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Objectives: Antimicrobial resistance is a growing concern and claims over 1 million lives per year. The discovery of new antimicrobial drugs is expensive and often generates low profitability, with very low success rates. One way to combat this is by the improvement of known antimicrobials, such as antimicrobial peptides (AMPs). The aim of this study was to improve the antimicrobial activities of two known AMPs, UyCT3 and indolicidin, with the use of peptide libraries and growth curves. Methods: Peptide permutation libraries were synthesized for two AMPs, indolicidin and UyCT3, which included 520 peptides. These peptides were subsequently tested against MG1655-K12, to which subsequent peptide design was performed, then tested against three clinically Gram-negative relevant drug-resistant isolates. Best-performing candidates were subjected to a haemolysis assay for toxicity validation. Results: Single amino acid permutations of UyCT3 and indolicidin were sufficient to inhibit growth of MG1655-K12, and subsequent generations of peptide design were able to inhibit growth of clinical isolates at concentrations as low as 5 µM. Our best-performing AMP, UyCT3I5A, W6Y, K10I, F13I, was not seen to be toxic towards sheep RBCs. Conclusions: The efficacy of the AMPs improved with the use of our peptide library technology, whereby an AMP was found that inhibited bacterial growth of clinical Gram-negative isolates 4-fold better than its WT counterpart.
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One of the main hallmarks of Parkinson's disease (PD) is abnormal alpha-synuclein (α-syn) aggregation which forms the main component of intracellular Lewy body inclusions. This short report used preformed α-syn fibrils, as well as an A53T mutant α-syn adenovirus to mimic conditions of pathological protein aggregation in dopaminergic human derived SH-SY5Y neural cells. Since there is evidence that the mTOR pathway and glutamatergic signaling each influence protein aggregation, we also assessed the impact of the mTOR inhibitor, rapamycin and the mGluR5 allosteric modulator, CTEP. We found that both rapamycin and CTEP induced a significant reduction of α-syn fibrils in SH-SY5Y cells and this effect was associated with a reduction in mTOR signaling and enhancement in autophagic pathway factors. These data support the possibility that CTEP (or rapamycin) might be a useful pharmacological approach to target abnormal α-syn accumulation by promoting intracellular degradation or enhanced clearance.
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Doença de Parkinson , Receptor de Glutamato Metabotrópico 5 , Serina-Treonina Quinases TOR , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Sirolimo/farmacologia , Receptor de Glutamato Metabotrópico 5/metabolismoRESUMO
Proteins play a critical role as key regulators in various biological systems, influencing crucial processes such as gene expression, cell cycle progression, and cellular proliferation. However, the functions of proteins can be further modified through post-translational modifications (PTMs), which expand their roles and contribute to disease progression when dysregulated. In this review, we delve into the methodologies employed for the characterization of PTMs, shedding light on the techniques and tools utilized to help unravel their complexity. Furthermore, we explore the prevalence of crosstalk and competition that occurs between different types of PTMs, specifically focusing on both histone and non-histone proteins. The intricate interplay between different modifications adds an additional layer of regulation to protein function and cellular processes. To gain insights into the competition for lysine residues among various modifications, computational systems such as MethylSight have been developed, allowing for a comprehensive analysis of the modification landscape. Additionally, we provide an overview of the exciting developments in the field of inhibitors or drugs targeting PTMs, highlighting their potential in combatting prevalent diseases. The discovery and development of drugs that modulate PTMs present promising avenues for therapeutic interventions, offering new strategies to address complex diseases. As research progresses in this rapidly evolving field, we anticipate remarkable advancements in our understanding of PTMs and their roles in health and disease, ultimately paving the way for innovative treatment approaches.
