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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Structural Characterization and Dynamics of AdhE Ultrastructures from Clostridium thermocellum Show a Containment Strategy for Toxic Intermediates

Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe; however, ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 A structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

09 BIOMASS FUELS↗
NASA NTRS2003

An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof

The Arabidopsis genome sequencing in 2000 gave to science the first blueprint of a vascular plant. Its successful completion also prompted the US National Science Foundation to launch the Arabidopsis 2010 initiative, the goal of which is to identify the function of each gene by 2010. In this study, an exhaustive analysis of The Institute for Genomic Research (TIGR) and The Arabidopsis Information Resource (TAIR) databases, together with all currently compiled EST sequence data, was carried out in order to determine to what extent the various metabolic networks from phenylalanine ammonia lyase (PAL) to the monolignols were organized and/or could be predicted. In these databases, there are some 65 genes which have been annotated as encoding putative enzymatic steps in monolignol biosynthesis, although many of them have only very low homology to monolignol pathway genes of known function in other plant systems. Our detailed analysis revealed that presently only 13 genes (two PALs, a cinnamate-4-hydroxylase, a p-coumarate-3-hydroxylase, a ferulate-5-hydroxylase, three 4-coumarate-CoA ligases, a cinnamic acid O-methyl transferase, two cinnamoyl-CoA reductases) and two cinnamyl alcohol dehydrogenases can be classified as having a bona fide (definitive) function; the remaining 52 genes currently have undetermined physiological roles. The EST database entries for this particular set of genes also provided little new insight into how the monolignol pathway was organized in the different tissues and organs, this being perhaps a consequence of both limitations in how tissue samples were collected and in the incomplete nature of the EST collections. This analysis thus underscores the fact that even with genomic sequencing, presumed to provide the entire suite of putative genes in the monolignol-forming pathway, a very large effort needs to be conducted to establish actual catalytic roles (including enzyme versatility), as well as the physiological function(s) for each member of the (multi)gene families present and the metabolic networks that are operative. Additionally, one key to identifying physiological functions for many of these (and other) unknown genes, and their corresponding metabolic networks, awaits the development of technologies to comprehensively study molecular processes at the single cell level in particular tissues and organs, in order to establish the actual metabolic context.

NASA Program Fundamental Space Biology↗
DOE OSTI2022

Examining Bioethanol-Producing Ultrastructures with Electron Microscopy and Molecular Dynamics

A longstanding goal in the biofuel sector is to increase the efficiency of the circularization of the use of materials (1). One such instance is the degradation of plant material, such as cellulose, and converting it to biofuel via bacterial digestion (2). The bifunctional aldehyde-alcohol dehydrogenase (AdhE) from the anaerobic thermophile C. thermocellum seems to be vital for the production and cellular tolerance of bioethanol; however, it lacks the efficiency to produce ethanol at industry standards (3). Therefore, to understand how C. thermocellum AdhE functions, we used cryo-electron microscopy (cryo-EM) to obtain a 3.2 A structure of the AdhE complex. When compared to previously published structures from E. coli (4-6), we identified potential regions that control the native conformation of the ultrastructure, as well as located channels that isolate the intermediate aldehyde from the cellular milieu. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into one example of substrate channeling and establishes a basis for future mutagenesis studies.

AdhE↗
DOE OSTI2023

Growth-uncoupled propanediol production in a Thermoanaerobacterium thermosaccharolyticum strain engineered for high ethanol yield

Abstract Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species Thermoanaerobacterium thermosaccharolyticum was engineered for high ethanol yield, but we found that the strains switched from growth-coupled production of ethanol to growth uncoupled production of acetate and 1,2-propanediol upon growth cessation, producing up to 6.7 g/L 1,2-propanediol from 60 g/L cellobiose. The unique capability of this species to make 1,2-propanediol from sugars was described decades ago, but the genes responsible were not identified. Here we deleted genes encoding methylglyoxal reductase, methylglyoxal synthase and glycerol dehydrogenase. Deletion of the latter two genes eliminated propanediol production. To understand how carbon flux is redirected in this species, we hypothesized that high ATP levels during growth cessation downregulate the activity of alcohol and aldehyde dehydrogenase activities. Measurements with cell free extracts show approximately twofold and tenfold inhibition of these activities by 10 mM ATP, supporting the hypothesized mechanism of metabolic redirection. This result may have implications for efforts to direct and maximize flux through alcohol dehydrogenase in other species.

