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DOE OSTI2020

A Group of O-Acetyltransferases Catalyze Xyloglucan Backbone Acetylation and Can Alter Xyloglucan Xylosylation Pattern and Plant Growth When Expressed in Arabidopsis

Abstract Xyloglucan is a major hemicellulose in plant cell walls and exists in two distinct types, XXXG and XXGG. While the XXXG-type xyloglucan from dicot species only contains O-acetyl groups on side-chain galactose (Gal) residues, the XXGG-type xyloglucan from Poaceae (grasses) and Solanaceae bears O-acetyl groups on backbone glucosyl (Glc) residues. Although O-acetyltransferases responsible for xyloglucan Gal acetylation have been characterized, the biochemical mechanism underlying xyloglucan backbone acetylation remains to be elucidated. In this study, we showed that recombinant proteins of a group of DUF231 members from rice and tomato were capable of transferring acetyl groups onto O-6 of Glc residues in cello-oligomer acceptors, indicating that they are xyloglucan backbone 6-O-acetyltransferases (XyBATs). We further demonstrated that XyBAT-acetylated cellohexaose oligomers could be readily xylosylated by AtXXT1 (Arabidopsis xyloglucan xylosyltransferase 1) to generate acetylated, xylosylated cello-oligomers, whereas AtXXT1-xylosylated cellohexaose oligomers were much less effectively acetylated by XyBATs. Heterologous expression of a rice XyBAT in Arabidopsis led to a severe reduction in cell expansion and plant growth and a drastic alteration in xyloglucan xylosylation pattern with the formation of acetylated XXGG-type units, including XGG, XGGG, XXGG, XXGG,XXGGG and XXGGG (G denotes acetylated Glc). In addition, recombinant proteins of two Arabidopsis XyBAT homologs also exhibited O-acetyltransferase activity toward cellohexaose, suggesting their possible role in mediating xyloglucan backbone acetylation in vivo. Our findings provide new insights into the biochemical mechanism underlying xyloglucan backbone acetylation and indicate the importance of maintaining the regular xyloglucan xylosylation pattern in cell wall function.

Cell Biology↗

NASA NTRS2004

Environmental impacts on the developing CNS: CD15, NCAM-L1, and GFAP expression in rat neonates exposed to hypergravity

We have previously reported that the developing rat cerebellum is affected by hypergravity exposure. The effect is observed during a period of both granule and glial cell proliferation and neuronal migration in the cerebellum and coincides with changes in thyroid hormone levels. The present study begins to address the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of cerebellar proteins that are known to be directly involved in cell-cell interactions [protein expressing 3-fucosyl-N-acetyl-lactosamine antigen (CD15), neuronal cell adhesion molecule (NCAM-L1)] and those that affect cell-cell interactions indirectly [glial fibrillary acidic protein (GFAP)] in rat neonates exposed to centrifuge-produced hypergravity. Cerebellar mass and protein expression in rat neonates exposed to hypergravity (1.5 G) from gestational day (G) 11 to postnatal day (P) 30 were compared at one of six time points between P6 and P30 against rat neonates developing under normal gravity. Proteins were analyzed by quantitative western blots of cerebellar homogenates prepared from male or female neonates. Cerebellar size was most clearly reduced in male neonates on P6 and in female neonates on P9, with a significant gender difference; differences in cerebellar mass remained significant even when change in total body mass was factored in. Densitometric analysis of western blots revealed both quantitative and temporal changes in the expression of selected cerebellar proteins that coincided with changes in cerebellar mass and were gender-specific. In fact, our data indicated certain significant differences even between male and female control animals. A maximal decrease in expression of CD15 was observed in HG females on P9, coinciding with maximal change in their cerebellar mass. A shift in the time-course of NCAM-L1 expression resulted in a significant increase in NCAM-L1 in HG males on P18, an isolated time at which cerebellar mass does not significantly differ between HG and SC neonates. A maximal decrease in expression of GFAP was observed in HG males on P6, coinciding with maximal change in their cerebellar mass. Altered expression of cerebellar proteins is likely to affect a number of developmental processes and contribute to the structural and functional alterations seen in the CNS developing under altered gravity. Our data suggest that both cerebellar development and its response to gravitational manipulations differ in males and females. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