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Lisina , Processamento de Proteína Pós-Traducional , Lisina/metabolismo , Acetilação , Histonas/metabolismoRESUMO
The mechanisms mediating the restricted growth in intrauterine growth restriction (IUGR) remain to be fully established. Mechanistic target of rapamycin (mTOR) signaling functions as a placental nutrient sensor, indirectly influencing fetal growth by regulating placental function. Increased secretion and the phosphorylation of fetal liver IGFBP-1 are known to markedly decrease the bioavailability of IGF-1, a major fetal growth factor. We hypothesized that an inhibition of trophoblast mTOR increases liver IGFBP-1 secretion and phosphorylation. We collected conditioned media (CM) from cultured primary human trophoblast (PHT) cells with a silenced RAPTOR (specific inhibition of mTOR Complex 1), RICTOR (inhibition of mTOR Complex 2), or DEPTOR (activates both mTOR Complexes). Subsequently, HepG2 cells, a well-established model for human fetal hepatocytes, were cultured in CM from PHT cells, and IGFBP-1 secretion and phosphorylation were determined. CM from PHT cells with either mTORC1 or mTORC2 inhibition caused the marked hyperphosphorylation of IGFBP-1 in HepG2 cells as determined by 2D-immunoblotting while Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) identified increased dually phosphorylated Ser169 + Ser174. Furthermore, using the same samples, PRM-MS identified multiple CK2 peptides coimmunoprecipitated with IGFBP-1 and greater CK2 autophosphorylation, indicating the activation of CK2, a key enzyme mediating IGFBP-1 phosphorylation. Increased IGFBP-1 phosphorylation inhibited IGF-1 function, as determined by the reduced IGF-1R autophosphorylation. Conversely, CM from PHT cells with mTOR activation decreased IGFBP-1 phosphorylation. CM from non-trophoblast cells with mTORC1 or mTORC2 inhibition had no effect on HepG2 IGFBP-1 phosphorylation. Placental mTOR signaling may regulate fetal growth by the remote control of fetal liver IGFBP-1 phosphorylation.
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Fator de Crescimento Insulin-Like I , Placenta , Feminino , Humanos , Gravidez , Disponibilidade Biológica , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação , Placenta/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
RNA is a vital component of the cell and is involved in a diverse range of cellular processes through a variety of functions. However, many of these functions cannot be performed without interactions with proteins. There are currently several techniques used to study protein-RNA interactions, such as electrophoretic mobility shift assay, fluorescence anisotropy, and filter binding. RNA-pulldown is a technique that uses biotinylated RNA probes to capture protein-RNA complexes of interest. First, the RNA probe and a recombinant protein are incubated to allow the in vitro interaction to occur. The fraction of bound protein is then captured by a biotin pull-down using streptavidin-agarose beads, followed by elution and immunoblotting for the recombinant protein with a His-tag-reactive probe. Overall, this method does not require specialized equipment outside what is typically found in a modern molecular laboratory and easily facilitates the maintenance of an RNase-free environment. This protocol was validated in: Nucleic Acids Res (2020), DOI: 10.1093/nar/gkaa029 Graphical abstract.
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In humans, coronaviruses are the cause of endemic illness and have been the causative agents of more severe epidemics. Most recently, SARS-CoV-2 was the causative agent of the COVID19 pandemic. Thus, there is a high interest in developing therapeutic agents targeting various stages of the coronavirus viral life cycle to disrupt viral propagation. Besides the development of small-molecule therapeutics that target viral proteases, there is also interest molecular tools to inhibit the initial event of viral attachment of the SARS-CoV-2 Spike protein to host ACE2 surface receptor. Here, we leveraged known structural information and peptide arrays to develop an in vitro peptide inhibitor of the Spike-ACE2 interaction. First, from previous co-crystal structures of the Spike-ACE2 complex, we identified an initial 24-residue long region (sequence: STIEEQAKTFLDKFNHEAEDLFYQ) on the ACE2 sequence that encompasses most of the known contact residues. Next, we scanned this 24-mer window along the ACE2 N-terminal helix and found that maximal binding to the SARS-CoV-2 receptor binding domain (CoV2-RBD) was increased when this window was shifted nine residues in the N-terminal direction. Further, by systematic permutation of this shifted ACE2-derived peptide we identified mutations to the wildtype sequence that confer increased binding of the CoV2-RBD. Among these peptides, we identified binding peptide 19 (referred to as BP19; sequence: SLVAVTAAQSTIEEQAKTFLDKFI) as an in vitro inhibitor of the Spike-ACE2 interaction with an IC50 of 2.08 ± 0.38 µM. Overall, BP19 adds to the arsenal of Spike-ACE2 inhibitors, and this study highlights the utility of systematic peptide arrays as a platform for the development of coronavirus protein inhibitors.
