either in the basal or in the chorionic plates or in both. Additional 18 proteins were associated according to the location within placenta indicating local variations in protein amounts. Calcineurin-like phosphoesterase domain-containing 1 (CPPED1), a phosphatase previously suggested dephosphorylating AKT1/PKB, was one of the identified proteins. qRT-PCR revealed the mRNA level of CPPED1 was higher in elective caesarean deliveries than in spontaneous births, while immunohistochemistry showed CPPED1 in cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts. Noteworthy, phosphorylation status of AKT1 did not differ between placentas from elective caesarean and spontaneous deliveries. Additionally, analyses of samples from infants indicated that single-nucleotide polymorphisms rs11643593 and rs8048866 of CPPED1 were associated with duration of term pregnancy. Finally, post-transcriptional silencing of CPPED1 in cultured HTR8/SVneo cells by siRNAs affected gene expression in pathways associated with inflammation and blood vessel development. We postulate that functions regulated by CPPED1 in trophoblasts at choriodecidual interphase have a role in the induction of term labour, but it may be independent of AKT1.


Assuntos
Calcineurina/metabolismo , Nascimento a Termo/metabolismo , Trofoblastos/metabolismo , Calcineurina/genética , Vilosidades Coriônicas/metabolismo , Parto Obstétrico , Feminino , Proteína Forkhead Box O1/metabolismo , Inativação Gênica , Idade Gestacional , Humanos , Recém-Nascido , Inflamação/genética , Neovascularização Fisiológica/genética , Fenótipo , Fosforilação , Placenta/metabolismo , Placenta/patologia , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Proteoma/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo
10.
Nature ; 483(7390): 484-8, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22343896

RESUMO

The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG, but not (S)-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy.


Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Dioxigenases/metabolismo , Glutaratos/química , Glutaratos/farmacologia , Proteínas Nucleares/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Dioxigenases/genética , Ativação Enzimática/efeitos dos fármacos , Glioma/enzimologia , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Glutaratos/metabolismo , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Proteínas Nucleares/genética , Oncogenes , Pró-Colágeno-Prolina Dioxigenase/genética
11.
Biochem J ; 474(5): 751-769, 2017 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-28093469

RESUMO

Collagen prolyl 4-hydroxylase (C-P4H), an α2ß2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The ß-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein-protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/ß-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/ß-subunits locate at the far ends of the ßααß complex. The PDI/ß-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB-CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.


Assuntos
Prolina/química , Prolil Hidroxilases/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Prolina/metabolismo , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Difração de Raios X
12.
Biochem Biophys Res Commun ; 491(4): 953-957, 2017 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-28756229

RESUMO

The Wnt proteins constitute a conserved family of secreted palmitoleate-containing signaling proteins that play important roles in development and tissue homeostasis. Their hydrophobic nature has raised the question of how the proteins are transported outside the cells. Accumulating evidence suggests that several different mechanisms, including transport by lipoprotein particles and exosomes, may contribute to this process. Here, we expressed epitope-tagged Wnt4 in HEK293 cells, and identified Mac-2 binding protein (Mac-2BP) as its binding partner in the serum-free conditioned medium. Serine-to-alanine substitution at the conserved fatty acid-conjugation site did not affect Mac-2BP binding. Subsequent studies showed that Mac-2BP may be a general Wnt interactor. It is found in the extracellular matrix (ECM) of various tissues, where it forms unusual oligomeric ring-like structures. Its functions appear to include interactions with cells and certain ECM components. Intriguingly, both Wnt signaling and Mac-2BP expression are upregulated in many types of cancer. Our studies on the four-domain Mac-2BP indicate a crucial role in Wnt binding for the C-terminal domain that bears no sequence similarity to any other protein. Mac-2BP may have a role in regulating the extracellular spreading and storage of the Wnts, thereby modulating their bioavailability and stability.


Assuntos
Antígenos de Neoplasias/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Wnt/metabolismo , Células HEK293 , Humanos
13.
Proteins ; 84(8): 1075-96, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27093562

RESUMO

Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cß2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Proteins 2016; 84:1075-1096. © 2016 Wiley Periodicals, Inc.