09 BIOMASS FUELS↗
DOE OSTI2026

Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

alcohol dehydrogenase↗
DOE OSTI2026

Genetic and metabolic drivers of membrane remodeling in Clostridium thermocellum under alcohol stress

Clostridium thermocellum is a leading candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels due to its native cellulolytic capabilities. Beyond ethanol, C. thermocellum is being developed as a platform for producing higher-chain alcohols such as isobutanol and n-butanol. However, its physiological adaptations to alcohol stress remain poorly understood. Here, we investigate how C. thermocellum remodels its membrane lipid composition in response to exogenous ethanol, n-butanol, isobutanol, and butyrate. Exposure to linear alcohols such as n-butanol or to organic acids like butyrate increased the proportion of straight-chain fatty acids in the membrane at the expense of branched-chain species, whereas exposure to the branched alcohol isobutanol produced the opposite effect. Isotope tracer experiments demonstrated that C. thermocellum directly incorporates the carbon backbones of exogenous alcohols and acids into fatty acids, providing a mechanistic basis for these contrasting shifts. We show that the bifunctional aldehyde/alcohol dehydrogenase AdhE is essential for the assimilation of exogenous alcohols into fatty acids, acting through its oxidative activity by first oxidizing alcohols to aldehydes and then converting them to acyl-CoA intermediates. Deletion of the pyruvate:ferredoxin oxidoreductase isozyme pfor4 abolished branched-chain fatty acid synthesis, but supplementation with isobutanol restored production, indicating that Pfor4 substitutes for the canonical branched-chain α-keto acid dehydrogenase complex. These findings reveal two distinct routes for branched-chain fatty acid production in C. thermocellum: a Pfor4-dependent pathway from α-keto acid intermediates derived from amino acid synthesis, and an AdhE-dependent salvage pathway that assimilates exogenous branched-chain alcohols.

Acetivibrio thermocellus↗
DOE OSTI2023

Pinoresinol rescues developmental phenotypes of Arabidopsis phenylpropanoid mutants overexpressing FERULATE 5-HYDROXYLASE

Most phenylpropanoid pathway flux is directed toward the production of monolignols, but this pathway also generates multiple bioactive metabolites. The monolignols coniferyl and sinapyl alcohol polymerize to form guaiacyl (G) and syringyl (S) units in lignin, components that are characteristic of plant secondary cell walls. Lignin negatively impacts the saccharification potential of lignocellulosic biomass. Although manipulation of its content and composition through genetic engineering has reduced biomass recalcitrance, in some cases, these genetic manipulations lead to impaired growth. Here, the reduced-growth phenotype is often attributed to poor water transport due to xylem collapse in low-lignin mutants, but alternative models suggest that it could be caused by the hyper- or hypoaccumulation of phenylpropanoid intermediates. In Arabidopsis thaliana, overexpression of FERULATE 5-HYDROXYLASE (F5H) shifts the normal G/S lignin ratio to nearly pure S lignin and does not result in substantial changes to plant growth. In contrast, when we overexpressed F5H in the low-lignin mutants cinnamyl dehydrogenase c and d (cadc cadd), cinnamoyl-CoA reductase 1, and reduced epidermal fluorescence 3, plant growth was severely compromised. In addition, cadc cadd plants overexpressing F5H exhibited defects in lateral root development. Exogenous coniferyl alcohol (CA) and its dimeric coupling product, pinoresinol, rescue these phenotypes. These data suggest that mutations in the phenylpropanoid pathway limit the biosynthesis of pinoresinol, and this effect is exacerbated by overexpression of F5H, which further draws down cellular pools of its precursor, CA. Overall, these genetic manipulations appear to restrict the synthesis of pinoresinol or a downstream metabolite that is necessary for plant growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Relative Activities of the β-Ketoacyl-CoA and Acyl-CoA Reductases Influence the Product Profile and Flux in a Reversed β-Oxidation Pathway