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Enzima de Conversão de Angiotensina 2 , Tratamento Farmacológico da COVID-19 , Humanos , SARS-CoV-2 , Ligação Proteica , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
The KDM5/JARID1 sub-family are 2-oxoglutarate and Fe(II)-dependent lysine-specific histone demethylases that are characterized by their Jumonji catalytic domains. The KDM5 family is known to remove tri-/di-methyl modifications from lysine-4 of histone H3 (i.e. H3-K4me2/3), a mark associated with active gene expression. As a result, studies to date have revolved around the influence of KDM5 on disease through their ability to regulate H3-K4me2/3. Recent evidence demonstrates that KDM5 may influence disease beyond H3-K4 demethylation, making it critical to further investigate KDM5-mediated demethylation of non-histone proteins. To help identify potential non-histone substrates for the KDM5 family, we developed a library of 180 permutated peptide substrates, with sequences that are systematically altered from the wild-type H3-K4me3 substrate. From this library, we characterized recombinant KDM5A/B/C/D substrate preference and developed recognition motifs for each KDM5 demethylase. The recognition motifs developed were used to predict potential substrates for KDM5A/B/C/D and profiled to generate a list of high-ranking and medium/low-ranking substrates for further in vitro validation. Through this approach, we identified 66 high-ranking substrates in which KDM5 demethylases displayed significant in vitro activity towards.
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Histonas , Lisina , Lisina/metabolismo , Histonas/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Domínio Catalítico , Peptídeos/metabolismoRESUMO
There are a vast number of molecular interactions that occur at the cellular level. Among these molecular interactions, interactions between multiple proteins are a widely studied area of research due to the importance of these interactions in cellular function and their potential in drug development. PeSA is a desktop application developed to facilitate the in vitro peptide study analysis to predict protein-protein interactions. PeSA can effortlessly generate visual outputs like motifs, bar charts, and visual matrices. Our implementation of PeSA version 2.0 includes additional tools, including the ability to further score peptide lists for consensus amongst interactions. The software is also able to design de novo peptides based on sequence motifs (sequence generator), which can be used to help design additional experiments for motif validation. Further, the efficacy of the sequence generator was validated using the lysine methyltransferase, SETD8, to identify new substrates of methylation based on motif-based predictions developed using PeSA2.0.
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Peptídeos , Software , Motivos de Aminoácidos , Peptídeos/química , Proteínas/química , Processamento de Proteína Pós-TraducionalRESUMO
Increased phosphorylation of decidual insulin-like growth factor-binding protein-1 (IGFBP-1) can contribute to intrauterine growth restriction (IUGR) by decreasing the bioavailability of insulin-like growth factor-1 (IGF-1). However, the molecular mechanisms regulating IGFBP-1 phosphorylation at the maternal-fetal interface are poorly understood. Protein kinase A (PKA) is required for normal decidualization. Consensus sequences for PKA are present in IGFBP-1. We hypothesized that the expression/interaction of PKA with decidual IGFBP-1 is increased in IUGR. Parallel reaction monitoring-mass spectrometry (PRM-MS) identified multiple PKA peptides (n=>30) co-immunoprecipitating with IGFBP-1 in decidualized primary human endometrial stromal cells (HESC). PRM-MS also detected active PKApThr197 and greater site-specific IGFBP-1 phosphorylation(pSer119), (pSer98+pSer101) (pSer169+pSer174) in response to hypoxia. Hypoxia promoted colocalization [dual immunofluorescence (IF)] of PKA with IGFBP-1 in decidualized HESC. Colocalization (IF) and interaction (proximity ligation assay) of PKA and IGFBP-1 were increased in decidua collected from placenta of human IUGR pregnancies (n=8) compared with decidua from pregnancies with normal fetal growth. Similar changes were detected in decidual PKA/IGFBP-1 using placenta from baboons subjected to maternal nutrient reduction (MNR) vs controls (n=3 each). In baboons, these effects were evident in MNR at gestational day 120 prior to IUGR onset. Increased PKA-mediated phosphorylation of decidual IGFBP-1 may contribute to decreased IGF-1 bioavailability in the maternal-fetal interface in IUGR.