Assuntos
Acetilcoenzima A/química , Acil Coenzima A/química , Aciltransferases/química , Malonatos/química , Proteínas Mitocondriais/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/enzimologia , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Metabolismo dos Lipídeos , Malonatos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trypanosoma brucei brucei/química
14.
Am J Physiol Lung Cell Mol Physiol ; 310(11): L1155-65, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27084846

RESUMO

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by irreversible airflow limitation. Cigarette smoking represents the main risk factor, but the specific mechanisms of COPD are not completely understood. Our aim was to identify COPD-specific proteomic changes involved in disease onset and severity. A comparative proteomic analysis of 51 lung tissues from nonsmokers, smokers, smokers with mild to moderate (stage I-II) COPD, severe to very severe COPD (stage III-IV), and patients with α-1-antitrypsin deficiency (AATD) and idiopathic pulmonary fibrosis (IPF) was performed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Selected COPD-specific changes were validated by immunoblotting and further by ELISA in 120 induced sputum and plasma samples from nonsmokers, smokers, and patients with COPD (stage I-III). Altogether 82 altered proteins were identified comprising COPD-, AATD-, and IPF-specific, overlapping, and unspecific changes. Cathepsin D (CTSD), dihydropyrimidinase-related protein 2 (DPYSL2), transglutaminase 2 (TGM2), and tripeptidyl-peptidase 1 (TPP1) were validated as COPD-specific. TGM2 was not associated with smoking and correlated with COPD severity in lung tissue. TGM2 levels in sputum and plasma were elevated in patients with COPD (stage II-III) and correlated with lung function. In conclusion, new proteins related to COPD onset and severity could be identified with TGM2 being a novel potential diagnostic and therapeutic target for COPD. Further studies in carefully characterized cohorts are required to validate the identified changes.


Assuntos
Proteínas de Ligação ao GTP/sangue , Pulmão/enzimologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Transglutaminases/sangue , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Proteoma/metabolismo , Proteômica , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumar/efeitos adversos , Fumar/sangue , Tripeptidil-Peptidase 1
15.
Oncogenesis ; 13(1): 37, 2024 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-39394169

RESUMO

Although there have been advances in understanding colorectal cancer (CRC) pathogenesis, significant gaps still exist, highlighting the need for deeper insights. Dysregulated protein homeostasis, including perturbations in the epidermal growth factor receptor (EGFR) pathway, remains a focal point in CRC pathogenesis. Within this context, the roles of ubiquitin ligases and deubiquitinases have attracted attention, but exploration of their precise contributions is still in its early stages. To address this gap, we investigated the involvement of the deubiquitinase USP10 in CRC. Our in vitro and in vivo study reveals a new paradigm in CRC biology and unravels a novel mechanistic axis, demonstrating for the first time the involvement of inositol polyphosphate 4-phosphatase type II B (INPP4B) in USP10-mediated CRC modulation. Specifically, our study demonstrates that the loss of USP10 results in reduced sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and osimertinib. This is accompanied by a decrease in the activation of the AKT1/PKB pathway upon EGF stimulation, which is mediated by INPP4B. Importantly, in vivo xenograft experiments validate these findings and highlight the crucial role of USP10, particularly in conjunction with INPP4B, in driving CRC progression. The findings enhance our understanding of CRC pathobiology and reveal a new regulatory axis involving USP10 and INPP4B in CRC progression. This unique insight identifies USP10 and INPP4B as potential therapeutic targets in CRC.

16.
J Dermatol Sci ; 113(3): 121-129, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38326167

RESUMO

BACKGROUND: Vildagliptin, a dipeptidyl peptidase-4 inhibitor (DPP-4i) is a widely used type 2 diabetes medication that is associated with an up-to 10-fold increased risk for the development of bullous pemphigoid (BP), an autoimmune skin disease. The mechanism by which vildagliptin promotes the development of BP remains unknown. OBJECTIVE: To elucidate effects of vildagliptin treatment on the mouse cutaneous proteome. METHODS: We analyzed the cutaneous proteome of nondiabetic mice treated for 12 weeks with vildagliptin using label-free shotgun mass spectrometry (MS), two-dimensional difference gel electrophoresis (2D-DIGE), immunohistochemistry, immunoblotting, and quantitative real-time polymerase chain reaction. RESULTS: Although vildagliptin treatment did not cause any clinical signs or histological changes in the skin, separate MS and 2D-DIGE analyses revealed altered cutaneous expression of several proteins, many of which were related to actin cytoskeleton remodeling. Altogether 18 proteins were increased and 40 were decreased in the vildagliptin-treated mouse skin. Both methods revealed increased levels of beta-actin and C->U-editing enzyme APOBEC2 in vildagliptin-treated mice. However, elevated levels of a specific moesin variant in vildagliptin-treated animals were only detected with 2D-DIGE. Immunohistochemical staining showed altered cutaneous expression of DPP-4, moesin, and galectin-1. The changed proteins detected by MS and 2D-DIGE were linked to actin cytoskeleton remodeling, transport, cell movement and organelle assembly. CONCLUSION: Vildagliptin treatment alters the cutaneous proteome of nondiabetic mice even without clinical signs in the skin. Cytoskeletal changes in the presence of other triggering factors may provoke a break of immune tolerance and further promote the development of BP.


