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1.
Sci Data ; 10(1): 375, 2023 06 10.
Article in English | MEDLINE | ID: mdl-37301912

ABSTRACT

Inflammation is associated with the adaptation of macrophages and endothelial cells, and the dysregulation of these differentiation processes has been directly linked to both acute and chronic disease states. As cells in constant contact with blood, macrophages and endothelial cells are also under the direct influence of immunomodulatory dietary components such as polyunsaturated fatty acids (PUFA). RNA sequencing analyses allow us to understand the global changes in gene expression occurring during cell differentiation, including both transcriptional (transcriptome) and post-transcriptional (miRNAs) levels. We generated a comprehensive RNA sequencing dataset of parallel transcriptome and miRNA profiles of PUFA-enriched and pro-inflammatory stimulated macrophages and endothelial cells aiming to uncover the underlying molecular mechanisms. PUFA concentrations and duration of supplementation were based on dietary ranges, allowing for metabolism and plasma membrane uptake of fatty acids. The dataset may serve as a resource to study transcriptional and post-transcriptional changes associated with macrophage polarisation and endothelial dysfunction in inflammatory settings and their modulation by omega-3 and omega-6 fatty acids.


Subject(s)
Fatty Acids, Omega-3 , MicroRNAs , Animals , Humans , Mice , Endothelial Cells/metabolism , Fatty Acids, Omega-3/metabolism , Fatty Acids, Unsaturated/metabolism , Macrophages/metabolism , MicroRNAs/genetics , Transcriptome
2.
Adv Exp Med Biol ; 1072: 227-231, 2018.
Article in English | MEDLINE | ID: mdl-30178350

ABSTRACT

Sepsis is an exaggerated immune reaction to an infection, which leads to organ dysfunction especially circulatory failure. This is based on cellular processes, which are regulated by post-transcriptional gene expression modulations including microRNAs (miRNAs). In order to elucidate the role miRNAs play in septic processes, monocytes and endothelial cells were grown in an inflammatory milieu. In addition, aortas from septic mice were investigated. Expression of miRNAs was analysed by both next generation sequencing (NGS) and NanoString technology, and miRNA targets were identified by in silico analysis. Clear alterations in miRNA expression profiles were found in monocytes, endothelial cells, and aortas exposed to septic conditions compared to the respective control. In silico analysis revealed several of the differentially expressed miRNAs to be involved in cellular response to hypoxia. In endothelial cells, for instance, miR-21-5p and miR-106b-5p emerged, which are known to interact with hypoxia inducible factor 1 alpha (HIF-1α), a major player in the process of angiogenesis. In line with this, in aortas expression changes were observed for miR-144-3p, which targets HIF-1α as well. Further validated target genes of differentially expressed miRNAs encompass the vascular endothelial growth factor receptor 1 (VEGFR1) and the vascular endothelial growth factor A (VEGFA), which represent essential mediators of angiogenesis. Moreover, several miRNAs impacting on genes encoding mediators of vasomotion were identified to be altered in their expression profiles in context of an inflammatory milieu. Altogether, the data indicate that miRNAs are an interesting starting point for functional and mechanistic sepsis research.


Subject(s)
Endothelial Cells/physiology , MicroRNAs/biosynthesis , Muscle, Smooth, Vascular/physiopathology , Neovascularization, Physiologic/physiology , Sepsis/physiopathology , Animals , Cell Hypoxia/physiology , Humans , Inflammation/metabolism , Inflammation/physiopathology , Macrophages/metabolism , Mice , RAW 264.7 Cells , Sepsis/metabolism
3.
Cell Chem Biol ; 25(2): 121-123, 2018 02 15.
Article in English | MEDLINE | ID: mdl-29452607

ABSTRACT

Although considerable knowledge of the biosynthetic machinery of thiopeptide antibiotics has been accumulated, the regulation of their production remains unclear. In this issue of Cell Chemical Biology, Li et al. (2018) have now characterized a key transcription factor and suggest its feedback regulation by biosynthesis intermediates and the final product.