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Retardo do Crescimento Fetal , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Decídua , Feminino , Humanos , Hipóxia/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , GravidezRESUMO
Engineering peptides to achieve a desired therapeutic effect through the inhibition of a specific target activity or protein interaction is a non-trivial task. Few of the existing in silico peptide design algorithms generate target-specific peptides. Instead, many methods produce peptides that achieve a desired effect through an unknown mechanism. In contrast with resource-intensive high-throughput experiments, in silico screening is a cost-effective alternative that can prune the space of candidates when engineering target-specific peptides. Using a set of FDA-approved peptides we curated specifically for this task, we assess the applicability of several sequence-based protein-protein interaction predictors as a screening tool within the context of peptide therapeutic engineering. We show that similarity-based protein-protein interaction predictors are more suitable for this purpose than the state-of-the-art deep learning methods publicly available at the time of writing. We also show that this approach is mostly useful when designing new peptides against targets for which naturally-occurring interactors are already known, and that deploying it for de novo peptide engineering tasks may require gathering additional target-specific training data. Taken together, this work offers evidence that supports the use of similarity-based protein-protein interaction predictors for peptide therapeutic engineering, especially peptide analogs.
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Algoritmos , Peptídeos , Peptídeos/metabolismo , Peptídeos/uso terapêuticoRESUMO
Gliomas are one of the most common and lethal brain tumors among adults. One process that contributes to glioma progression and recurrence is the epithelial to mesenchymal transition (EMT). EMT is regulated by a set of defined transcription factors which tightly regulate this process, among them is the basic helix-loop-helix family member, TWIST1. Here we show that TWIST1 is methylated on lysine-33 at chromatin by SETD6, a methyltransferase with expression levels correlating with poor survival in glioma patients. RNA-seq analysis in U251 glioma cells suggested that both SETD6 and TWIST1 regulate cell adhesion and migration processes. We further show that TWIST1 methylation attenuates the expression of the long-non-coding RNA, LINC-PINT, thereby promoting EMT in glioma. Mechanistically, TWIST1 methylation represses the transcription of LINC-PINT by increasing the occupancy of EZH2 and the catalysis of the repressive H3K27me3 mark at the LINC-PINT locus. Under un-methylated conditions, TWIST1 dissociates from the LINC-PINT locus, allowing the expression of LINC-PINT which leads to increased cell adhesion and decreased cell migration. Together, our findings unravel a new mechanistic dimension for selective expression of LINC-PINT mediated by TWIST1 methylation.
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Glioma , Proteínas Metiltransferases , RNA Longo não Codificante , Proteína 1 Relacionada a Twist , Humanos , Transição Epitelial-Mesenquimal , Proteínas Nucleares/genética , Proteínas Metiltransferases/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Glioma/metabolismo , Glioma/patologia , RNA Longo não Codificante/metabolismo , Linhagem Celular TumoralRESUMO
Jumonji C (JmjC) lysine demethylases (KDMs) catalyze the removal of methyl (-CH3) groups from modified lysyl residues. Several JmjC KDMs promote cancerous properties and these findings have primarily been in relation to histone demethylation. However, the biological roles of these enzymes are increasingly being shown to also be attributed to non-histone demethylation. Notably, KDM3A has become relevant to tumour progression due to recent findings of this enzyme's role in promoting cancerous phenotypes, such as enhanced glucose consumption and upregulated mechanisms of chemoresistance. To aid in uncovering the mechanism(s) by which KDM3A imparts its oncogenic function(s), this study aimed to unravel KDM3A substrate specificity to predict high-confidence substrates. Firstly, substrate specificity was assessed by monitoring activity towards a peptide permutation library of histone H3 di-methylated at lysine-9 (i.e., H3K9me2). From this, the KDM3A recognition motif was established and used to define a set of high-confidence predictions of demethylation sites from within the KDM3A interactome. Notably, this led to the identification of three in vitro substrates (MLL1, p300, and KDM6B), which are relevant to the field of cancer progression. This preliminary data may be exploited in further tissue culture experiments to decipher the avenues by which KDM3A imparts cancerous phenotypes.
