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1.
Mol Pharmacol ; 97(5): 304-313, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32132133

RESUMO

Free-fatty acid receptor-4 (FFA4), previously termed GPR120, is a G protein-coupled receptor (GPCR) for medium and long-chained fatty acids, agonism of which can regulate a myriad of metabolic, sensory, inflammatory, and proliferatory signals. Two alternative splice isoforms of FFA4 exist that differ by the presence of an additional 16 amino acids in the longer (FFA4-L) transcript, which has been suggested to be an intrinsically ß-arrestin-biased GPCR. Although the shorter isoform (FFA4-S) has been studied more extensively, very little is known about mechanisms of regulation or signaling of the longer isoform. Because ß-arrestin recruitment is dependent on receptor phosphorylation, in the current study, we used the endogenous agonist docosahexaenoic acid (DHA) to examine the mechanisms of FFA4-L phosphorylation, as well as DHA-dependent ß-arrestin recruitment and DHA-dependent extracellular-signal regulated kinase-1/2 (ERK1/2) signaling in human embryonic kidney 293 cells. Our results reveal differences in basal phosphorylation of the two FFA4 isoforms, and we show that DHA-mediated phosphorylation of FFA4-L is primarily regulated by G protein-coupled receptor kinase 6, whereas protein kinase-C can also contribute to agonist-induced and heterologous phosphorylation. Moreover, our data demonstrate that FFA4-L phosphorylation occurs on the distal C terminus and is directly responsible for recruitment and interactions with ß-arrestin-2. Finally, using CRISPR/Cas9 genome-edited cells, our data reveal that unlike FFA4-S, the longer isoform is unable to facilitate phosphorylation of ERK1/2 in cells that are devoid of ß-arrestin-1/2. Together, these results are the first to demonstrate phosphoregulation of FFA4-L as well as the effects of loss of phosphorylation sites on ß-arrestin recruitment and ERK1/2 activation. SIGNIFICANCE STATEMENT: Free-fatty acid receptor-4 (FFA4) is a cell-surface G protein-coupled receptor for medium and long-chained fatty acids that can be expressed as distinct short (FFA4-S) or long (FFA4-L) isoforms. Although much is known about FFA4-S, the longer isoform remains virtually unstudied. Here, we reveal the mechanisms of docosahexaenoic acid-induced phosphorylation of FFA4-L and subsequent ß-arrestin-2 recruitment and extracellular-signal regulated kinase-1/2 activity.


Assuntos
Processamento Alternativo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestina 2/metabolismo , Processamento Alternativo/efeitos dos fármacos , Sequência de Aminoácidos , Ácidos Docosa-Hexaenoicos/farmacologia , Quinases de Receptores Acoplados a Proteína G/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/efeitos dos fármacos
2.
Subcell Biochem ; 94: 499-520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189313

RESUMO

C-reactive protein (CRP) is an evolutionary highly conserved member of the pentraxin superfamily of proteins. CRP is widely used as a marker of inflammation, infection and for risk stratification of cardiovascular events. However, there is now a large body of evidence, that continues to evolve, detailing that CRP directly mediates inflammatory reactions and the innate immune response in the context of localised tissue injury. These data support the concept that the pentameric conformation of CRP dissociates into pro-inflammatory CRP isoforms termed pCRP* and monomeric CRP. These pro-inflammatory CRP isoforms undergo conformational changes that facilitate complement binding and immune cell activation and therefore demonstrate the ability to trigger complement activation, activate platelets, monocytes and endothelial cells. The dissociation of pCRP occurs on the surface of necrotic, apoptotic, and ischaemic cells, regular ß-sheet structures such as ß-amyloid, the membranes of activated cells (e.g., platelets, monocytes, and endothelial cells), and/or the surface of microparticles, the latter by binding to phosphocholine. Therefore, the deposition and localisation of these pro-inflammatory isoforms of CRP have been demonstrated to amplify inflammation and tissue damage in a broad range of clinical conditions including ischaemia/reperfusion injury, Alzheimer's disease, age-related macular degeneration and immune thrombocytopaenia. Given the potentially broad relevance of CRP to disease pathology, the development of inhibitors of CRP remains an area of active investigation, which may pave the way for novel therapeutics for a diverse range of inflammatory diseases.


