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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
NASA NTRS1986

Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays

Indole-3-acetyl-myo-inositol galactoside labeled with 3H in the indole and 14C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [3H]indole-3-acetyl-myo-inositol and [3H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumption concerning the equilibration of applied [3H]indole-3-acetyl-myo-inositol-[U-14C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [3H]indole-3-acetyl-myo-inositol-[14C]galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [3H]indole-3-acetyl-myo-inositol-[14C]galactose supplies appreciable amounts of 14C to the shoot and both 14C and 3H to an uncharacterized insoluble fraction of the endosperm.

NASA Discipline Number 40-30↗
DOE OSTI2012

Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study

Highlights: Black-Right-Pointing-Pointer Fibrinogen was incubated in vitro with glucose or aspirin. Black-Right-Pointing-Pointer Acetylations and glycations were found at twelve lysine sites by mass spectrometry. Black-Right-Pointing-Pointer The labeling by aspirin and glucose occurred dose-dependently. Black-Right-Pointing-Pointer No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called 'aspirin resistance'. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to 'aspirin resistance' in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the {alpha}-chain: {alpha}K191, {alpha}K208, {alpha}K224, {alpha}K429, {alpha}K457, {alpha}K539, {alpha}K562, in the {beta}-chain: {beta}K233, and in the {gamma}-chain: {gamma}K170 and {gamma}K273. Glycations were found at {beta}K133 and {gamma}K75, alternatively {gamma}K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [{sup 14}C-acetyl]salicylic acid and [{sup 14}C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 {mu}M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of fibrinogen.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

TBL10 is required for O -acetylation of pectic rhamnogalacturonan-I in Arabidopsis thaliana

O -Acetylated pectins are plentiful in the primary cell wall of plants and growing evidence suggests they have important roles in plant cell growth and interaction with the environment. Despite their importance, genes required for O -acetylation of pectins are still largely unknown. Here, we demonstrate that TRICHOME BIREFRINGENCE LIKE 10 (AT3G06080) is involved in O -acetylation of pectins in Arabidopsis ( Arabidopsis thaliana ). The activity of the TBL10 promoter was strong in tissues where pectins are highly abundant (e.g. leaves). Two homozygous knock-out mutants of Arabidopsis, tbl10-1 and tbl10-2 , were isolated and shown to exhibit reduced levels of wall-bound acetyl esters, equivalent of ~50% of the wild-type level in pectin-enriched fractions derived from leaves. Further fractionation revealed that the degree of acetylation of the pectin rhamnogalacturonan-I (RG-I) was reduced in the tbl10 mutant compared to the wild type, whereas the pectin homogalacturonan (HG) was unaffected. The degrees of acetylation in hemicelluloses (i.e. xyloglucan, xylan and mannan) were indistinguishable between the tbl10 mutants and the wild type. The mutant plants contained normal trichomes in leaves and exhibited a similar level of susceptibility to the phytopathogenic microorganisms Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea ; while they displayed enhanced tolerance to drought. These results indicate that TBL10 is required for O -acetylation of RG-I, possibly as an acetyltransferase, and suggest that O -acetylated RG-I plays a role in abiotic stress responses in Arabidopsis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth. Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. In conclusion, they also demonstrate how to increase bacterial growth in complex media.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Final report: The Systems Biology of Protein Acetylation in Fuel-Producing Microorganisms

Our goal is to determine how protein acetylation affects metabolism in engineered microorganisms. Lysine acetylation is a common post-translational modification that eukaryotes, archaea, and bacteria employ to regulate protein activity. Multiple studies have recently shown that lysine acetylation predominantly targets metabolic enzymes – in fact, most metabolic enzymes are subject to lysine acetylation. Still, we know very little about lysine acetylation, particularly in the case of bacteria. We hypothesize that bacteria employ lysine acetylation as a global mechanism to regulate metabolism in response to their energy and redox status. Our previous work suggests that lysine acetylation may be an attractive and innovative target for metabolic engineering. We propose to investigate how lysine acetylation affects fuel production in engineered microorganisms. The significance of this work is that it will address a fundamental gap in our understanding of bacterial metabolism and identify new approaches for overcoming the problems associated with the production of advanced biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73 degrees C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA beta-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula.

Hawkins, AB↗
DOE OSTI2011

Chitosan Molecular Structure as a Function of N-Acetylation

Molecular dynamics simulations have been carried out to characterize the structure and solubility of chitosan nanoparticle-like structures as a function of the deacetylation level (0, 40, 60, and 100%) and the spatial distribution of the N-acetyl groups in the particles. The polysaccharide chains of highly N-deacetylated particles where the N-acetyl groups are uniformly distributed present a high flexibility and preference for the relaxed two-fold helix and five-fold helix motifs. When these groups are confined to a given region of the particle, the chains adopt preferentially a two-fold helix with f and w values close to crystalline chitin. Nanoparticles with up to 40% acetylation are moderately soluble, forming stable aggregates when the N-acetyl groups are unevenly distributed. Systems with 60% or higher N-acetylation levels are insoluble and present similar degrees of swelling regardless the distribution of their N-acetyl groups. Overall particle solvation is highly affected by electrostatic forces resulting from the degree of acetylation. The water mobility and orientation around the polysaccharide chains affects the stability of the intramolecular O3- HO3(n) ... O5(n+ 1) hydrogen bond, which in turn controls particle aggregation.

