scholarly journals The enzyme activity of mitochondrial trifunctional protein is not altered by lysine acetylation or lysine succinylation

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0256619
Author(s):  
Yuxun Zhang ◽  
Eric Goetzman
Keyword(s):  
Enzyme Activity ◽  
Bound State ◽  
Loss Of Function ◽  
Substrate Channeling ◽  
Post Translational Modifications ◽  
Lysine Residues ◽  
Membrane Bound ◽  
Mitochondrial Trifunctional Protein ◽  
Long Chain

Mitochondrial trifunctional protein (TFP) is a membrane-associated heterotetramer that catalyzes three of the four reactions needed to chain-shorten long-chain fatty acids inside the mitochondria. TFP is known to be heavily modified by acetyllysine and succinyllysine post-translational modifications (PTMs), many of which are targeted for reversal by the mitochondrial sirtuin deacylases SIRT3 and SIRT5. However, the functional significance of these PTMs is not clear, with some reports showing TFP gain-of-function and some showing loss-of-function upon increased acylation. Here, we mapped the known SIRT3/SIRT5-targeted lysine residues onto the recently solved TFP crystal structure which revealed that many of the target sites are involved in substrate channeling within the TFPα subunit. To test the effects of acylation on substate channeling through TFPα, we enzymatically synthesized the physiological long-chain substrate (2E)-hexadecenoyl-CoA. Assaying TFP in SIRT3 and SIRT5 knockout mouse liver and heart mitochondria with (2E)-hexadecenoyl-CoA revealed no change in enzyme activity. Finally, we investigated the effects of lysine acylation on TFP membrane binding in vitro. Acylation did not alter recombinant TFP binding to cardiolipin-containing liposomes. However, the presence of liposomes strongly abrogated the acylation reaction between succinyl-CoA and TFP lysine residues. Thus, TFP in the membrane-bound state may be protected against lysine acylation.

scholarly journals TSC1 loss-of-function increases risk for tauopathy by inducing tau acetylation and preventing autophagy-mediated tau clearance

2020 ◽  
Author(s):  
Carolina Alquezar ◽  
Kathleen M Schoch ◽  
Ethan G Geier ◽  
Eliana Marisa Ramos ◽  
Aurora Scrivo ◽  
...  
Keyword(s):  
Genetic Model ◽  
Loss Of Function ◽  
Therapeutic Modalities ◽  
Risk Variants ◽  
Lysine Residues ◽  
Monoallelic Mutation ◽  
Mutant Forms ◽  
Chaperone Mediated Autophagy ◽  
Tau Acetylation

AbstractAge-associated neurodegenerative disorders demonstrating tau-laden intracellular inclusions, including Alzheimer’s disease (AD), frontotemporal lobar degeneration (FTLD) and progressive supranuclear palsy (PSP), are collectively known as tauopathies. The vast majority of human tauopathies accumulate non-mutant tau rather than mutant forms of the protein, yet cell and animal models for non-mutant tauopathies are lacking. We previously linked a monoallelic mutation in the TSC1 gene to tau accumulation and FTLD. Now, we have identified new variants in TSC1 that predisposed to other tauopathies such as AD and PSP. These new TSC1 risk variants significantly decreased the half-life of TSC1/hamartin in vitro. Cellular and murine models of TSC1 haploinsufficiency (TSC1+/-) accumulated tau protein that exhibited aberrant acetylation on six lysine residues. Tau acetylation hindered its lysosomal degradation via chaperone-mediated autophagy leading to neuronal tau accumulation. Enhanced tau acetylation in TSC1+/- models was achieved through both an increase in p300 acetyltransferase activity and a decrease in SIRT1 deacetylase levels. Pharmacological modulation of either enzyme restored tau levels. Together, these studies substantiate TSC1 as a novel tauopathy risk gene and advance TSC1 haploinsufficiency as a new genetic model for tauopathy. In addition, these results promote acetylated tau as a rational target for diagnostic and therapeutic modalities in multiple tauopathies.

scholarly journals Finding the gas pedal on a slow sirtuin

2020 ◽  
Vol 295 (5) ◽  
pp. 1400-1401
Author(s):  
Alexander L. Nielsen ◽  
Christian A. Olsen
Keyword(s):  
Fatty Acids ◽  
Histone Deacetylase ◽  
Fatty Acyl ◽  
Allosteric Modulators ◽  
Kinetic Investigation ◽  
Class Iii ◽  
Histone Lysine ◽  
Lysine Residues ◽  
Long Chain