Assuntos
Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Sequência Conservada , Evolução Molecular , Inflamação/metabolismo , Inflamação/patologia , Biomarcadores/química , Biomarcadores/metabolismo , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
3.
Gene ; 742: 144538, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32184168

RESUMO

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.


Assuntos
Ciclídeos/metabolismo , Proteínas de Peixes/metabolismo , Neuraminidase/metabolismo , Animais , Ciclídeos/genética , Ciclídeos/crescimento & desenvolvimento , Clonagem Molecular , Evolução Molecular , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Neuraminidase/química , Neuraminidase/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Especificidade por Substrato/genética
4.
Food Chem ; 315: 126318, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32035317

RESUMO

To better understand the contribution of myosin light chain (MLC) isoforms to sensory defects in Jinhua ham, dipeptidyl peptidase (DPP) activities, peptide fragments, cleavage sites and the potential of DPP to develop sensory defects of dry-cured ham were evaluated and discussed in normal and defective hams. Higher residual activities of DPP I were found in defective ham compared with normal ham; approximate 3-fold peptide fragments were identified in defective ham than in normal ham. These regions of positions 11-35 and 116-141 in MLC 1, 13-53 and 139-156 in MLC 2, and 18-50 in MLC 3 contributed to the intense generation of peptide fragments in defective ham. PLS-DA further revealed DPP I showing intense response to degrade peptides. Cleavage sites including Glu-128, Tyr-132 and Glu-133 were responsible for the intense release of dipeptides in defective ham. These cleavages could play key role in discriminating taste attributes between defective and normal hams.


Assuntos
Produtos da Carne/análise , Cadeias Leves de Miosina/química , Carne de Porco/análise , Animais , Cadeias Leves de Miosina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteólise , Suínos
5.
PLoS One ; 15(1): e0228325, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999773

RESUMO

Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 PcH subunit isoforms, in contrast with other gastropods that usually have 2 or 3. Each isoform has the typical Keyhole limpet-type hemocyanin architecture, comprising a string of eight globular functional units (FUs). Correspondingly, genes are organized in eight FUs coding regions. All FUs in the 4 genes are encoded by more than one exon, a feature not found in non- caenogastropods. Transmission electron microscopy images of PcH showed a cylindrical structure organized in di, tri and tetra-decamers with an internal collar structure, being the di and tri-decameric cylinders the most abundant ones. PcH is N-glycosylated with high mannose and hybrid-type structures, and complex-type N-linked glycans, with absence of sialic acid. Terminal ß-N-GlcNAc residues and nonreducing terminal α-GalNAc are also present. The molecule lacks O-linked glycosylation but presents the T-antigen (Gal-ß1,3-GalNAc). Using an anti-PcH polyclonal antibody, no cross-immunoreactivity was observed against other gastropod hemocyanins, highlighting the presence of clade-specific structural differences among gastropod hemocyanins. This is, to the best of our knowledge, the first gene structure study of a Caenogastropoda hemocyanin.


Assuntos
Gastrópodes/genética , Gastrópodes/metabolismo , Hemocianinas/química , Hemocianinas/genética , Animais , Evolução Molecular , Gastrópodes/química , Perfilação da Expressão Gênica , Genômica , Hemocianinas/metabolismo , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteômica
6.
PLoS One ; 15(1): e0226382, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929539

RESUMO

Klotho is an age-extending, cognition-enhancing protein found to be down-regulated in aged mammals when age-related diseases start to appear. Low levels of Klotho occur in neurodegenerative diseases, kidney disease and many cancers. Many normal and pathologic processes involve the proteolytic shedding of membrane proteins. Transmembrane (TM) Klotho contains two homologous domains, KL1 and KL2 with homology to glycosidases. After shedding by ADAM 10 and 17, a shed Klotho isoform is released into serum and urine by the kidney, and into the CSF by the choroid plexus. We previously reported that human Klotho contains two major cleavage sites. However, the exact cleavage site responsible for the cleavage between the KL1 and KL2 domains remains unknown for the human Klotho, and both sites are unknown for mouse Klotho. In this study, we aimed to identify the cleavage sites leading to the shed forms of human and mouse Klotho. Mutations in the region close to the TM domain of mouse Klotho result in the reduced shedding of the 130 kD (KL1+KL2) and 70 kD (KL1) fragments, suggesting that the cleavage site lies within the mutated region. We further identified the cleavage sites responsible for the cleavage between KL1 and KL2 of human and mouse Klotho. Moreover, mutated Klotho proteins have similar subcellular localization patterns as wild type Klotho. Finally, in an FGF23 functional assay, all Klotho mutants with a nine amino acid deletion can also function as an FGFR1 co-receptor for FGF23 signaling, however, the signaling activity was greatly reduced. The study provides new and important information on Klotho shedding, and paves the way for studies aimed to distinguish between the distinct roles of the various isoforms of Klotho.