Subject(s)
Peptides , Thiazoles , Anti-Bacterial Agents
4.
PeerJ ; 6: e4212, 2018.
Article in English | MEDLINE | ID: mdl-29312832

ABSTRACT

BACKGROUND: Toll like receptors (TLRs) are an important and evolutionary conserved class of pattern recognition receptors associated with innate immunity. The recognition of Gram-positive cell wall constituents strongly depends on TLR2. In order to be functional, TLR2 predominantly forms a heterodimer with TLR1 or TLR6 within specialized membrane microdomains, the lipid rafts. The membrane lipid composition and the physicochemical properties of lipid rafts are subject to modification by exogenous fatty acids. Previous investigations of our group provide evidence that macrophage enrichment with polyunsaturated fatty acids (PUFA) induces a reordering of lipid rafts and non-rafts based on the incorporation of supplemented PUFA as well as their elongation and desaturation products. METHODS: In the present study we investigated potential constraining effects of membrane microdomain reorganization on the clustering of TLR2 with its co-receptors TLR1 and TLR6 within lipid rafts. To this end, RAW264.7 macrophages were supplemented with either docosahexaenoic acid (DHA) or arachidonic acid (AA) and analyzed for receptor expression and microdomain localization in context of TLR stimulation. RESULTS AND CONCLUSIONS: Our analyses showed that receptor levels and microdomain localization were unchanged by PUFA supplementation. The TLR2 pathway, in contrast to the TLR4 signaling cascade, is not affected by exogenous PUFA at the membrane level.

5.
Int J Mol Sci ; 18(2)2017 Jan 28.
Article in English | MEDLINE | ID: mdl-28134837

ABSTRACT

Alteration of miRNAs and dietary polyunsaturated fatty acids (PUFAs) underlies vascular inflammation. PUFAs are known to be incorporated into the cell membrane of monocytes/macrophages or endothelial cells, the major cellular players of vascular diseases, thereby affecting cellular signal transduction. Nevertheless, there are no investigations concerning the PUFA impact on miRNA expression by these cells. With regard to the key role miRNAs play for overall cellular functionality, this study aims to elucidate whether PUFAs affect miRNA expression profiles. To this end, the monocyte/macrophage cell line RAW264.7 and the endothelial cell line TIME were enriched with either docosahexaenoic acid (DHA; n3-PUFA) or arachidonic acid (AA; n6-PUFA) until reaching a stable incorporation into the plasma membrane and, at least in part, exposed to an inflammatory milieu. Expressed miRNAs were determined by deep sequencing, and compared to unsupplemented/unstimulated controls. Data gained clearly show that PUFAs in fact modulate miRNA expression of both cell types analyzed regardless the presence/absence of an inflammatory stimulator. Moreover, certain miRNAs already linked to vascular inflammation were found to be affected by cellular PUFA enrichment. Hence, vascular inflammation appears to be influenced by dietary fatty acids, inter alia, via PUFA-mediated modulation of the type and amount of miRNAs synthesized by cells involved in the inflammatory process.


Subject(s)
Endothelial Cells/metabolism , Fatty Acids, Unsaturated/pharmacology , Gene Expression Profiling , Macrophages/metabolism , MicroRNAs/metabolism , Monocytes/metabolism , Animals , Cluster Analysis , Computer Simulation , Cytokines/pharmacology , Endothelial Cells/drug effects , Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Mice , MicroRNAs/genetics , Monocytes/drug effects , Pilot Projects , RAW 264.7 Cells , Reproducibility of Results
6.
Sci Rep ; 6: 22643, 2016 Mar 04.
Article in English | MEDLINE | ID: mdl-26940860

ABSTRACT

Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1-6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M(-1)s(-1). These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM).


