scholarly journals Effect of lysine acetylation on the regulation of Trypanosoma brucei glycosomal aldolase activity

2020 ◽  
Vol 477 (9) ◽  
pp. 1733-1744 ◽  
Author(s):  
Ariely Barbosa Leite ◽  
Antoniel Augusto Severo Gomes ◽  
Ana Caroline de Castro Nascimento Sousa ◽  
Marcos Roberto de Mattos Fontes ◽  
Sergio Schenkman ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Environmental Changes ◽  
In Silico Analysis ◽  
Lysine Acetylation ◽  
Lysine Residues ◽  
Acetyl Group ◽  
Aldolase Activity ◽  
Fructose 1,6 Bisphosphate

Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to Nε-amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei, the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei, expanding the factors responsible for regulating important pathways in this parasite.

scholarly journals Characterization of Lysine Acetyltransferase Activity of Recombinant Human ARD1/NAA10

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 588 ◽  
Author(s):  
Tam Thuy Lu Vo ◽  
Ji-Hyeon Park ◽  
Eun Ji Lee ◽  
Yen Thi Kim Nguyen ◽  
Byung Woo Han ◽  
...  
Keyword(s):  
Size Exclusion ◽  
Lysine Acetylation ◽  
Monomeric Form ◽  
Experimental Conditions ◽  
Lysine Residues ◽  
Lysine Acetyltransferase ◽  
Over Time

Arrest defective 1 (ARD1), also known as N(alpha)-acetyltransferase 10 (NAA10) was originally identified as an N-terminal acetyltransferase (NAT) that catalyzes the acetylation of N-termini of newly synthesized peptides. After that, mammalian ARD1/NAA10 expanded its’ role to lysine acetyltransferase (KAT) that post-translationally acetylates internal lysine residues of proteins. ARD1/NAA10 is the only enzyme with both NAT and KAT activities. However, recent studies on the role of human ARD1/NAA10 (hARD1/NAA10) in lysine acetylation are contradictory, as crystal structure and in vitro acetylation assay results revealed the lack of KAT activity. Thus, the role of hARD1/NAA10 in lysine acetylation is still debating. Here, we found a clue that possibly explains these complicated and controversial results on KAT activity of hARD1/NAA10. Recombinant hARD1/NAA10 exhibited KAT activity, which disappeared soon in vitro. Size-exclusion analysis revealed that most recombinant hARD1/NAA10 formed oligomers over time, resulting in the loss of KAT activity. While oligomeric recombinant hARD1/NAA10 lost its ability for lysine acetylation, its monomeric form clearly exhibited lysine acetylation activity in vitro. We also characterized the KAT activity of hARD1/NAA10 that was influenced by several experimental conditions, including concentration of reactants and reaction time. Taken together, our study proves that recombinant hARD1/NAA10 exhibits KAT activity in vitro but only under accurate conditions, including reactant concentrations and reaction duration.

scholarly journals The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro

2000 ◽  
Vol 267 (12) ◽  
pp. 3461-3468 ◽  
Author(s):  
Xiao-Song Gong ◽  
Jiang Qi Wen ◽  
Nicholas E. Fisher ◽  
Simon Young ◽  
Christopher J. Howe ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Cytochrome F ◽  
Lysine Residues

scholarly journals PATH-65. MOLECULAR SIGNATURE OF FAT1 RELATED MOLECULES IN GLIOMAS IN THE CONTEXT OF THE WHO 2016 CLASSIFICATION

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi158-vi158
Author(s):  
Kunzang Chosdol ◽  
Manvi Arora ◽  
Nargis Malik ◽  
Prerana Jha ◽  
Jyotsna Singh ◽  
...  
Keyword(s):  
Molecular Markers ◽  
In Silico Analysis ◽  
Molecular Signature ◽  
World Health ◽  
Low Grade ◽  
Who Grade ◽  
Inflammatory Microenvironment ◽  
Molecular Features