NASA Program Fundamental Space Biology↗

DOE OSTI2023

Chemical identity of carbon substrates drives differences in denitrification and N 2 O reduction within agricultural soils

Rates of nitrous oxide (N 2 O) production from agricultural soils are highly variable across space and time. Improving predictions of N 2 O emissions will require improving our understanding of the drivers of denitrification and the sources of variability in the rates of N 2 O production between soils and over time. While the amount of available carbon (C) is a known control on denitrification and N 2 O reduction, relatively little attention has been paid to the effect of the chemical identity of C substrates on rates of denitrification and N 2 O reduction. We investigated the effects of twelve different C-substrate additions on the production and reduction of N 2 O in five soils taken from two distinct agricultural locations in Michigan under multiple land uses. We provided additions of glucose, cellulose, N-acetyl-glucosamine, chitin, amino acids, protein, vanillyl alcohol, lignin, citrate, succinate, methanol, and water in laboratory denitrification potential assays to determine the effects of denitrifier C preference on denitrification rates. We found that amino acids, protein, and organic acids stimulated the greatest rates of denitrification potential across all land uses. Similarly, we found these same substrates caused the most N 2 O reduction, resulting in the lowest net concentrations of N 2 O. Soils from agricultural rotations without cover crops had overall lower rates of denitrifier activity, leading to less net N 2 O production compared to soils from other land uses. In general, C-utilization patterns were similar among all soils, and C-substrate identity had a much stronger effect than land use. Here, we demonstrate that the chemical identity of available C gives rise to wide variability in rates of denitrification and N 2 O reduction.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

The Role of Sugar Signaling in Regulating Plant Fatty Acid Synthesis

Photosynthates such as glucose, sucrose, and some of their derivatives play dual roles as metabolic intermediates and signaling molecules that influence plant cell metabolism. Such sugars provide substrates for de novo fatty acid (FA) biosynthesis. However, compared with the well-defined examples of sugar signaling in starch and anthocyanin synthesis, until recently relatively little was known about the role of signaling in regulating FA and lipid biosynthesis. Recent research progress shows that trehalose 6-phosphate and 2-oxoglutarate (2-OG) play direct signaling roles in the regulation of FA biosynthesis by modulating transcription factor stability and enzymatic activities involved in FA biosynthesis. Specifically, mechanistic links between sucrose non-fermenting−1–related protein kinase 1 (SnRK1)–mediated trehalose 6-phosphate (T6P) sensing and its regulation by phosphorylation of WRI1 stability, diacylglycerol acyltransferase 1 (DGAT1) enzyme activity, and of 2-OG–mediated relief of inhibition of acetyl-CoA carboxylase (ACCase) activity by protein PII are exemplified in detail in this review.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP’s enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP’s role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Cytochrome c lysine acetylation regulates cellular respiration and cell death in ischemic skeletal muscle

Skeletal muscle is more resilient to ischemia-reperfusion injury than other organs. Tissue specific post-translational modifications of cytochrome c (Cytc) are involved in ischemia-reperfusion injury by regulating mitochondrial respiration and apoptosis. Here, we describe an acetylation site of Cytc, lysine 39 (K39), which was mapped in ischemic porcine skeletal muscle and removed by sirtuin5 in vitro. Using purified protein and cellular double knockout models, we show that K39 acetylation and acetylmimetic K39Q replacement increases cytochrome c oxidase (COX) activity and ROS scavenging while inhibiting apoptosis via decreased binding to Apaf-1, caspase cleavage and activity, and cardiolipin peroxidase activity. These results are discussed with X-ray crystallography structures of K39 acetylated (1.50 Å) and acetylmimetic K39Q Cytc (1.36 Å) and NMR dynamics. We propose that K39 acetylation is an adaptive response that controls electron transport chain flux, allowing skeletal muscle to meet heightened energy demand while simultaneously providing the tissue with robust resilience to ischemia-reperfusion injury.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Plant Cell Wall Polysaccharide O-Acetyltransferases