Assuntos
Glucuronidase/metabolismo , Proteína ADAM10/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/química , Glucuronidase/genética , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Mutagênese , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Alinhamento de Sequência , Transdução de Sinais
7.
RNA ; 26(1): 19-28, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31624092

RESUMO

Direct RNA sequencing holds great promise for the de novo identification of RNA modifications at single-coordinate resolution; however, interpretation of raw sequencing output to discover modified bases remains a challenge. Using Oxford Nanopore's direct RNA sequencing technology, we developed a random forest classifier trained using experimentally detected N 6-methyladenosine (m6A) sites within DRACH motifs. Our software MINES (m6A Identification using Nanopore Sequencing) assigned m6A methylation status to more than 13,000 previously unannotated DRACH sites in endogenous HEK293T transcripts and identified more than 40,000 sites with isoform-level resolution in a human mammary epithelial cell line. These sites displayed sensitivity to the m6A writer, METTL3, and eraser, ALKBH5, respectively. MINES (https://github.com/YeoLab/MINES.git) enables m6A annotation at single coordinate-level resolution from direct RNA nanopore sequencing.


Assuntos
Sequenciamento por Nanoporos/métodos , Isoformas de Proteínas/química , RNA Mensageiro/química , Software , Células HEK293 , Humanos , Metilação , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos
8.
J Chromatogr A ; 1611: 460608, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31629491

RESUMO

Mechanistic modeling of protein adsorption has gained increasing importance in the development of ion-exchange (IEX) chromatography processes. The most common adsorption models use a stoichiometric representation of the adsorption process based on the law of mass action. Despite the importance of these models in model-based development, the stoichiometric representation of the adsorption process is not accurate for the description of long-range electrostatic interactions in IEX chromatography, limiting the application and mechanistic extension of these models. In this work an adsorption model is introduced describing the non-stoichiometric electrostatic interaction in IEX chromatography based on the linear Poisson-Boltzmann equation and a simplified colloidal representation of the protein. In contrast to most recent non-stoichiometric models, the introduced model accounts for charge regulation during the adsorption process. Its capability of describing the adsorption equilibrium is demonstrated by simulating partitioning coefficients of multiple proteins on different adsorber systems as a function of ionic strength and pH. Despite model simplifications the physical meaning and predictive value of the model could be preserved. By transferring model parameters of a monoclonal antibody (mAb) from one adsorber system to another, it could be demonstrated that protein parameters are theoretically not only valid on a specific adsorber system but freely transferable to other adsorbers. The predictive value of the mechanistic model on the new adsorber system was highlighted by predicting the elution behavior of charge variants of the mAb.


Assuntos
Cromatografia por Troca Iônica/métodos , Coloides/química , Proteínas/química , Eletricidade Estática , Adsorção , Ligantes , Isoformas de Proteínas/química , Software
9.
Biochim Biophys Acta Biomembr ; 1862(2): 183055, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31487493