Abstract Glioblastoma (GBM, WHO grade-IV) being the most malignant and aggressive form of glioma remains a major clinical challenge, with an overall 5-year survival rate of only 9.8%. Till recently, glioma diagnosis and grading were solely dependent on the phenotypic and histological features. However, with the advancement in the understanding of the molecular biology of glioma several molecules have been identified. The importance of these molecular/genotypic features of the tumor became evident by the inclusion of these molecular features by World Health Organization (WHO) in 2016 in glioma sub-grouping. Our lab is focused on studying the role of FAT1 gene (human ortholog of Drosophila tumor suppressor gene, fat) in glioma biology and aggressiveness. We observed FAT1 gene to have an oncogenic role in glioma where it has been found to upregulate migration/invasion, inflammatory microenvironment of the tumors, HIF1α expression/activity in the tumor-cells under severe hypoxia and in regulating EMT/stemness properties of GBM-cells under hypoxia. Here, we have characterized the molecular relationship between FAT1 related molecules and known- molecular markers of glioma with the hope of identifying glioma subgroup with a molecular signature of clinical significance by (i) analyzing the expression correlation of FAT1 and FAT1 regulated pro-inflammatroy molecules like COX2, IL1b and IL6 with the known- molecular markers of glioma like p53, IDH1, MGMT, EGFR, TERT in low-grade (grade-II) and high-grade (grade-III/IV) gliomas (n=50) by real-time PCR, sequencing, immunohistochemistry and in-silico analysis of TCGA-GBM-data (ii) Analyzed the regulatory role of FAT1 on the above known markers by siRNA mediated knockdown of FAT1 in in-vitro cell-culture system and (iii) further analyzed the identified molecular signature for their correlation with the patients prognosis/survival in the follow up patients. We observed a novel molecular signature with significant correlation with patients’ clinical outcome. Therapeutic targetting of FAT1 may benefit patients with high FAT1 expressing tumors.

scholarly journals Inositol 1,4-bisphosphate is an allosteric activator of muscle-type 6-phosphofructo-1-kinase

1989 ◽  
Vol 259 (2) ◽  
pp. 463-470 ◽  
Author(s):  
G W Mayr
Keyword(s):  
Inositol Phosphates ◽  
Head Group ◽  
Muscle Type ◽  
Allosteric Effectors ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties ◽  
Allosteric Activator

The allosteric effects of various inositol biphosphate (InsP2) isomers and other inositol phosphates, of glycerophosphoinositol phosphates (GroPInsPx) and of phosphoinositides (PtdInsPx) on muscle-type 6-phosphofructo-1-kinase (PFK) were investigated. The binding of these substances to PFK was indirectly estimated by their ability to stabilize the tetrameric enzyme. At near-physiological concentrations of other allosteric effectors, muscle PFK was activated AMP-dependently by Ins(1,4)P2 (Ka = 43 microM), Ins(2,4)P2 (Ka = 70 microM) and GroPIns4P (Ka = 20 microM). These compounds activated PFK by a mechanism similar to that established for activating hexose bisphosphates. Indirect binding experiments indicated minimal Kd,app. values of about 5 microM for the binding of Ins(1,4)P2 in the presence of 0.1 mM-AMP at pH 7.4. This apparent affinity was comparable with that of fructose 1,6-bisphosphate and glucose 1,6-bisphosphate at identical conditions. The enzyme was also found to interact specifically with PtdIns4P (Kd,app. = 37 microM), the inositol phospholipid carrying Ins(1,4)P2 as its head group. The regulatory behaviour of muscle-type PFK in vitro and the concentrations of Ins(1,4)P2 in vivo (between 4 and greater than 50 nmol/g wet wt. of tissue) are consistent with the hypothesis that there is a functional interaction in vivo. Furthermore, a role of PtdIns4P in membrane compartmentation of PFK is suggested. Comparative experiments with liver PFK indicate that these regulatory properties may be relatively specific for the muscle isoform. Unlike muscle PFK, the liver isoform was slightly activated by sub-micromolar concentrations of Ins(1,4,5)P3.

scholarly journals Lysine Acetylation in the Proteome of Renal Tubular Epithelial Cells in Diabetic Nephropathy

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiayi Wan ◽  
Mingyang Hu ◽  
Ziming Jiang ◽  
Dongwei Liu ◽  
Shaokang Pan ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Epithelial Cells ◽  
High Resolution Mass Spectrometry ◽  
Microvascular Complications ◽  
Lysine Acetylation ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Renal Tubular