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Lisina , Neoplasias , Desmetilação , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Processamento de Proteína Pós-TraducionalRESUMO
Within the realm of lysine methylation, the discovery of lysine methyltransferase (KMTs) substrates has been burgeoning because of established systematic substrate screening protocols. Here, we describe a protocol enabling the systematic identification of JmjC KDM substrate preference and in vitro substrates. Systematically designed peptide libraries containing methylated lysine residues are used to characterize enzyme-substrate preference and identify new candidate substrates in vitro. For complete details on the use and execution of this protocol, please refer to Hoekstra and Biggar (2021).
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Histona Desmetilases com o Domínio Jumonji , Lisina , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/química , Lisina/química , MetilaçãoRESUMO
A major regulatory influence over gene expression is the dynamic post translational methylation of histone proteins, with major implications from both lysine methylation and demethylation. The KDM5/JARID1 sub-family of Fe(II)/2-oxoglutarate dependent lysine-specific demethylases is, in part, responsible for the removal of tri/dimethyl modifications from lysine 4 of histone H3 (i.e., H3K4me3/2), a mark associated with active gene expression. Although the relevance of KDM5 activity to disease progression has been primarily established through its ability to regulate gene expression via histone methylation, there is evidence that these enzymes may also target non-histone proteins. To aid in the identification of new non-histone substrates, we examined KDM5A in vitro activity towards a library of 180 permutated peptide substrates derived from the H3K4me3 sequence. From this data, a recognition motif was identified and used to predict candidate KDM5A substrates from the methyllysine proteome. High-ranking candidate substrates were then validated for in vitro KDM5A activity using representative trimethylated peptides. Our approach correctly identified activity towards 90% of high-ranked substrates. Here, we have demonstrated the usefulness of our method in identifying candidate substrates that is applicable to any Fe(II)- and 2-oxoglutarate dependent demethylase.
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Proteína 2 de Ligação ao Retinoblastoma/análise , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Humanos , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Especificidade por SubstratoRESUMO
Insulin-like growth factor-1 (IGF-1) bioavailability in pregnancy is governed by IGF binding protein (IGFBP-1) and its phosphorylation, which enhances the affinity of IGFBP-1 for the growth factor. The decidua is the predominant source of maternal IGFBP-1; however, the mechanisms regulating decidual IGFBP-1 secretion/phosphorylation are poorly understood. Using decidualized primary human endometrial stromal cells (HESCs) from first-trimester placenta, we tested the hypothesis that mTORC1 signaling mechanistically links hypoxia to decidual IGFBP-1 secretion/phosphorylation. Hypoxia inhibited mechanistic target of rapamycin (mTORC1) (p-P70-S6K/Thr389, -47%, p = 0.038; p-4E-BP1/Thr70, -55%, p = 0.012) and increased IGFBP-1 (total, +35%, p = 0.005; phosphorylated, Ser101/+82%, p = 0.018; Ser119/+88%, p = 0.039; Ser 169/+157%, p = 0.019). Targeted parallel reaction monitoring-mass spectrometry (PRM-MS) additionally demonstrated markedly increased dual IGFBP-1 phosphorylation (pSer98+Ser101; pSer169+Ser174) in hypoxia. IGFBP-1 hyperphosphorylation inhibited IGF-1 receptor autophosphorylation/ Tyr1135 (-29%, p = 0.002). Furthermore, silencing of tuberous sclerosis complex 2 (TSC2) activated mTORC1 (p-P70-S6K/Thr389, +68%, p = 0.038; p-4E-BP1/Thr70, +30%, p = 0.002) and reduced total/site-specific IGFBP-1 phosphorylation. Importantly, TSC2 siRNA prevented inhibition of mTORC1 and the increase in secretion/site-specific IGFBP-1 phosphorylation in hypoxia. PRM-MS indicated concomitant changes in protein kinase autophosphorylation (CK2/Tyr182; PKC/Thr497; PKC/Ser657). Overall, mTORC1 signaling mechanistically links hypoxia to IGFBP-1 secretion/phosphorylation in primary HESC, implicating decidual mTORC1 inhibition as a novel mechanism linking uteroplacental hypoxia to fetal growth restriction.