RESUMO

The search of antimicrobial peptides (AMP) as candidates for the development of antibiotics is an active research field. In this paper we investigated the role of charged residues in antimicrobial activity by using as a template the previously characterized crabrolin peptide. Mutant peptides in which the charge was diminished (Crabrolin Minus) or increased (Crabrolin Plus) were assayed for their ability to inhibit bacterial growth and to bind model bacterial membranes or lipopolysaccharide (LPS). Structural analysis of both peptides by means of CD, NMR and Molecular Dynamics was also performed and correlated to the biological data. Although native Crabrolin (WT) displays smaller efficacy than other antibacterial peptides with similar length, Crabrolin Plus displays a significant antimicrobial activity while Crabrolin Minus is not active, thus confirming the key role of the positive charge for interacting with the bacterial membrane. Moreover, our results show that charge position has no effect on the helical propensity of the peptides but drastically affects their antimicrobial activity. Antimicrobial activity versus Gram-positive and Gram-negative bacteria, as well as specific interaction with LPS, suggest multiple binding modes for the active peptide.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Íons/química , Venenos de Vespas/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sítios de Ligação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Lipopolissacarídeos , Estrutura Molecular , Engenharia de Proteínas/métodos , Isoformas de Proteínas/química , Venenos de Vespas/farmacologia
10.
Food Chem Toxicol ; 136: 110977, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31759068

RESUMO

Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.


Assuntos
Antifúngicos/toxicidade , Inseticidas/toxicidade , Proteínas de Plantas/toxicidade , Medição de Risco , Urease/toxicidade , Animais , Antifúngicos/química , Canavalia/enzimologia , Biologia Computacional , Fungos/efeitos dos fármacos , Insetos/efeitos dos fármacos , Inseticidas/química , Proteínas de Plantas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/toxicidade , Proteólise , Urease/química
11.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140330, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31756432

RESUMO

Knotted proteins are some of the most fascinating examples of how linear polypeptide chains can achieve intricate topological arrangements efficiently and spontaneously. The entanglements of polypeptide chains could potentially enhance their folding stabilities. We recently reported the unprecedented mechanostability of the Gordian (52) knotted family of human ubiquitin C-terminal hydrolases (UCHs) in the context of withstanding the mechanical unfolding of the bacterial AAA+ proteasome, ClpXP; a green fluorescence protein (GFP) was fused to the N-terminus of various UCHs as a reporter of the unfolding and degradation of these topologically knotted substrates, but it also limited the ability to examine the effect of untying the knotted topology via N-terminal truncation. In this study, we directly monitored the ClpXP-mediated degradation of UCH variants by electrophoresis and quantitative imaging analyses. We demonstrated that untying of the 52 knot in UCHL1 via N-terminal truncation (UCHL1Δ11) significantly reduces its mechanostability. We further quantified the ATP expenditures of degrading different UCH variants by ClpXP. The unknotted UCHL1Δ11 underwent accelerated ClpXP-dependent proteolysis, with a 30-fold reduction in ATP consumption compared to the knotted wild type. Unlike all other known ClpXP substrates, UCHL5, which is the most resilient substrate known to date, significantly slowed down the ATP turnover rate by ClpXP. Furthermore, UCHL5 required 1000-fold more ATP to be fully degraded by ClpXP compared to GFP. Our results underscored how the complex, knotted folding topology in UCHs may interfere with the mechano-unfolding processes of the AAA+ unfoldase, ClpX.


Assuntos
Trifosfato de Adenosina/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Endopeptidase Clp/genética , Metabolismo Energético , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteólise , Especificidade por Substrato , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo
12.
J Plant Physiol ; 245: 153082, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31862648

RESUMO

Indole-3-acetic acid (IAA) conjugation is one of the mechanisms responsible for auxin homeostasis. IAA ester conjugates biosynthesis has been studied during development of maize seeds where IAA-inositol (IAInos) and its glycosidic forms make up about 50 % of its ester conjugates pool. 1-O-indole-3-acetyl-ß-d-glucose (IAGlc) synthase and indole-3-acetyl transferase (IAInos synthase) are key enzymes in a two-step pathway of IAInos synthesis. In the first reaction, IAA is glucosylated to a high energy acetal, 1-O-indole-3-acetyl-ß-d-glucose by IAGlc synthase, whereas in the second step, IAInos synthase transfers IAA moiety to myo-inositol forming a stable auxin ester, indole-3-acetyl-myo-inositol (IAInos). It should be mentioned that IAGlc synthase catalyzes a reversible reaction with unfavourable equilibrium that delivers IAGlc for favourable transacylation to IAInos. This is the first study where IAGlc synthase and IAInos synthase are simultaneously analyzed by enzymatic activity assay and quantitative RT-PCR in maize seeds at four stages of development (13, 26, 39 and 52 Days After Flowering). Activity of IAGlc/IAInos synthases as well as their expression profiles during seed development were different. While both enzymatic activities and ZmIAIn expression were the highest in seeds at 26 DAF, the highest expression of ZmIAGlc was observed at 13 DAF. Protein gel blot analysis showed that IAInos synthase exists as a mixture of several isoforms at a similar protein level at particular stages of seed development. Neither of other ester conjugates of IAA (IAA-mannose) nor IAA-amino acids were detected at the stages studied. Catalytic activity of l-tryptophan aminotransferase involved in IAA biosynthesis as well as UDPG pyrophosphorylase, synthesizing UDPG as a substrate for IAGlc synthase, were also analyzed. l-tryptophan aminotransferase activity was the highest at 26 DAF. Changes in enzyme activity of UDPG pyrophosphorylase are difficult to interpret. Expression levels of ZmIPS and ZmIPP encoding two enzymes of myo-inositol biosynthesis pathway: inositol-x-phosphate synthase (IPS) and inositol-x-phosphate phosphatase (IPP), respectively, were analyzed. 26 DAF seeds displayed the highest expression level of ZmIPS, whereas transcription of ZmIPP was the highest at 13 DAF.