08 HYDROGEN↗
DOE OSTI2015

Oxygen-dependent acetylation and dimerization of the corepressor CtBP2 in neural stem cells

The transcriptional corepressor CtBP2 is essential for proper development of the nervous system. The factor exerts its repression by interacting in complexes with chromatin-modifying factors such as histone deacetylases (HDAC) 1/2 and the histone demethylase LSD1/KDM1. Notably, the histone acetyl transferase p300 acetylates CtBP2 and this is an important regulatory event of the activity and subcellular localization of the protein. We recently demonstrated an essential role for CtBPs as sensors of microenvironmental oxygen levels influencing the differentiation potential of neural stem cells (NSCs), but it is not known whether oxygen levels influence the acetylation levels of CtBP factors. Here we show by using proximity ligation assay (PLA) that CtBP2 acetylation levels increased significantly in undifferentiated, proliferating NSCs under hypoxic conditions. CtBP2 interacted with the class III HDAC Sirt1 but this interaction was unaltered in hypoxic conditions, and treatment with the Sirt1 inhibitor Ex527 did not result in any significant change in total CtBP2 acetylation levels. Instead, we revealed a significant decrease in PLA signal representing CtBP2 dimerization in NSCs under hypoxic conditions, negatively correlating with the acetylation levels. Our results suggest that microenvironmental oxygen levels influence the dimerization and acetylation levels, and thereby the activity, of CtBP2 in proliferating NSCs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex

Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

GATA3 acetylation at K119 by CBP inhibits cell migration and invasion in lung adenocarcinoma

Highlights: • GATA3 interacts with and is acetylated by the acetyltransferase CBP. • The major acetylated site of GATA3 in lung adenocarcinoma cells is lysine 119. • GATA3 is deacetylated by Class I deacetylases HDAC1, HDAC2 and HDAC3. • The acetylation status of GATA3-K119 is critical to lung adenocarcinoma progression. GATA3 is a transcriptional factor involved in the development of multiple organs. Post translational modifications of GATA3 are critical to its function. Here, we report that GATA3 interacts with and is acetylated by the acetyltransferase CBP. Class I deacetylases HDAC1, HDAC2 and HDAC3 deacetylate GATA3. The major acetylated site of GATA3 in lung adenocarcinoma cells was determined at lysine 119 (AcK119). Functionally, GATA3-acetylation mimics K119Q mutant was found to inhibit lung adenocarcinoma cell migration and invasion with concomitant downregulation of EMT-controlling transcriptional factors Slug, Zeb1 and Zeb2. Taken together, we demonstrated that GATA3 acetylation at lysine 119 by CBP hinders the migration and invasion of lung adenocarcinoma cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Ancient Origin of Acetyltransferases Catalyzing O -acetylation of Plant Cell Wall Polysaccharides

Abstract Members of the domain of unknown function 231/trichome birefringence–like (TBL) family have been shown to be O-acetyltransferases catalyzing the acetylation of plant cell wall polysaccharides, including pectins, mannan, xyloglucan and xylan. However, little is known about the origin and evolution of plant cell wall polysaccharide acetyltransferases. Here, we investigated the biochemical functions of TBL homologs from Klebsormidium nitens, a representative of an early divergent class of charophyte green algae that are considered to be the closest living relatives of land plants, and Marchantia polymorpha, a liverwort that is an extant representative of an ancient lineage of land plants. The genomes of K. nitens and Marchantia polymorpha harbor two and six TBL homologs, respectively. Biochemical characterization of their recombinant proteins expressed in human embryonic kidney 293 cells demonstrated that the two K. nitens TBLs exhibited acetyltransferase activities acetylating the pectin homogalacturonan (HG) and hence were named KnPOAT1 and KnPOAT2. Among the six M. polymorpha TBLs, five (MpPOAT1 to 5) possessed acetyltransferase activities toward pectins and the remaining one (MpMOAT1) catalyzed 2-O- and 3-O-acetylation of mannan. While MpPOAT1,2 specifically acetylated HG, MpPOAT3,4,5 could acetylate both HG and rhamnogalacturonan-I. Consistent with the acetyltransferase activities of these TBLs, pectins isolated from K. nitens and both pectins and mannan from M. polymorpha were shown to be acetylated. These findings indicate that the TBL genes were recruited as cell wall polysaccharide O-acetyltransferases as early as in charophyte green algae with activities toward pectins and they underwent expansion and functional diversification to acetylate various cell wall polysaccharides during evolution of land plants.