Assuntos
Diabetes Mellitus Tipo 2 , Penfigoide Bolhoso , Camundongos , Animais , Vildagliptina/efeitos adversos , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/tratamento farmacológico , Proteoma , Proteômica , Penfigoide Bolhoso/induzido quimicamente , Citoesqueleto de Actina
17.
Tob Induc Dis ; 222024.
Artigo em Inglês | MEDLINE | ID: mdl-38496254

RESUMO

INTRODUCTION: Mesenchymal stromal cells (MSCs) play a crucial role in promoting tissue regeneration and healing, particularly in bone tissue. Both smoking and nicotine use are known to delay and inhibit the healing process in patients. This study aims at delineating these cellular effects by comparing the impact of nicotine alone to cigarette smoke with equivalent nicotine content, and shedding light on potential differences in the healing process. METHODS: We examined how cigarette smoke and nicotine affect the migration, proliferation, and osteogenic differentiation of human patient-derived MSCs in vitro, as well as the secretion of cytokines IL-6 and IL-8. We measured nicotine concentration of the cigarette smoke extract (CSE) to clarify the role of the nicotine in the effect of the cigarette smoke. RESULTS: MSCs exposed to nicotine-concentration-standardized CSE exhibited impaired wound healing capability, and at high concentrations, increased cell death. At lower concentrations, CSE dose-dependently impaired migration, proliferation, and osteogenic differentiation, and increased IL-8 secretion. Nicotine impaired proliferation and decreased PINP secretion. While there was a trend for elevated IL-6 levels by nicotine in undifferentiated MSCs, these changes were not statistically significant. Exposure of MSCs to equivalent concentrations of nicotine consistently elicited stronger responses by CSE and had a more pronounced effect on all studied parameters. Our results suggest that the direct effect of cigarette smoke on MSCs contributes to impaired MSC function, that adds to the nicotine effects. CONCLUSIONS: Cigarette smoke extract reduced the migration, proliferation, and osteogenic differentiation in MSCs in vitro, while nicotine alone reduced proliferation. Cigarette smoke impairs the osteogenic and regenerative ability of MSCs in a direct cytotoxic manner. Cytotoxic effect of nicotine alone impairs regenerative ability of MSCs, but it only partly explains cytotoxic effects of cigarette smoke. Direct effect of cigarette smoke, and partly nicotine, on MSCs could contribute to the smoking-related negative impact on long-term bone health, especially in bone healing.

18.
Front Cell Dev Biol ; 11: 1250000, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38020884

RESUMO

Hypoxia-inducible factors (HIFs) are best known for their roles in the adaptation to low oxygen environments. Besides hypoxia, HIF-1/2 α-subunits are also regulated by various non-hypoxic stimuli including insulin which can act via the PI3K/protein kinase B (PKB) signaling pathway. However, with respect to insulin little is known about HIF-3α. We aimed to investigate this relationship and found that insulin stimulates HIF-3α expression under both normal and low oxygen conditions. Blocking PKB activity reversed the effects of insulin, indicating that HIF-3α is a direct target of PKB. We identified serine 524, located in the oxygen-dependent degradation domain of HIF-3α, as a phosphorylation site of PKB. Mutating serine 524 impaired binding of PKB to HIF-3α and its ubiquitination, suggesting that PKB regulates HIF-3α stability through phosphorylation, thereby affecting important cellular processes such as cell viability and cell adhesion. Importantly, we discovered that this phosphorylation site also influenced insulin-dependent cell migration. These findings shed light on a novel mechanism by which insulin affects PKB-dependent HIF-3α expression and activity, with potential implications in metabolic diseases and cancer.