Assuntos
Glucosiltransferases/metabolismo , Ácidos Indolacéticos/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Zea mays/enzimologia , Zea mays/crescimento & desenvolvimento , Vias Biossintéticas/genética , Vias Biossintéticas/fisiologia , Catálise , Glucosiltransferases/genética , Homeostase/genética , Homeostase/fisiologia , Indóis/metabolismo , Inositol/metabolismo , Inositol Polifosfato 5-Fosfatases/metabolismo , Liases Intramoleculares/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Triptofano Transaminase/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Uridina Difosfato Glucose/metabolismo , Zea mays/metabolismo
13.
Int J Mol Sci ; 20(24)2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31835844

RESUMO

p53, first described four decades ago, is now established as a master regulator of cellular stress response, the "guardian of the genome". p53 contributes to biological robustness by behaving in a cellular-context dependent manner, influenced by several factors (e.g., cell type, active signalling pathways, the type, extent and intensity of cellular damage, cell cycle stage, nutrient availability, immune function). The p53 isoforms regulate gene transcription and protein expression in response to the stimuli so that the cell response is precisely tuned to the cell signals and cell context. Twelve isoforms of p53 have been described in humans. In this review, we explore the interactions between p53 isoforms and other proteins contributing to their established cellular functions, which can be both tumour-suppressive and oncogenic in nature. Evidence of p53 isoform in human cancers is largely based on RT-qPCR expression studies, usually investigating a particular type of isoform. Beyond p53 isoform functions in cancer, it is implicated in neurodegeneration, embryological development, progeroid phenotype, inflammatory pathology, infections and tissue regeneration, which are described in this review.


Assuntos
Neoplasias/metabolismo , Fenômenos Fisiológicos , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência Conservada , Humanos , Mutação/genética , Neoplasias/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética
14.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801293

RESUMO

The gastric secretory trefoil factor family (TFF) peptides xP1 and xP4 are the Xenopus laevis orthologs of mammalian TFF1 and TFF2, respectively. The aim of this study was to analyze the molecular forms of xP1 and xP4 in the X. laevis gastric mucosa by FPLC. xP1 mainly occurred in a monomeric low-molecular-mass form and only a minor subset is associated with the mucus fraction. The occurrence of monomeric xP1 is unexpected because of its odd number of cysteine residues. Probably a conserved acidic residue flanking Cys55 allows monomeric secretion. Furthermore, Cys55 is probably post-translationally modified. For the first time, we hypothesize that the free thiol of monomeric xP1-and probably also its mammalian ortholog TFF1-could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. In contrast, xP4 mainly occurs in a high-molecular-mass form and is non-covalently bound to a mucin similarly as TFF2. In vitro binding studies with radioactively labeled porcine TFF2 even showed binding to X. laevis gastric mucin. Thus, xP4 is expected to bind as a lectin to an evolutionary conserved sugar epitope of the X. laevis ortholog of mucin MUC6 creating a tight mucus barrier. Taken together, xP1 and xP4 appear to have different gastric protective functions.