Cell Biology↗
DOE OSTI2016

Concurrent acetylation of FoxO1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated HepG2 cells

Post-translational modifications i.e. phosphorylation and acetylation are pivotal requirements for proper functioning of eukaryotic proteins. The current study aimed to decode the impact of acetylation/deacetylation of non-histone targets i.e. FoxO1/3a and p53 of sirtuins (NAD{sup +} dependent enzymes with lysine deacetylase activity) in berberine treated human hepatoma cells. Berberine (100 μM) inhibited sirtuins significantly (P < 0.05) at transcriptional level as well as at translational level. Combination of nicotinamide (sirtuin inhibitor) with berberine potentiated sirtuins inhibition and increased the expression of FoxO1/3a and phosphorylation of p53 tumor suppressor protein. As sirtuins deacetylate non-histone targets including FoxO1/3a and p53, berberine increased the acetylation load of FoxO1/3a and p53 proteins. Acetylated FoxO and p53 proteins transcriptionally activate BH3-only proteins Bim and PUMA (3.89 and 3.87 fold respectively, P<0.001), which are known as direct activator of pro-apoptotic Bcl-2 family protein Bax that culminated into mitochondria mediated activation of apoptotic cascade. Bim/PUMA knock-down showed no changes in sirtuins' expression while cytotoxicity induced by berberine and nicotinamide was curtailed up to 28.3% (P < 0.001) and it restored pro/anti apoptotic protein ratio in HepG2 cells. Sirtuins inhibition was accompanied by decline in NAD{sup +}/NADH ratio, ATP generation, enhanced ROS production and decreased mitochondrial membrane potential. TEM analysis confirmed mitochondrial deterioration and cell damage. SRT-1720 (1–10 μM), a SIRT-1 activator, when pre-treated with berberine (25 μM), reversed sirtuins expression comparable to control and significantly restored the cell viability (P < 0.05). Thus, our findings suggest that berberine mediated sirtuins inhibition resulting into FoxO1/3a and p53 acetylation followed by BH3-only protein Bim/PUMA activation may in part be responsible for mitochondria-mediated apoptosis. - Highlights: • Berberine inhibits sirtuins at transcriptional as well as at translational level. • Sirtuins inhibition leads to acetylation of non-histone proteins, FoxO1/3a and p53. • Acetylated FoxO and p53 transcriptionally upregulate BH3-only proteins Bim and PUMA respectively. • BH3-only proteins trigger mitochondrial dysfunction culminating into apoptosis. • SRT-1720 (SIRT-1 activator) partially restores Bax/Bcl-2 ratio and reduces berberine induced cytotoxicity in HepG2 cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30{sup II} accessory protein and the induction of oncogenic cellular transformation by p30{sup II}/c-MYC

The human T-cell leukemia retrovirus type-1 (HTLV-1) p30{sup II} protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30{sup II} interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30{sup II} and c-MYC remain to be completely understood. Herein we demonstrate that p30{sup II} induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30{sup II} in c-myc{sup −/−} HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30{sup II} is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30{sup II} inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30{sup II}/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. - Highlights: • Acetylation of c-MYC is required for oncogenic transformation by HTLV-1 p30{sup II}/c-MYC. • Acetylation-defective c-MYC mutants are impaired for foci-formation by p30{sup II}/c-MYC. • The HTLV-1 p30{sup II} protein induces lysine-acetylation of c-MYC. • p30{sup II} is present in c-MYC nucleoprotein complexes in HTLV-1-transformed T-cells. • HTLV-1 p30{sup II} inhibits apoptosis in c-MYC-expressing proliferating cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids

Here, the ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and acyltransferases were retransformed with the Euonymus alatus gene for diacylglycerol acetyltransferase (EaDAcT) that synthesises acetyl-TAGs. Concomitant RNAi suppression of acyl-CoA:diacylglycerol acyltransferase increased the levels of acetyl-TAG, with up to 77 mole percent in the best lines. However, the total oil content was reduced. Analysis of the composition of the acetyl-TAG molecular species using electrospray ionisation mass spectrometry demonstrated the successful synthesis of acetyl-TAG containing MCFAs. Field growth of high-yielding plants generated enough oil for quantification of viscosity. As part of an ongoing design–test–learn cycle, these results, which include not only the synthesis of ‘designer’ lipids but also their functional analysis, will lead to the future production of such molecules tailored for specific applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Genome-scale analysis of regulatory protein acetylation enzymes from photosynthetic eukaryotes