19.
J Mol Cell Cardiol ; 53(6): 906-15, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23036824

RESUMO

Reactive oxygen species (ROS) have been implicated in many aspects of tissue/cellular metabolic signaling and pathology, including cardioprotection against ischemia-reperfusion damage. Recent reports of enhanced ROS production under global or simulated ischemia in intact heart or isolated cardiomyocytes, respectively, and its decrease again upon reperfusion are paradoxical. Mechanisms for increasing ROS production with decreasing reactant (oxygen) concentration remain elusive, making it important to critically evaluate the experimental methods used to measure ROS production. In the present paper superoxide production in isolated perfused rat hearts was monitored by lucigenin chemiluminescence or dihydroethidine (DHE) oxidation product fluorescence in parallel with redox state of flavin and cytochrome oxidase. Lucigenin luminescence decreased in ischemia and increased again upon reperfusion, transiently reaching values eightfold the control value coincidently with an overshoot of mitochondrial oxygen concentration. Hypoxic perfusion decreased lucigenin chemiluminescence in spite of coronary flow increase, whereas change in lucigenin concentration in the perfusate had negligible effect. In contrast to lucigenin luminescence, the fluorescence of the DHE oxidation product increased continuously during a 30-min global ischemia and decreased precipitously upon reperfusion, this change is coincident with absorption changes of the oxygen-binding protein myoglobin. The time course of DHE oxidation product fluorescence during ischemia and reperfusion was similar to that of the mitochondrial membrane potential probe safranin as shown in perfused heart previously [Ylitalo KV, Ala-Rämi A, Liimatta EV, Peuhkurinen KJ, Hassinen IE. J Mol Cell Cardiol 2000;32:1223-38]. In solution under high oxygen partial pressure DHE was mainly oxidized to a product, whose fluorescence, absorbance and mass spectra were similar to ethidium, and this product behaved like a mitochondrial membrane potential probe in isolated mitochondria. As a membrane permeable cation it accumulates into the mitochondria when the membrane potential is high (high intramitochondrial concentration quenches fluorescence) and then is released (increased fluorescence) during hypoxia/ischemia. Upon reperfusion it is re-accumulated in the mitochondria as the membrane potential recovers. The non-specific oxidation of DHE makes this dye less suitable for superoxide detection in experiments on isolated perfused hearts that necessitate high oxygen partial pressure in the perfusate. The time course of lucigenin luminescence during ischemia/reperfusion is consistent with decreased ROS production during ischemia/hypoxia, while the oxygen concentration is decreased, followed by an overshoot when the heart tissue is reperfused and the oxygen pressures return to normal or above normal.


Assuntos
Traumatismo por Reperfusão Miocárdica/metabolismo , Superóxidos/metabolismo , Acridinas , Animais , Circulação Coronária , Dicarbetoxi-Di-Hidrocolidina/análogos & derivados , Dicarbetoxi-Di-Hidrocolidina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Flavoproteínas/metabolismo , Técnicas In Vitro , Fígado/metabolismo , Substâncias Luminescentes , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mioglobina/metabolismo , Oxirredução , Consumo de Oxigênio , Ratos
20.
J Proteome Res ; 11(2): 599-608, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22053820

RESUMO

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms leading to COPD and its progression are still poorly understood. In this study, induced sputum was analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis. The comparison of nonsmokers, smokers, and smokers with moderate COPD revealed 15 changed proteins with the majority, including polymeric immunoglobulin receptor (PIGR), being elevated in smokers and subjects with COPD. PIGR, which is involved in specific immune defense and inflammation, was further studied in sputum, lung tissue, and plasma by Western blot, immunohistochemistry/image analysis, and/or ELISA. Sputum PIGR was characterized as glycosylated secretory component (SC). Lung PIGR was significantly elevated in the bronchial and alveolar epithelium of smokers and further increased in the alveolar area in mild to moderate COPD. Plasma PIGR was elevated in smokers and smokers with COPD compared to nonsmokers with significant correlation to obstruction. In conclusion, new proteins in smoking-related chronic inflammation and COPD could be identified, with SC/PIGR being one of the most prominent not only in the lung but also in circulating blood.


Assuntos
Proteoma/análise , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores de Imunoglobulina Polimérica/análise , Fumar/metabolismo , Escarro/química , Eletroforese em Gel Bidimensional , Humanos , Imuno-Histoquímica , Proteoma/metabolismo , Proteômica/métodos , Alvéolos Pulmonares/química , Alvéolos Pulmonares/metabolismo , Doença Pulmonar Obstrutiva Crônica/sangue , Receptores de Imunoglobulina Polimérica/sangue , Receptores de Imunoglobulina Polimérica/metabolismo , Fumar/sangue , Escarro/metabolismo
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