Assuntos
Proteínas de Anfíbios/química , Depuradores de Radicais Livres/química , Mucosa Gástrica/metabolismo , Substâncias Protetoras/química , Processamento de Proteína Pós-Traducional , Fator Trefoil-1/química , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/metabolismo , Proteínas de Anfíbios/farmacologia , Animais , Depuradores de Radicais Livres/isolamento & purificação , Depuradores de Radicais Livres/metabolismo , Depuradores de Radicais Livres/farmacologia , Peso Molecular , Mucinas/química , Mucinas/metabolismo , Substâncias Protetoras/isolamento & purificação , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Espécies Reativas de Nitrogênio/antagonistas & inibidores , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Suínos , Fator Trefoil-1/isolamento & purificação , Fator Trefoil-1/metabolismo , Fator Trefoil-1/farmacologia , Xenopus laevis/fisiologia
15.
BMC Mol Cell Biol ; 20(1): 50, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31726976

RESUMO

BACKGROUND: Olfactomedin-1 (Olfm1; also known as Noelin or Pancortin) is a highly-expressed secreted brain and retina protein and its four isoforms have different roles in nervous system development and function. Structural studies showed that the long Olfm1 isoform BMZ forms a disulfide-linked tetramer with a V-shaped architecture. The tips of the Olfm1 "V" each consist of two C-terminal ß-propeller domains that enclose a calcium binding site. Functional characterisation of Olfm1 may be aided by new biochemical tools derived from these core structural elements. RESULTS: Here we present the production, purification and structural analysis of three novel monomeric, dimeric and tetrameric forms of mammalian Olfm1 for functional studies. We characterise these constructs structurally by high-resolution X-ray crystallography and small-angle X-ray scattering. The crystal structure of the Olfm1 ß-propeller domain (to 1.25 Å) represents the highest-resolution structure of an olfactomedin family member to date, revealing features such as a hydrophilic tunnel containing water molecules running into the core of the domain where the calcium binding site resides. The shorter Olfactomedin-1 isoform BMY is a disulfide-linked tetramer with a shape similar to the corresponding region in the longer BMZ isoform. CONCLUSIONS: These recombinantly-expressed protein tools should assist future studies, for example of biophysical, electrophysiological or morphological nature, to help elucidate the functions of Olfm1 in the mature mammalian brain. The control over the oligomeric state of Olfm1 provides a firm basis to better understand the role of Olfm1 in the (trans-synaptic) tethering or avidity-mediated clustering of synaptic receptors such as post-synaptic AMPA receptors and pre-synaptic amyloid precursor protein. In addition, the variation in domain composition of these protein tools provides a means to dissect the Olfm1 regions important for receptor binding.


Assuntos
Proteínas da Matriz Extracelular , Glicoproteínas , Neurobiologia/métodos , Animais , Sítios de Ligação , Encéfalo/citologia , Encéfalo/metabolismo , Cristalografia por Raios X , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/isolamento & purificação , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Células HEK293 , Humanos , Camundongos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Transfecção
16.
BMC Genomics ; 20(1): 804, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31684883

RESUMO

BACKGROUND: Stability is one of the most fundamental intrinsic characteristics of proteins and can be determined with various methods. Characterization of protein properties does not keep pace with increase in new sequence data and therefore even basic properties are not known for far majority of identified proteins. There have been some attempts to develop predictors for protein stabilities; however, they have suffered from small numbers of known examples. RESULTS: We took benefit of results from a recently developed cellular stability method, which is based on limited proteolysis and mass spectrometry, and developed a machine learning method using gradient boosting of regression trees. ProTstab method has high performance and is well suited for large scale prediction of protein stabilities. CONCLUSIONS: The Pearson's correlation coefficient was 0.793 in 10-fold cross validation and 0.763 in independent blind test. The corresponding values for mean absolute error are 0.024 and 0.036, respectively. Comparison with a previously published method indicated ProTstab to have superior performance. We used the method to predict stabilities of all the remaining proteins in the entire human proteome and then correlated the predicted stabilities to protein chain lengths of isoforms and to localizations of proteins.