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Decídua/patologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Adulto , Caseína Quinase II/metabolismo , Hipóxia Celular , Forma Celular , Células Cultivadas , Feminino , Inativação Gênica , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fosforilação , Gravidez , Primeiro Trimestre da Gravidez/fisiologia , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Adulto JovemRESUMO
Hypoxia increases fetal hepatic insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR-mediated IGFBP-1 hyperphosphorylation in response to hypoxia requires hypoxia-inducible factor-1α (HIF-1α) and regulated in development and DNA-damage responses-1 (REDD-1) signaling. Western blotting (n = 6) and immunohistochemistry (n = 3) using fetal liver indicated greater expression of HIF-1α, REDD-1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2 ) (n = 3) induced REDD-1, inhibited mTOR complex-1 (mTORC1) activity and increased IGFBP-1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF-1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia-mediated inhibition of mTORC1 and induction of IGFBP-1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF-1α and also REDD-1 expression, inhibited mTORC1 and increased IGFBP-1 secretion/phosphorylation. Induction of HIF-1α (DMOG) and REDD-1 by Compound 3 inhibited mTORC1, increased IGFBP-1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF-1α induction, increased REDD-1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP-1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia-responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.
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Técnicas de Cultura de Células , Modelos Animais de Doenças , Retardo do Crescimento Fetal/metabolismo , Feto/metabolismo , Hipóxia/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Animais , Eritropoetina/metabolismo , Peso Fetal , Feto/química , Células Hep G2 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Microscopia de Fluorescência , Tamanho do Órgão , Papio , Fosforilação , Proteína Quinase C-alfa/metabolismo , Receptores da Eritropoetina/metabolismo , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Fetal growth restriction (FGR) is associated with decreased nutrient availability and reduced insulin-line growth factor (IGF)-I bioavailability via increased IGF binding protein (IGFBP)-1 phosphorylation. While protein kinase C (PKC) is implicated in IGFBP-1 hyperphosphorylation in nutrient deprivation, the mechanisms remain unclear. We hypothesised that the interaction of PKCα with protein kinase CK2ß and activation of PKCα under leucine deprivation (L0) mediate fetal hepatic IGFBP-1 hyperphosphorylation. Parallel Reaction Monitoring Mass Spectrometry (PRM-MS) followed by PKCα knockdown demonstrated the PKCα isoform interacts with IGFBP-1 and CK2ß under L0. Pharmacological PKCα activation with phorbol 12-myristate 13-acetate (PMA) increased whereas inhibition with bisindolylmaleimide II (Bis II) decreased IGFBP-1 phosphorylation (Ser101/119/169, Ser98 + 101 and Ser169 + 174), respectively. Furthermore, PMA mimicked L0-induced PKCα translocation and IGFBP-1 expression. PKCα expression was increased in baboon fetal liver in FGR, providing biological relevance in vivo. In summary, we report a novel nutrient-sensitive mechanism for PKCα in mediating IGFBP-1 hyperphosphorylation in FGR.
