Biomedical subjects

James M Fulcher

Publications and source records attributed to James M Fulcher.

4 recordsLinked to original sources

PubMed2026

The anaerobic fungus Caecomyces churrovis produces H2 via a non-bifurcating NADH-dependent enzyme complex.

UNLABELLED: Hydrogenosomes are mitochondrion-derived organelles that produce ATP and H2 to support energy metabolism in anaerobic eukaryotes. H2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H2 production in anaerobic eukaryotes remain incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P)+ and link electron transfer to H2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H2 production in the AF Caecomyces churrovis. Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H2:NAD+ oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE and NuoF) in C. churrovis and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme known from anaerobic prokaryotes. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex for H2 production in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H2 production and altering fungal metabolism. IMPORTANCE: H2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H2 production in Neocallimastix was first reported, the molecular mechanism for hydrogenosomal H2 production and redox balance remains unclear. We demonstrate that AF produce H2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H2-producing systems.

Hydrogen↗

PubMed2026

Addressing lignin composition and content via Arabidopsis arogenate dehydratase knockout and over-expression genotypes.

Following the down-selection of 14 Arabidopsis thaliana arogenate dehydratase (ADT) knockout and over-expression (OE) genotypes, the most highly contrasting quadruple knockout adt3/4/5/6 and ADT OE genotypes were subjected to proteomics, metabolomics, and scanning electron microscopy (SEM) analyses as needed, with results compared to Columbia wild-type (WT). The basal adt3/4/5/6 stem cross-sections, ∼70% lignin content reduced, exhibited buckled vessel cell walls and partially detached xylary fibers, in contrast to WT and ADT4m/5 m OE genotypes that did not. Anatomical defects primarily resulted from guaiacyl lignin level reductions in vessels with concomitant increased stem syringyl:guaiacyl (S/G) ratios. Phenylpropanoid and various upstream shikimate-chorismate pathway enzyme abundances, as well as specific monolignol oxidases (laccases/peroxidases), generally increased in adt3/4/5/6 at different stem and rosette leaf growth/development stages, relative to WT. Opposite effects were largely observed with the ADT5m OE genotype. By contrast, flavonoid and glucosinolate pathway enzyme amounts varied. Such enzyme abundance increases were overall unproductive as adt3/4/5/6 was unable to restore WT, ADT4 OE, ADT5 OE, ADT5m OE, and ADT4m/5 m OE secondary metabolite (lignin, phenylpropanoid, lignan, flavonoid, phenolic acid, and glucosinolate) levels. Conversely, ADT OE genotypes did not significantly increase programmed lignin levels or alter S/G compositions. In sum, proteomics analyses of adt3/4/5/6 and adt5 'perceived' that lignin and low molecular weight secondary metabolite amounts were not at 'programmed' levels as for WT and ADT OE genotypes but observed increases in relevant pathway protein abundances were futile. Notably though, proteomics analyses did not lead to predicting that lignin and associated biochemical pathways would have reduced metabolite levels, relative to WT and ADT OE genotypes. Genotype adt3/4/5/6, possibly the highest lignin level reduced genotype reported, did not utilize other phenolics to compensate. By contrast, the differential temporal and spatial deposition of cell wall oxidases again indicate the exquisite control over lignin deposition, and our lack of knowledge of precise lignin structure and assembly in subcellular regions of the lignified cell walls.

Lignin↗

PubMed2026

Unbiased Spatial Proteomics Uncovers Hepatic in Situ Regulation in Alcohol-Associated Hepatitis.

Alcohol-associated hepatitis (AH) is an acute inflammatory form of alcohol-associated liver disease. Previous studies have explored molecular mechanisms associated with AH pathogenesis through bulk liver tissue analysis; however, the heterogeneity of liver tissue and hence the spatial regulation within the AH liver microenvironment remained unaddressed. Here, an unbiased spatial proteomics analysis on the pathologic regions (PRs) of AH liver tissue is presented, including immune cell infiltration foci, lipid droplets, chicken-wire fibrosis, and fibrotic bands. Through combining a highly efficient nanodroplet processing in one pot for trace samples platform with ultrasensitive liquid chromatography-mass spectrometry, this study identified and quantified a total of 5186 unique proteins from PRs isolated in 200-μm-long × 200-μm-wide × 10-μm-thick areas. This in-depth spatial proteome coverage allowed us to discover mechanistic regulations within individual PRs, including compromised resolution of inflammation with infiltrated neutrophils at infiltration foci, increase of mitochondrial and peroxisomal fatty acid β-oxidation at lipid droplets, and differential cellular and extracellular regulations between chicken-wire fibrosis and fibrotic bands. Overall, this study demonstrated a new capability for AH research, revealed the significance of understanding spatial regulation within AH liver tissue, and further facilitated the development of therapeutic strategies at high resolution.

Proteomics↗

PubMed2026

High-throughput single-cell proteomics and transcriptomics from same cells with a nanoliter-scale, spin-transfer approach.

Single-cell multiomic platforms provide a comprehensive snapshot of cellular states and cell types by offering critical insights into the spatiotemporal regulation of biomolecular networks at a systems level, thereby defining the basis of multicellularity. Here, we introduce nanoSPINS, an advanced platform that enables high-throughput profiling and integrative analysis of the transcriptome and proteome from the same single cells using RNA sequencing and isobaric labeling LC-MS-based proteomics, respectively. NanoSPINS can efficiently transfer mRNA-containing droplets across two microarrays via a centrifugation-based approach, while proteins are retained on the initial platform. Benchmarking of nanoSPINS on two cell lines demonstrates its ability to generate global proteomic and transcriptomic profiles that align well with previously established methodologies/platforms. The incorporation of isobaric TMTpro labeling into this single-cell multiomics platform significantly enhances the throughput of single-cell proteomic analyses. Through the high-throughput quantification of the proteome and transcriptome, nanoSPINS not only facilitates the identification of molecular features at both mRNA and protein level but also provides larger sample sizes for improved statistical power in clustering and differential abundance. Given the broad applicability of single-cell multiomics in biological research and clinical settings, we believe nanoSPINS represents a powerful platform for the characterization of heterogeneous cell populations.

Single-Cell Analysis↗