Diabetic nephropathy is considered one of the most common microvascular complications of diabetes and the pathophysiology involves multiple factors. Progressive diabetic nephropathy is believed to be related to the structure and function of the tubular epithelial cells in the kidney. However, the role of lysine acetylation in lesions of the renal tubular epithelial cells arising from hyperglycemia is poorly understood. Consequently, in this study, we cultured mouse renal tubular epithelial cells in vitro under high glucose conditions and analyzed the acetylation levels of proteins by liquid chromatography-high-resolution mass spectrometry. We identified 48 upregulated proteins and downregulated 86 proteins. In addition, we identified 113 sites with higher acetylation levels and 374 sites with lower acetylation levels. Subcellular localization analysis showed that the majority of the acetylated proteins were located in the mitochondria (43.17%), nucleus (28.57%) and cytoplasm (16.19%). Enrichment analysis indicated that these acetylated proteins are primarily associated with oxidative phosphorylation, the citrate cycle (TCA cycle), metabolic pathways and carbon metabolism. In addition, we used the MCODE plug-in and the cytoHubba plug-in in Cytoscape software to analyze the PPI network and displayed the first four most compact MOCDEs and the top 10 hub genes from the differentially expressed proteins between global and acetylated proteomes. Finally, we extracted 37 conserved motifs from 4915 acetylated peptides. Collectively, this comprehensive analysis of the proteome reveals novel insights into the role of lysine acetylation in tubular epithelial cells and may make a valuable contribution towards the identification of the pathological mechanisms of diabetic nephropathy.

scholarly journals Yoda1 Activates Piezo1 in Vitro to Simulate the Upregulation of Piezo1 in the Infected Brain: Piezo1 Participates in the Immune Activation of Microglia.

2021 ◽  
Author(s):  
Heguo Luo ◽  
Hailin Liu ◽  
Wengong Bian ◽  
Bochao Chen ◽  
Dongxia Yang ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune Activation ◽  
Environmental Changes ◽  
Microglial Cells ◽  
Bv2 Cells ◽  
Bacterial Lipopolysaccharides ◽  
Multiple Signaling ◽  
Lps Treatment

Abstract Aim: The expression of Piezo1 in reactive glial cells in the peripherally infected patient's brain was upregulated. This study aimed to determine whether Piezo1 is involved in the immune activation of microglial cells induced by bacterial lipopolysaccharides.Materials and methods: BV2 cells were used as a model of brain microglia. In vitro, Yoda1 was used to activate Piezo1 in BV2 cells, and Piezo1 was simulated for LPS-induced Piezo1 activation to evaluate the role of Piezo1 in microglial inflammatory activation.Key findings: In vitro, LPS upregulates the expression of Piezo1 in microglial cells through TLR4. In the absence of LPS, Yoda1 treatment of microglia produced similar immune function changes as LPS treatment. This indicates that Piezo1 plays a role in LPS-induced microglial immune activation. Specifically, Piezo1-mediated Ca2+ signals are involved in the immune activation of microglia. Piezo1-mediated Ca2+ regulates multiple signaling mechanisms downstream of TLR4, including the JNK1, mTOR and NF-κB signaling pathways, which are related to the immune activation of microglia.Significance: Piezo1 is involved in the immune response of microglia to LPS. Changes in Piezo1 activity may play an indispensable role in the immune response of microglia, and mechanical environmental changes may affect neuroinflammatory progression through Piezo1.

scholarly journals Cardiac miRNAs were involved in the regulation of pathophysiological mechanisms underlying HF in pediatric patients after ventricular assist device implantation

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
R Ragusa ◽  
A Di Molfetta ◽  
S Del Turco ◽  
G Basta ◽  
M Rizzo ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
Real Time ◽  
Mirna Expression ◽  
Real Time Pcr ◽  
In Silico ◽  
In Silico Analysis ◽  
Adiponectin Receptors ◽  
Silico Analysis