Assuntos
Células/metabolismo , Biologia Computacional/métodos , Proteoma/química , Proteoma/metabolismo , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estabilidade Proteica
17.
PLoS One ; 14(11): e0225775, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31770404

RESUMO

The Sonic Hedgehog (Shh) signalling pathway plays multiple roles during embryonic development and under pathological conditions. Although the core components of the Shh pathway are conserved, the regulation of signal transduction varies significantly among species and cell types. Protein kinases Ulk3 and Pka are involved in the Shh pathway as modulators of the activities of Gli transcription factors, which are the nuclear mediators of the signal. Here, we investigate the regulation and activities of two GLI1 isoforms, full-length GLI1 (GLI1FL) and GLI1ΔN. The latter protein lacks the first 128 amino acids including the conserved phosphorylation cluster and the binding motif for SUFU, the key regulator of GLI activity. Both GLI1 isoforms are co-expressed in all human cell lines analysed and possess similar DNA binding activity. ULK3 potentiates the transcriptional activity of both GLI1 proteins, whereas PKA inhibits the activity of GLI1ΔN, but not GLI1FL. In addition to its well-established role as a transcriptional activator, GLI1FL acts as a repressor by inhibiting transcription from the early promoters of human papillomavirus type 18 (HPV18). Additionally, compared to GLI1ΔN, GLI1FL is a more potent suppressor of replication of several HPV types. Altogether, our data show that the N-terminal part of GLI1FL is crucial for the realization of its full potential as a transcriptional regulator.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Papillomaviridae/fisiologia , Proteínas Repressoras/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , DNA/metabolismo , Humanos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/química , Alinhamento de Sequência , Ativação Transcricional , Replicação Viral , Proteína GLI1 em Dedos de Zinco/química , Proteína GLI1 em Dedos de Zinco/genética
18.
Int J Mol Sci ; 20(23)2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31779209

RESUMO

The human leucocyte antigen (HLA)-G, which consists of seven splice variants, is a tolerogenic immune checkpoint molecule. It plays an important role in the protection of the fetus from the maternal immune response by binding to inhibitory receptors, including leukocyte Ig-like receptors (LILRs). Recent studies have also revealed that HLA-G is involved in the progression of cancer cells and the protection from autoimmune diseases. In contrast to its well characterized isoform, HLA-G1, the binding activities of other major HLA-G isoforms, such as HLA-G2, toward available anti-HLA-G antibodies are only partially understood. Here, we investigate the binding specificities of anti-HLA-G antibodies by using surface plasmon resonance. MEM-G9 and G233 showed strong affinities to HLA-G1, with a nM range for their dissociation constants, but did not show affinities to HLA-G2. The disulfide-linker HLA-G1 dimer further exhibited significant avidity effects. On the other hand, 4H84 and MEM-G1, which can be used for the Western blotting of HLA-G isoforms, can bind to native HLA-G2, while MEM-G9 and G233 cannot. These results reveal that HLA-G2 has a partially intrinsically disordered structure. Furthermore, MEM-G1, but not 4H84, competes with the LILRB2 binding of HLA-G2. These results provide novel insight into the functional characterization of HLA-G isoforms and their detection systems.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos HLA-G/genética , Antígenos HLA-G/imunologia , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Sítios de Ligação , Dissulfetos/química , Antígenos HLA-G/química , Humanos , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores Imunológicos/metabolismo , Ressonância de Plasmônio de Superfície
19.
J Phys Chem Lett ; 10(23): 7333-7339, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31714784

RESUMO

Due to the poor aqueous solubility of retinoids, evolution has tuned their binding to cellular proteins to address specialized physiological roles by modulating uptake, storage, and delivery to specific targets. With the aim to disentangle the structure-function relationships in these proteins and disclose clues for engineering selective carriers, the binding mechanism of the two most abundant retinol-binding isoforms was explored by using enhanced sampling molecular dynamics simulations and surface plasmon resonance. The distinctive dynamics of the entry portal site in the holo species was crucial to modulate retinol dissociation. Remarkably, this process is controlled to a large extent by the replacement of Ile by Leu in the two isoforms, thus suggesting that fine control of ligand release can be achieved through a rigorous selection of conservative mutations in accessory sites.


Assuntos
Proteínas Celulares de Ligação ao Retinol/metabolismo , Vitamina A/metabolismo , Sítios de Ligação , Humanos , Isomerismo , Cinética , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Celulares de Ligação ao Retinol/química , Termodinâmica , Vitamina A/química
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