The class III histone deacetylase sirtuin 6 (SIRT6) modulates numerous functions in the cell by deacetylating histone lysine residues. Interestingly, SIRT6's efficiency in in vitro experiments is far greater against substrates carrying long-chain fatty acyl modifications such as myristoylated lysine compared with acetylated counterparts, but the deacetylase activity can be stimulated by fatty acids and small-molecule allosteric modulators. A new study helps to explain this puzzling activation using a novel activator, thorough kinetic investigation, and mutagenesis studies. These data help elucidate the molecular requirements for activation of SIRT6 and provide a foundation for development of activators for therapeutic purposes.

scholarly journals Structure-Based Design of Nucleoside-Derived Analogues as Sulfotransferase Inhibitors

2019 ◽  
Author(s):  
Neil Kershaw ◽  
Dominic Byrne ◽  
Hollie Parsons ◽  
Neil G Berry ◽  
David Fernig ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Methyl Esters ◽  
Cell Signalling ◽  
In Vitro Assays ◽  
Protein Protein Interactions ◽  
Tyrosine Sulfation ◽  
Post Translational Modifications ◽  
Relative Specificity ◽  
Membrane Bound

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer’s disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

scholarly journals Crystal structure of human mitochondrial trifunctional protein, a fatty acid β-oxidation metabolon

2019 ◽  
Vol 116 (13) ◽  
pp. 6069-6074 ◽  
Author(s):  
Chuanwu Xia ◽  
Zhuji Fu ◽  
Kevin P. Battaile ◽  
Jung-Ja P. Kim
Keyword(s):  
Crystal Structure ◽  
Active Sites ◽  
Acyl Chain ◽  
Protein A ◽  
Membrane Integrity ◽  
Fatty Acyl ◽  
Substrate Channeling ◽  
Mitochondrial Inner Membrane ◽  
Membrane Bound ◽  
Mitochondrial Trifunctional Protein

Membrane-bound mitochondrial trifunctional protein (TFP) catalyzes β-oxidation of long chain fatty acyl-CoAs, employing 2-enoyl-CoA hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3-ketothiolase (KT) activities consecutively. Inherited deficiency of TFP is a recessive genetic disease, manifesting in hypoketotic hypoglycemia, cardiomyopathy, and sudden death. We have determined the crystal structure of human TFP at 3.6-Å resolution. The biological unit of the protein is α2β2. The overall structure of the heterotetramer is the same as that observed by cryo-EM methods. The two β-subunits make a tightly bound homodimer at the center, and two α-subunits are bound to each side of the β2dimer, creating an arc, which binds on its concave side to the mitochondrial innermembrane. The catalytic residues in all three active sites are arranged similarly to those of the corresponding, soluble monofunctional enzymes. A structure-based, substrate channeling pathway from the ECH active site to the HAD and KT sites is proposed. The passage from the ECH site to the HAD site is similar to those found in the two bacterial TFPs. However, the passage from the HAD site to the KT site is unique in that the acyl-CoA intermediate can be transferred between the two sites by passing along the mitochondrial inner membrane using the hydrophobic nature of the acyl chain. The 3′-AMP-PPi moiety is guided by the positively charged residues located along the “ceiling” of the channel, suggesting that membrane integrity is an essential part of the channel and is required for the activity of the enzyme.

scholarly journals Hat1 acetylates histone H4 and modulates the transcriptional program in Drosophila embryogenesis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Júlia Varga ◽  
Szabina Korbai ◽  
Alexandra Neller ◽  
Nóra Zsindely ◽  
László Bodai
Keyword(s):  
Rna Seq ◽  
Histone H4 ◽  
Loss Of Function ◽  
Post Translational Modifications ◽  
Cell Functions ◽  
Drosophila Embryogenesis ◽  
Developmental Program ◽  
Lysine Residues ◽  
Transcriptional Changes

AbstractPost-translational modifications of histone proteins play a pivotal role in DNA packaging and regulation of genome functions. Histone acetyltransferase 1 (Hat1) proteins are conserved enzymes that modify histones by acetylating lysine residues. Hat1 is implicated in chromatin assembly and DNA repair but its role in cell functions is not clearly elucidated. We report the generation and characterization of a Hat1 loss-of-function mutant in Drosophila. Hat1 mutants are viable and fertile with a mild sub-lethal phenotype showing that Hat1 is not essential in fruit flies. Lack of Hat1 results in the near complete loss of histone H4 lysine (K) 5 and K12 acetylation in embryos, indicating that Hat1 is the main acetyltransferase specific for these marks in this developmental stage. We found that Hat1 function and the presence of these acetyl marks are not required for the nuclear transport of histone H4 as histone variant His4r retained its nuclear localization both in Hat1 mutants and in His4r-K5R-K12R double point mutants. RNA-seq analysis of embryos indicate that in Hat1 mutants over 2000 genes are dysregulated and the observed transcriptional changes imply a delay in the developmental program of gene expression.