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Retardo do Crescimento Fetal/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Leucina/deficiência , Fígado/embriologia , Proteína Quinase C-alfa/metabolismo , Animais , Caseína Quinase II/metabolismo , Retardo do Crescimento Fetal/genética , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Indóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Maleimidas/farmacologia , Espectrometria de Massas , Modelos Biológicos , Papio , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/genética , Transporte Proteico , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Following the decoding of the first human genome, researchers have vastly improved their understanding of cell biology and its regulation. As a result, it has become clear that it is not merely genetic information, but the aberrant changes in the functionality and connectivity of its encoded proteins that drive cell response to periods of stress and external cues. Therefore, proper utilization of refined methods that help to describe protein signalling or regulatory networks (i.e., functional connectivity), can help us understand how change in the signalling landscape effects the cell. However, given the vast complexity in 'how and when' proteins communicate or interact with each other, it is extremely difficult to define, characterize, and understand these interaction networks in a tangible manner. Herein lies the challenge of tackling the functional proteome; its regulation is encoded in multiple layers of interaction, chemical modification and cell compartmentalization. To address and refine simple research questions, modern reductionist strategies in protein biochemistry have successfully used peptide-based experiments; their summation helping to simplify the overall complexity of these protein interaction networks. In this way, peptides are powerful tools used in fundamental research that can be readily applied to comparative biochemical research. Understanding and defining how proteins interact is one of the key aspects towards understanding how the proteome functions. To date, reductionist peptide-based research has helped to address a wide range of proteome-related research questions, including the prediction of enzymes substrates, identification of posttranslational modifications, and the annotation of protein interaction partners. Peptide arrays have been used to identify the binding specificity of reader domains, which are able to recognise the posttranslational modifications; forming dynamic protein interactions that are dependent on modification state. Finally, representing one of the fastest growing classes of inhibitor molecules, peptides are now begin explored as "disruptors" of protein-protein interactions or enzyme activity. Collectively, this review will discuss the use of peptides, peptide arrays, peptide-oriented computational biochemistry as modern reductionist strategies in deconvoluting the functional proteome.
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Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteoma/metabolismo , Animais , Humanos , Fragmentos de Peptídeos/química , Mapas de Interação de Proteínas , Proteínas/químicaRESUMO
BACKGROUND: Understanding the disease pathogenesis of the novel coronavirus, denoted SARS-CoV-2, is critical to the development of anti-SARS-CoV-2 therapeutics. The global propagation of the viral disease, denoted COVID-19 ("coronavirus disease 2019"), has unified the scientific community in searching for possible inhibitory small molecules or polypeptides. A holistic understanding of the SARS-CoV-2 vs. human inter-species interactome promises to identify putative protein-protein interactions (PPI) that may be considered targets for the development of inhibitory therapeutics. METHODS: We leverage two state-of-the-art, sequence-based PPI predictors (PIPE4 & SPRINT) capable of generating the comprehensive SARS-CoV-2 vs. human interactome, comprising approximately 285,000 pairwise predictions. Three prediction schemas (all, proximal, RP-PPI) are leveraged to obtain our highest-confidence subset of PPIs and human proteins predicted to interact with each of the 14 SARS-CoV-2 proteins considered in this study. Notably, the use of the Reciprocal Perspective (RP) framework demonstrates improved predictive performance in multiple cross-validation experiments. RESULTS: The all schema identified 279 high-confidence putative interactions involving 225 human proteins, the proximal schema identified 129 high-confidence putative interactions involving 126 human proteins, and the RP-PPI schema identified 539 high-confidence putative interactions involving 494 human proteins. The intersection of the three sets of predictions comprise the seven highest-confidence PPIs. Notably, the Spike-ACE2 interaction was the highest ranked for both the PIPE4 and SPRINT predictors with the all and proximal schemas, corroborating existing evidence for this PPI. Several other predicted PPIs are biologically relevant within the context of the original SARS-CoV virus. Furthermore, the PIPE-Sites algorithm was used to identify the putative subsequence that might mediate each interaction and thereby inform the design of inhibitory polypeptides intended to disrupt the corresponding host-pathogen interactions. CONCLUSION: We publicly released the comprehensive sets of PPI predictions and their corresponding PIPE-Sites landscapes in the following DataVerse repository: https://www.doi.org/10.5683/SP2/JZ77XA. The information provided represents theoretical modeling only and caution should be exercised in its use. It is intended as a resource for the scientific community at large in furthering our understanding of SARS-CoV-2.
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Protein lysine methylation mediates a variety of biological processes, and their dysregulation has been established to play pivotal roles in human disease. A number of these sites constitute attractive drug targets. However, systematic identification of methylation sites is challenging and resource intensive. Here, we present a protocol combining MethylSight, a machine learning model trained to identify promising lysine methylation sites, and mass spectrometry for subsequent validation. Our approach can reduce the time and investment required to identify novel methylation sites. For complete information on the use and execution of this protocol, please refer to Biggar et al. (2020).