Plant cell walls are largely composed of polysaccharide polymers, including cellulose, hemicelluloses (xyloglucan, xylan, mannan, and mixed-linkage β-1,3/1,4-glucan), and pectins. Among these cell wall polysaccharides, xyloglucan, xylan, mannan, and pectins are often O-acetylated, and polysaccharide O-acetylation plays important roles in cell wall assembly and disease resistance. Genetic and biochemical analyses have implicated the involvement of three groups of proteins in plant cell wall polysaccharide O-acetylation: trichome birefringence-like (TBL)/domain of unknown function 231 (DUF231), reduced wall acetylation (RWA), and altered xyloglucan 9 (AXY9). Although the exact roles of RWAs and AXY9 are yet to be identified, members of the TBL/DUF231 family have been found to be O-acetyltransferases responsible for the O-acetylation of xyloglucan, xylan, mannan, and pectins. Here, we provide a comprehensive overview of the occurrence of O-acetylated cell wall polysaccharides, the biochemical properties, structural features, and evolution of cell wall polysaccharide O-acetyltransferases, and the potential biotechnological applications of manipulations of cell wall polysaccharide acetylation. Further in-depth studies of the biochemical mechanisms of cell wall polysaccharide O-acetylation will not only enrich our understanding of cell wall biology, but also have important implications in engineering plants with increased disease resistance and reduced recalcitrance for biofuel production.

Plant Sciences↗

DOE OSTI2024

The lowdown on breakdown: Open questions in plant proteolysis

Abstract Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Eckardt, Nancy A. (ORCID:0000000316581412)↗

DOE OSTI2026

Integrative SP3 Workflow for Multi-PTM Proteomics Profiling (TZ-DP0)

The goal of the experiment was to demonstrate that the optimized multiplexed multi-PTM profiling workflow can comprehensively and quantitatively capture dynamic changes in protein abundance, cysteine oxidation, phosphorylation, and acetylation in cytokine-induced inflammatory stress in mouse pancreatic ß-cells. Global proteomic, redox proteomic, phosphoproteomic, and acetylomic were data collected from mouse Beta-TC-6 pancreatic Beta-cells, untreated (mock) and cytokine-treated Beta-cells at 4, 8, and 24 hours with 4 biological replicates. Samples were digested with trypsin and Lys-C, then analyzed by LC-MS/MS. Data were searched with MS-GF+, MASIC, and MaxQuant using PNNL's DMS processing pipeline.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk

Background. Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). Methods. We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples. Results. By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. Conclusions. Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation

Abstract In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli , we found a reduction in the phosphorylated population of CopR II when magnesium was added to the medium, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta , a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylation state of proteins when heterologously expressed, since their biochemical and physiological properties can be dependent on post-translational modification.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Regulation of Translation by Lysine Acetylation in Escherichia coli

Numerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from central metabolite acetyl-coenzyme A or acetyl phosphate to a lysine residue in a protein.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Discovery and Development of a Small-Molecule Inhibitor Targeting the GAS41 YEATS Domain in Nonsmall Cell Lung Cancer

Abstract GAS41 is frequently overexpressed in Non-Small Cell Lung Cancer (NSCLC). GAS41 contains a YEATS domain, which recognizes acetylated lysine residues on histones to recruit protein complexes and facilitate transcription. Suppression of GAS41 in NSCLC models inhibits cellular proliferation and markedly reduces tumor growth in mouse xenografts, justifying the development of small-molecule inhibitors. We have employed structure-based design and medicinal chemistry optimization to discover DLG-41, a submicromolar inhibitor binding to the GAS41 YEATS domain. DLG-41 potently disrupts the association of GAS41 YEATS with chromatin in mammalian cells and inhibits the proliferation of NSCLC cell lines with submicromolar potency without significantly affecting normal lung fibroblasts. DLG-41 induces more effective growth inhibition in A549 versus GAS41-knockout cells, demonstrating on-target activity. DLG-41 treatment upregulates the CDKN1A gene and downregulates pathways associated with lung cancer cell identity, tumor migration, and invasion. DLG-41 is a promising chemical probe for targeting GAS41 protein in NSCLC models and has potential for future development.