Subject(s)
Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/isolation & purification , Sirtuins/antagonists & inhibitors , Sirtuins/analysis , Humans
7.
J Med Chem ; 58(18): 7217-23, 2015 Sep 24.
Article in English | MEDLINE | ID: mdl-26308971

ABSTRACT

Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes like control of metabolic pathways, DNA repair, and stress response. Modulators of sirtuin activity are needed as tools for uncovering the biological function of these enzymes and as potential therapeutics. Systematic discovery of such modulators is hampered by the lack of efficient and simple continuous activity assays running at low sirtuin concentrations in microtiter plates. Here we describe an improved continuous sirtuin 5 assay based on the coupling of the sirtuin reaction to a proteolytic cleavage using internally fluorescence-quenched substrates. Systematic optimization of a carbamoyl phosphate synthetase 1 derived, glutarylated peptide yielded a Sirt5 substrate with k(cat)/K(M) value of 337,000 M(-1) s(-1), which represents the best sirtuin substrate described so far. These extraordinary substrate properties allowed reliable determination of Ki values for different inhibitors in the presence of only 10 nM sirtuin in microtiter plate format. Assay conditions could be transferred effectively to other lysine deacetylases, like sirtuin 2 and sirtuin 3, which now enables more efficient development of sirtuin targeting drugs.


Subject(s)
Carbamoyl-Phosphate Synthase (Ammonia)/chemistry , Histone Deacetylases/chemistry , Oligopeptides/chemistry , Sirtuins/chemistry , Enzyme Assays/methods , Histone Deacetylase Inhibitors/chemistry , Kinetics , Proteolysis , Sirtuins/antagonists & inhibitors , Substrate Specificity
8.
Nat Commun ; 6: 6263, 2015 Feb 12.
Article in English | MEDLINE | ID: mdl-25672491

ABSTRACT

Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.


Subject(s)
Sirtuin 2/antagonists & inhibitors , Sirtuin 2/chemistry , Acetamides/chemistry , Acetamides/pharmacology , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Kinetics , Ligands , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Thiazoles/chemistry , Thiazoles/pharmacology
9.
Angew Chem Int Ed Engl ; 53(40): 10728-32, 2014 Sep 26.
Article in English | MEDLINE | ID: mdl-25111069

ABSTRACT

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5.


Subject(s)
Peptides/chemistry , Peptides/pharmacology , Sirtuins/antagonists & inhibitors , Sirtuins/chemistry , Acylation , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Drug Design , Humans , Models, Molecular , Peptides/metabolism , Protein Conformation , Sirtuins/metabolism , Substrate Specificity
10.
Expert Opin Drug Discov ; 9(2): 183-99, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24382304

ABSTRACT

INTRODUCTION: Sirtuins are an evolutionarily conserved family of NAD(+)-dependent protein lysine deacylases. In mammals, 7 Sirtuin isoforms control various functions in metabolism, stress responses and aging processes. Sirtuins are considered attractive therapeutic targets for metabolic and aging-related diseases, such as metabolic syndrome and neurodegenerative disorders. Extensive development efforts on small-molecule Sirtuin inhibitors and activators have yielded few potent and selective compounds, partly due to shortcomings of available assays and a lack of mechanistic characterization of identified compounds. AREAS COVERED: The authors describe the developments in analyzing Sirtuin activity and conceptual advances in the identification, improvement and design of Sirtuin-modulating compounds. They also review the application of these methods and concepts for the development and mechanistic characterization of Sirtuin modulators. EXPERT OPINION: Novel assays and experimental approaches for studying Sirtuin activity have been instrumental for major progress in understanding functions of Sirtuins and how these enzymes can be modulated with drugs. The improved tools and mechanistic insights now enable a more efficient development of Sirtuin modulators for in vivo studies and therapeutic applications.


Subject(s)
Drug Discovery , Sirtuins/metabolism , Animals , Biological Assay , Humans , Sirtuins/chemistry
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