Abstract Background VAD use in heart failure (HF) children have undergone rapid progress in the last three decades through pump technological innovation and improvement of perioperative care. Studies in HF adults showed that VAD put native heart at rest and lead to molecular changes in cardiac muscle, including at microRNA (miRNA) level. However, little is known on changes induced by VAD implant in cardiac miRNA expression and their putative targets in HF children. Purpose The aims of this study were to evaluate: 1) modification of miRNA expression in cardiac muscle from HF children after VAD support; 2) the putative targets of selected miRNAs by in silico analysis; 2) the role of the identify miRNAs on putative targets by in vitro study. Methods Cardiac biopsies were collected from HF children at the moment of VAD implant [n=8; 20 (7.5–64.5) months, 2 males; 19 (15.75–32.25) LVEF%] and at the time of heart transplant after VAD support [n=5; 32 (5–204) months; 4 males; 13.5 (10–18) LVEF%]. Cardiac miRNA expression was evaluated by NGS. The potential miRNA targets were identified by bioinformatics analyses and their cardiac expression by real-time PCR was evaluated. HL-1 cell line was used for testing the regulatory role of selected miRNA on predicted targets by miRNA mimic transfection study. Results At NGS, 465 miRNA were found on average in each sample and the cardiac expression levels of miR19a-3p, miR-1246 and miR-199b-5p decreased in HF children after VAD support compared to pre-implant (Fig. 1A-B). In silico analysis showed that more than 5000 potential gene targets regulated by miR-19a-3p, miR-1246 and miR-199b-5p. Among them, adiponectin receptors (AdipoR1, AdipoR2, T-CAD) were identified as common targets for 3 miRNAs. Real-time PCR data showed that levels of all adiponectin receptors increased significantly whilst the expression of 3 miRNAs decreased after VAD support (Fig. 1C). Moreover, AdipoR2 and T-CAD were inversely related to miRNA levels (Fig. 1D). In vitro studies confirmed the regulatory role of miR-1246 and miR-199b-5p on AdipoR2 (Fig. 1E-F), whilst only miR-199b-5p reduced the expression of T-CAD (Fig. 1G). Finally, AdipoR1 expression levels are not modified compared to control by miRNAs mimic transfection (data not shown). Conclusion In HF children the use of VAD could modify the expression of several miRNAs potentially involved in the regulation of several pathophysiological mechanisms underlying HF. Specifically, the reductions of miR-1246, mir-19a-3p, miR-199b-5p were associated with an increase of the adiponectin receptors AdipoR2 and T-CAD mRNA, suggesting the existence of a miRNAs related fine tuning of the adiponectin system at cardiac tissue level by VAD implant, able to favour the protective effect of adiponectin in HF cardiac muscle. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): FP7-ICT-2009 Project, Grant Agreement 24863 Figure 1

scholarly journals Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

2020 ◽  
Vol 48 (15) ◽  
pp. 8704-8723
Author(s):  
Joseph T Smith Jr. ◽  
Eva Doleželová ◽  
Brianna Tylec ◽  
Jonathan E Bard ◽  
Runpu Chen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
High Throughput Sequencing ◽  
Environmental Changes ◽  
Developmental Regulation ◽  
Complex Life Cycle ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Life Cycle Stages

Abstract Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3′ to 5′ progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

Molecules with O-acetyl group protect protein glycation by acetylating lysine residues

RSC Advances ◽  
2016 ◽  
Vol 6 (70) ◽  
pp. 65572-65578 ◽  
Author(s):  
Garikapati Vannuruswamy ◽  
Mashanipalya G. Jagadeeshaprasad ◽  
K. Kashinath ◽  
Suresh K. Kesavan ◽  
Shweta Bhat ◽  
...  
Keyword(s):  
Protein Glycation ◽  
Lysine Residues ◽  
Acetyl Group

In-vitro and in-vivo chemical proteomic studies of acetyl group molecules revealed that, O-acetyl molecules competitively inhibits the protein glycation by acetylating the lysine residues.

scholarly journals "Identification of Mechanisms By Which Mesenchymal Stem/Stromal Cells Contribute to Acute Myeloid Leukemia"

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5194-5194
Author(s):  
Allolo Aldreiwish ◽  
Gauri Muradia ◽  
Remi Gagne ◽  
Marc Beal ◽  
Carole Yauk ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
In Silico Analysis ◽  
Leukemic Cells ◽  
Silico Analysis ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a blood malignancy resulting in abnormal hematopoiesis that is reported to be associated with alterations in the bone marrow microenvironment (BME). Current treatments for this heterogeneous disease, which target the leukemic cells but not the BME, are largely unsuccessful for the majority of AML subtypes. By better understanding the mechanisms by which the BME contributes to leukemogenesis, it may be possible to introduce more effective treatments for AML. Mesenchymal stem cells (MSCs) are an essential component of the BME that have been shown to support normal hematopoiesis. Therefore, MSCs may have several roles in the alteration of the BME, leukemogenesis, and AML relapse and can provide an excellent model for studying the BME in vitro. While some studies have characterized AML-derived MSCs (AML-MSCs), their exact role in the disease remains unclear. Our RNAseq analysis of AML-MSCs (n=30), and healthy donor MSCs (HD-MSCs) (n=8) identified that, among 7655 genes, 21 genes were significantly differentially expressed in AML-MSCs. Through in silico analysis of this gene set, genes of interest were identified as having the potential to directly alter the BME and affect AML pathogenesis through BMP/TGF-β pathways. Current work is focusing on investigation of the effects of selected genes with biological relevance on MSCs intrinsic and extrinsic functional properties. This study will improve our understanding of the role of MSCs in AML BME and help in the discovery of new therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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