Listunov, Dymytrii [University of Michigan , , , ,↗

NASA NTRS2023

Design of a Minimal di-Nickel Hydrogenase Peptide

The most ancient processes for energy production in the evolution of life involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13 amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe]-hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase (ACS), two ancient, extant proteins central to metabolism. These experimental results clearly demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth. One Sentence Summary: Small metal-binding peptides were the likely precursors of modern enzymes.

Joshua A. Mancini↗

DOE OSTI2022

Structural insights into acetylated histone ligand recognition by the $\mathrm{BDP1}$ bromodomain of Plasmodium falciparum

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to regulate gene expression. The P. falciparum Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

C/EBP-α induces autophagy by binding to Beclin1 through its own acetylation modification in activated hepatic stellate cells

The activation of hepatic stellate cells (HSCs) plays a key role in the occurrence of liver fibrosisand promoting the apoptosis of activated HSCs or reducing the number of activated HSCs can reverse the development of liver fibrosis. In our previous studies, we have demonstrated that the CCAAT/enhancer binding protein α (C/EBP-α) played an important role in promoting the apoptosis of activated HSCs, thereby exerting an anti-liver fibrosis effect. Unlike apoptosis, autophagy, as a caspase-independent programmed cell death, can promptly remove the abnormal accumulation of substances or damaged organelles in cells and play a key role in regulating the homeostasis of intracellular environment. However, it is still unclear whether C/EBP-α participates in the occurrence of autophagy in HSCs. Therefore, in this study, we firstly used the methods of Western blot and immunofluorescence to characterize the consequence of C/EBP-α overexpression on the expression of proteins LC3B, P62, ATG5 and Beclin1 which were related to autophagy in HSCs. Subsequently, we performed Western blot and site-directed mutagenesis methods to clarify the type and related mechanism of autophagy which was induced by C/EBP-α. Here we show that C/EBP-α promotes the occurrence of autophagy in HSCs and the autophagy induced by C/EBP-α belongs to mitophagy. The stability of C/EBP-α protein regulates the level of autophagy in HSCs. In addition, acetylation of C/EBP-α also regulates the occurrence of autophagy in HSCs. Acetylation of lysine at positions K298, K302 and K326 of C/EBP-α promotes its binding to Beclin1. In conclusion, our study uncovers the role of C/EBP-α in regulating autophagy in HSCs, thereby providing a new strategy for clinical treatment of liver fibrosis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein’s function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε -acetyl- l -lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Structural, functional, and regulatory evaluation of a cysteine post-translationally modified Gcn5-related N -acetyltransferase

Polyamines within the cell are tightly regulated by spermidine/spermine N-acetyltransferase (SSAT) enzymes. While several SSATs have been investigated in different bacterial species, there is still a significant gap in knowledge about which proteins are functional SSATs in many organisms. For example, while it is known that Pseudomonas aeruginosa synthesizes the polyamine spermidine, the SSAT that acetylates this molecule and its importance in regulating intracellular polyamines remains unknown. We previously identified a candidate Gcn5-related N-acetyltransferase (GNAT) protein from P. aeruginosa (PA2271) that could fulfill this role since it acetylates spermidine, but no further studies were conducted. Here, we explored the structure/function relationship of the PA2271 protein by determining its X-ray crystal structure and performing enzyme kinetics assays. We also identified active site residues that are essential for catalysis and substrate binding. As the study progressed, we encountered results that led us to explore the importance of four cysteine residues on enzyme activity and disulfide bond formation or modification of cysteine residues. We found these cysteine residues in PA2271 are important for protein solubility and activity, and there is an interrelationship between cysteine residues that contribute to these effects. Furthermore, we also found disulfide bonds could form between C 121 and C 165 and speculate that these residues may contribute to redox regulation of PA2271 protein activity.

59 BASIC BIOLOGICAL SCIENCES↗