Background: Reversible protein acetylation occurring on Lys-Ne has emerged as a key regulatory post-translational modification in eukaryotes. It is mediated by two groups of enzymes: lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) that catalyze the addition and removal of acetyl groups from target proteins. Estimates indicate that protein acetylation is second to protein phosphorylation in abundance, with thousands of acetylated sites now identified in different subcellular compartments. Considering the important regulatory role of protein phosphorylation, elucidating the diversity of KATs and KDACs across photosynthetic eukaryotes is essential in furthering our understanding of the impact of reversible protein acetylation on plant cell processes. Results: We report a genome-scale analysis of lysine acetyltransferase (KAT)- and lysine deacetylase (KDAC)-families from 53 photosynthetic eukaryotes. KAT and KDAC orthologs were identified in sequenced genomes ranging from glaucophytes and algae to land plants and then analyzed for evolutionary relationships. Based on consensus molecular phylogenetic and subcellular localization data we found new sub-classes of enzymes in established KAT- and KDAC-families. Specifically, we identified a non-photosynthetic origin of the HD-tuin family KDACs, a new monocot-specific Class I HDA-family sub-class, and a phylogenetically distinct Class II algal/heterokont sub-class which maintains an ankyrin domain not conserved in land plant Class II KDACs. Protein structure analysis showed that HDA- and SRT-KDACs exist as bare catalytic subunits with highly conserved median protein length, while all KATs maintained auxiliary domains, with CBP- and TAFII250-KATs displaying protein domain gain and loss over the course of photosynthetic eukaryote evolution in addition to variable protein length. Lastly, promoter element enrichment analyses across species revealed conserved cis-regulatory sequences that support KAT and KDAC involvement in the regulation of plant development, cold/drought stress response, as well as cellular processes such as the circadian clock. Conclusions: Our results reveal new evolutionary, structural, and biological insights into the KAT- and KDAC-families of photosynthetic eukaryotes, including evolutionary parallels to protein kinases and protein phosphatases. Further, we provide a comprehensive annotation framework through our extensive phylogenetic analysis, from which future research investigating aspects of protein acetylation in plants can use to position new findings in a broader context.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The Effect of Acetylation on Iron Uptake and Diffusion in Water Saturated Wood Cell Walls and Implications for Decay

Acetylation is widely used as a wood modification process that protects wood from fungal decay. The mechanisms by which acetylation protects wood are not fully understood. With these experiments, we expand upon the literature and test whether previously observed differences in iron uptake by wood were a result of decreased iron binding capacity or slower diffusion. We measured the concentration of iron in 2 mm thick wood sections at 0, 10, and 20% acetylation as a function of time after exposure to iron solutions. The iron was introduced either strongly chelated with oxalate or weakly chelated with acetate. The concentrations of iron and oxalate in solution were chosen to be similar to those found during brown rot decay, while the concentration of iron and acetate matched previous work. The iron content of oxalate-exposed wood increased only slightly and was complete within an hour, suggesting little absorption and fast diffusion, or only slight surface adsorption. The increase in iron concentration from acetate solutions with time was consistent with Fickian diffusion, with a diffusion coefficient on the order of 10 -16 m 2 s -1 . The rather slow diffusion rate was likely due to significant binding of iron within the wood cell wall. The diffusion coefficient did not depend on the acetylation level; however, the capacity for iron absorption from acetate solution was greatly reduced in the acetylated wood, likely due to the loss of OH groups. We explored several hypotheses that might explain why the diffusion rate appears to be independent of the acetylation level and found none of them convincing. Implications for brown rot decay mechanisms and future research are discussed.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

Acetylation of lysine residues in the recombinant nucleoprotein and VP40 matrix protein of Zaire Ebolavirus by eukaryotic histone acetyltransferases

Acetylation of histones and other proteins plays crucial roles in transcriptional regulation, chromatin organization, and other biological processes. It has been recently reported that the nucleoprotein (NP) of influenza virus is acetylated in infected cells, and this modification contributes to the RNA polymerization activity of the virus. As the influenza virus, the Ebolavirus contains single-stranded negative-sense RNA as its viral genome, which interacts with NP and other viral proteins. In this study, we performed a series of biochemical experiments and revealed that the recombinant Ebolavirus NP and the viral matrix protein VP40, which binds with NP, were acetylated by eukaryotic histone acetyltransferases, such as P300/CREB-binding protein (P300/CBP) and P300/CBP-associated factor (PCAF), in vitro. Mass spectrometry was used to identify the lysine residues that were potential acetylation targets in NP and VP40. The identified lysine residues in NP were located in the RNA-binding cleft and the VP35-binding domain. Potentially acetylated lysine targets in VP40 were identified in the basic patch, which is necessary for constructing oligomers. These results suggest that the acetylation of these lysine residues is involved in the interactions between viral proteins.

60 APPLIED LIFE SCIENCES↗