The β-oxidation pathway, normally involved in the catabolism of fatty acids, can be functionally made to act as a fermentative, iterative, elongation pathway when driven by the activity of a trans-enoyl-CoA reductase (TER). Here, the terminal acyl-CoA reduction to alcohol can occur on substrates with varied chain lengths, leading to a broad distribution of fermentation products in vivo. Tight control of the average chain length and product profile is desirable, as the chain length greatly influences the molecular properties and the commercial value. Lacking a termination enzyme with a narrow chain length preference, we sought alternative factors that could influence the product profile and pathway flux in the iterative pathway. In this study, we reconstituted the reversed β-oxidation (R-βox) pathway in vitro with a purified tri-functional complex (FadBA) responsible for the thiolase, enoyl-CoA hydratase, and hydroxyacyl-CoA dehydrogenase activities, a TER, and an acyl-CoA reductase. Using this system, we determined the rate-limiting step of the elongation cycle and demonstrated that by controlling the ratio of these three enzymes and the ratio of NADH and NADPH, we can influence the average chain length of the alcohol product profile.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Tnseq to identify catabolism of b-5 linked aromatics by Novosphingobium aromaticivorans

This study investigates the catabolism of the b-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans. We used genome wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidates elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans.

Noguera, Daniel R.↗
DOE OSTI2024

Tnseq to identify catabolism of b-5 linked aromatics by Novosphingobium aromaticivorans

This study investigates the catabolism of the b-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans. We used genome wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidates elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans.

aromatic compounds↗
DOE OSTI2024

Catabolism of β-5 linked aromatics by Novosphingobium aromaticivorans

ABSTRACT Aromatic compounds are an important source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the β-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans . We used genome-wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidate genes elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans . Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers vanillin and 5-formylferulate (5-FF). We also showed that the aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in the β-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans . A comparative genomic analysis predicted that the newly discovered β-5 aromatic catabolic pathway is common within the order Sphingomonadales. IMPORTANCE In the transition to a circular bioeconomy, the plant polymer lignin holds promise as a renewable source of industrially important aromatic chemicals. However, since lignin contains aromatic subunits joined by various chemical linkages, producing single chemical products from this polymer can be challenging. One strategy to overcome this challenge is using microbes to funnel a mixture of lignin-derived aromatics into target chemical products. This approach requires strategies to cleave the major inter-unit linkages of lignin to release monomers for funneling into valuable products. In this study, we report newly discovered aspects of a pathway by which the Novosphingobium aromaticivorans DSM12444 catabolizes aromatics joined by the second most common inter-unit linkage in lignin, the β-5 linkage. This work advances our knowledge of aromatic catabolic pathways, laying the groundwork for future metabolic engineering of this and other microbes for optimized conversion of lignin into products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Formate dependent heterodisulfide reduction in a Methanomicrobiales archaeon

Hydrogenotrophic methanogens produce CH 4 using H 2 as an electron donor to reduce CO 2 . In the absence of H 2 , many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H 2 , but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the Methanomicrobiales, we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus. Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus co-purified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr associated hydrogenase, and H 2 could not function as an electron donor, even with Hdr purified from cells grown on H 2 . Here, we found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order Methanomicrobiales.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

Abstract R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l -arabinose and d -xylose metabolism proceeding through d -arabitol and d -ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d -arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d -arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d -xylose to d -arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d -ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l -ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Crystallographic Fragment Screening of a Bifunctional Proline Catabolic Enzyme Reveals New Inhibitor Templates for Proline Dehydrogenase and L-Glutamate-γ-semialdehyde Dehydrogenase