scholarly journals Structure-Based Design of Nucleoside-Derived Analogues as Sulfotransferase Inhibitors

2019 ◽  
Author(s):  
Neil Kershaw ◽  
Dominic Byrne ◽  
Hollie Parsons ◽  
Neil G Berry ◽  
David Fernig ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Methyl Esters ◽  
Cell Signalling ◽  
In Vitro Assays ◽  
Protein Protein Interactions ◽  
Tyrosine Sulfation ◽  
Post Translational Modifications ◽  
Relative Specificity ◽  
Membrane Bound

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer’s disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

scholarly journals Sit4p/PP6 regulates ER-to-Golgi traffic by controlling the dephosphorylation of COPII coat subunits

2013 ◽  
Vol 24 (17) ◽  
pp. 2727-2738 ◽  
Author(s):  
Deepali Bhandari ◽  
Jinzhong Zhang ◽  
Shekar Menon ◽  
Christopher Lord ◽  
Shuliang Chen ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Loss Of Function ◽  
Serine Threonine Kinase ◽  
Vesicle Budding ◽  
Threonine Kinase ◽  
Binding Partners ◽  
Copii Vesicle ◽  
Membrane Bound

Traffic from the endoplasmic reticulum (ER) to the Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p-Sec24p complex to ER membranes. The Sec23p-Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p-Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screen all known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identifies the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.

scholarly journals Action of long-chain fatty acids in vitro on Ca2+-stimulatable, Mg2+-dependent ATPase activity in human red cell membranes

1987 ◽  
Vol 248 (2) ◽  
pp. 511-516 ◽  
Author(s):  
F B Davis ◽  
P J Davis ◽  
S D Blas ◽  
M Schoenl
Keyword(s):  
Fatty Acids ◽  
Enzyme Activity ◽  
Oleic Acid ◽  
Thyroid Hormone ◽  
Atpase Activity ◽  
Red Cell ◽  
Long Chain Fatty Acids ◽  
Long Chain ◽  
Chain Fatty Acids

Human red cell membrane Ca2+-stimulatable, Mg2+-dependent adenosine triphosphatase (Ca2+-ATPase) activity and its response to thyroid hormone have been studied following exposure of membranes in vitro to specific long-chain fatty acids. Basal enzyme activity (no added thyroid hormone) was significantly decreased by additions of 10(-9)-10(-4) M-stearic (18:0) and oleic (18:1 cis-9) acids. Methyl oleate and elaidic (18:1 trans-9), palmitic (16:0) and lauric (12:0) acids at 10(-6) and 10(-4) M were not inhibitory, nor were arachidonic (20:4) and linolenic (18:3) acids. Myristic acid (14:0) was inhibitory only at 10(-4) M. Thus, chain length of 18 carbon atoms and anionic charge were the principal determinants of inhibitory activity. Introduction of a cis-9 double bond (oleic acid) did not alter the inhibitory activity of the 18-carbon moiety (stearic acid), but the trans-9 elaidic acid did not cause enzyme inhibition. While the predominant effect of fatty acids on erythrocyte Ca2+-ATPase in situ is inhibition of basal activity, elaidic, linoleic (18:2) and palmitoleic (16:1) acids at 10(-6) and 10(-4) M stimulated the enzyme. Methyl elaidate was not stimulatory. These structure-activity relationships differ from those described for fatty acids and purified red cell Ca2+-ATPase reconstituted in liposomes. Thyroid hormone stimulation of Ca2+-ATPase was significantly decreased by stearic and oleic acids (10(-9)-10(-4) M), but also by elaidic, linoleic, palmitoleic and myristic acids. Arachidonic, palmitic and lauric acids were ineffective, as were the methyl esters of oleic and elaidic acids. Thus, inhibition of the iodothyronine effect on Ca2+-ATPase by fatty acids has similar, but not identical, structure-activity relationships to those for basal enzyme activity. To examine mechanisms for these fatty acid effects, we studied the action of oleic and stearic acids on responsiveness of the enzyme to purified calmodulin, the Ca2+-binding activator protein for Ca2+-ATPase. Oleic and stearic acids (10(-9)-10(-4) M) progressively inhibited, but did not abolish, enzyme stimulation by calmodulin (10(-9) M). Double-reciprocal analysis of the effect of oleic acid on calmodulin stimulation indicated noncompetitive inhibition. Addition of calmodulin to membranes in the presence of equimolar oleic acid restored basal enzyme activity. Oleic acid also reduced 125I-calmodulin binding to membranes, but had no effect on the binding of [125I]T4 by ghosts. The mechanism of the decrease by long chain fatty acids of Ca2+-ATPase activity in situ in human red cell ghosts thus is calmodulin-dependent and involves reduction in membrane binding of calmodulin.

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