The proline catabolic pathway consisting of proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde (GSAL) dehydrogenase (GSALDH) catalyzes the four-electron oxidation of L-proline to L-glutamate. Chemical probes to these enzymes are of interest for their role in cancer and inherited metabolic disease. Here, we report the results of a crystallographic fragment-screening campaign targeting both enzymes. A unique aspect of our approach is the screening of both enzymes simultaneously using crystals of the bifunctional PRODH-GSALDH enzyme, proline utilization A (PutA). A 288-fragment library from Zenobia was screened in crystallo in cocktails of six fragments. Validation X-ray crystallography with individual fragments identified seven crystal hits distributed in the PRODH active site, GSALDH aldehyde substrate-binding site, and GSALDH NAD+ adenine-binding site. The fragment bound in the PRODH active site, 4-methoxybenzyl alcohol, is structurally distinct from all known PRODH inhibitors as it lacks an anionic anchor and stabilizes open conformations of the active site, motivating the study of eighteen analogs. In total, thirteen crystal structures with resolutions ranging from 1.32 Å to 1.80 Å were determined, resolving the poses and interactions of seven fragments from the Zenobia library and five analogs of 4-methoxybenzyl alcohol. These results expand the chemical space of probes targeting proline catabolic enzymes and provide new structural information for further inhibitor development.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production

Abstract Clostridium tyrobutyricumis an anaerobe known for its ability to produce short‐chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine‐tuning gene expression inC. tyrobutyricum. Synthetic inducible promoters were created by employing anEscherichia coli lacoperator to regulate the thiolase promoter (PCathl) fromClostridium acetobutylicum, with the best one (LacI‐Pto4s) showing a 5.86‐fold dynamic range with isopropyl β‐d‐thiogalactoside (IPTG) induction. A LT‐Pt7 system with a dynamic range of 11.6‐fold was then created by combining LacI‐Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lacpromoter. Furthermore, two inducible expression systems BgaR‐PbgaLA and BgaR‐PbgaLB with a dynamic range of ~40‐fold were developed by optimizing a lactose‐inducible expression system fromClostridium perfringenswith modified 5′ untranslated region (5′ UTR) and ribosome‐binding site (RBS). BgaR‐PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded byadhE2 and butyryl‐CoA/acetate Co‐A transferase encoded bycat1 inC. tyrobutyricumwild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulatedcat1 expression also confirmed that the Cat1‐catalyzed reaction was responsible for acetate assimilation inC. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression inC. tyrobutyricumfor industrial applications.

Biotechnology & Applied Microbiology↗
DOE OSTI2024

Steroid responsiveness in alcohol-associated hepatitis is linked to glucocorticoid metabolism, mitochondrial repair, and heat shock proteins

Alcohol-associated hepatitis (AH) is one of the clinical presentations of alcohol-associated liver disease. AH has poor prognosis, and corticosteroids remain the mainstay of drug therapy. However, ~40% of patients do not respond to this treatment, and the mechanisms underlying the altered response to corticosteroids are not understood. The current study aimed to identify changes in hepatic protein expression associated with responsiveness to corticosteroids and prognosis in patients with AH. Patients with AH were enrolled based on the National Institute on Alcohol Abuse and Alcoholism inclusion criteria for acute AH and further confirmed by a diagnostic liver biopsy. Proteomic analysis was conducted on liver samples acquired from patients with AH grouped as nonresponders (AH-NR, n = 7) and responders (AH-R, n = 14) to corticosteroids, and nonalcohol-associated liver disease controls (n = 10). The definition of responders was based on the clinical prognostic model, the Lille Score, where a score < 0.45 classified patients as AH-R and a score > 0.45 as AH-NR. Primary outcomes used to assess steroid response were Lille Score (eg, improved liver function) and survival at 24 weeks. Reduced levels of the glucocorticoid receptor and its transcriptional co-activator, glucocorticoid modulatory element-binding protein 2, were observed in the hepatic proteome of AH-NR versus AH-R. The corticosteroid metabolizing enzyme, 11-beta-hydroxysteroid dehydrogenase 1, was increased in AH-NR versus AH-R along with elevated mitochondrial DNA repair enzymes, while several proteins of the heat shock pathway were reduced. Analysis of differentially expressed proteins in AH-NR who survived 24 weeks relative to AH-NR nonsurvivors revealed several protein expression changes, including increased levels of acute phase proteins, elevated coagulation factors, and reduced mast cell markers. This study identified hepatic proteomic changes that may predict responsiveness to corticosteroids and mortality in patients with AH.

59 BASIC BIOLOGICAL SCIENCES↗