Biology subjects

Cunningham, J. T.

Publications and source records attributed to Cunningham, J. T..

6 recordsLinked to original sources

bioRxiv2026

Divergent evolution of the PRPS enzymes across the tree of life

The phosphoribosyl pyrophosphate synthetase (PRPS) enzyme plays a central role in core biochemical pathways across all life, reflecting its deep evolutionary significance. Here, we present a pan-domain analysis of more than 35,000 non-redundant protein sequences that defines the fundamental features of PRPS at the roots of both domains of life and at critical branchpoints in the tree of life, including during early eukaryogenesis. Combining protein language modeling with maximum likelihood phylogenetic analysis, we identify entirely new PRPS enzyme classes and reveal how neofunctionalization of the canonical class I (bacteria-derived) or class III (archaea-derived) enzymes proceeds via genetic drift or gene duplication. We further demonstrate that multiple PRPS classes from distinct bacterial ancestries were transferred to the eukaryotic genome prior to supergroup radiation, and we provide biochemical and structural characterization of representative examples to clarify their roles in eukaryotic metabolism. Finally, we identify over 30 independent instances of PRPS pseudoenzyme formation across nearly all major eukaryotic lineages and PRPS orthologs, revealing a widespread but underappreciated mechanism of PRPS regulation. Together, this systems-level investigation resolves some of the earliest genetic events shaping life on Earth and offers detailed insight into the evolutionary mechanisms that sculpt enzyme structure and function.

evolutionary biology↗

bioRxiv2025

Dynamic assessment of the allocation of copper to cytochrome c oxidase using size-exclusion chromatography (SEC) combined with inductively coupled plasma mass spectrometry (ICP-MS)

Copper (Cu) is an essential trace element required for mitochondrial respiration via its incorporation into cytochrome c oxidase (CuCOX), the terminal enzyme of the electron transport chain. In this study, we employed size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS), UV-Vis spectroscopy, and immunoblotting to identify and validate a high-molecular-weight Cu-containing peak in SEC-ICP-MS chromatogram as representative of CuCOX activity. We demonstrate that this CuCOX peak is enhanced under metabolic conditions favoring oxidative phosphorylation, such as high Cu supplementation or galactose-containing media, and correlates with increased mitochondrial respiration. By tracing exogenously supplied 63Cu, we characterized the time- and dose-dependent incorporation of newly acquired Cu into CuCOX. Functional RNA interference (RNAi) experiments targeting key Cu transporters revealed that CuCOX formation is independent of the high-affinity Cu importer CTR1, but instead relies on alternative transporters including DMT1, LAT1, and the mitochondrial carrier SLC25A3. These findings offer new insight into the cellular pathways governing Cu trafficking and allocation to mitochondria under physiologically relevant conditions. Furthermore, our work establishes SEC-ICP-MS as a sensitive and specific method for quantifying CuCOX and assessing mitochondrial metabolism. This platform holds promise for the identification of Cu-related biomarkers and therapeutic targets, particularly in the context of diseases such as renal cell carcinoma (RCC), where dysregulated Cu homeostasis plays a critical role.

biochemistry↗

bioRxiv2025

PRPS activity tunes redox homeostasis in Myc-driven lymphoma

Myc hyperactivation coordinately regulates numerous metabolic processes to drive lymphomagenesis. Here, we elucidate the temporal and functional relationships between the medley of pathways, factors, and mechanisms that cooperate to control redox homeostasis in Myc-overexpressing B cell lymphomas. We find that Myc overexpression rapidly stimulates the oxidative pentose phosphate pathway (oxPPP), nucleotide synthesis, and mitochondrial respiration, which collectively steers cellular equilibrium to a more oxidative state. We identify Myc-dependent hyperactivation of the phosphoribosyl pyrophosphate synthetase (PRPS) enzyme as a primary regulator of redox status in lymphoma cells. Mechanistically, we show that genetic inactivation of the PRPS2 isozyme, but not PRPS1, in MYC-driven lymphoma cells leads to elevated NADPH levels and reductive stress-mediated death. Employing a pharmacological screen, we demonstrate how targeting PRPS1 or PRPS2 elicits opposing sensitivity or resistance, respectively, to chemotherapeutic agents affecting the thioredoxin and glutathione network, thus providing a therapeutic blueprint for treating MYC-driven lymphomas.

cancer biology↗

bioRxiv2024

Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

The phosphoribosyl pyrophosphate synthetase (PRPS) enzyme conducts a chokepoint reaction connecting central carbon metabolism and nucleotide production pathways, making it essential for life1,2. Here, we show that the presence of multiple PRPS-encoding genes is a hallmark trait of eukaryotes, and we trace the evolutionary origins and define the individual functions of each of the five mammalian PRPS homologs - three isozymes (one testis-restricted)3,4 and two non-enzymatic associated proteins (APs)5,6 - which we demonstrate operate together as a large molecular weight complex capable of attaining a heterogeneous array of functional multimeric configurations. Employing a repertoire of isogenic fibroblast clones in all viable individual or combinatorial assembly states, we define preferential interactions between subunits, and we show that cells lacking PRPS2, PRPSAP1, and PRPSAP2 render PRPS1 into aberrant homo-oligomeric assemblies with diminished metabolic flux and impaired proliferative capacity. We demonstrate how numerous evolutionary innovations in the duplicated genes have created specialized roles for individual complex members and identify translational control mechanisms that enable fine-tuned regulation of PRPS assembly and activity, which provide clues into the positive and negative selective pressures that facilitate metabolic flexibility and tissue specialization in advanced lifeforms. Collectively, our study demonstrates how evolution has transformed a single PRPS gene into a multimeric complex endowed with functional and regulatory features that govern cellular biochemistry.

biochemistry↗

bioRxiv2024

Avian influenza virus circulation and immunity in a wild urban duck population prior to and during a highly pathogenic H5N1 outbreak

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses were first detected in St. Johns, Newfoundland, Canada in late 2021, with the virus rapidly spreading across the western hemisphere over the next year. To investigate the patterns of avian influenza virus (AIV) infection and immune responses subsequent to the arrival of H5N1, we sampled the wild urban duck population in St. Johns for a period of 16 months after the start of the outbreak and compared these findings to archived samples. Antibody seroprevalence was relatively stable before the outbreak (2011-2014) at 27.6% and 3.9% for anti-AIV (i.e., NP) and H5-specific antibodies, respectively. During the winter of 2022, AIV-NP and H5-specific antibody seroprevalence both reached 100%, signifying a population-wide infection event. As expected, population-level immunity waned over time, and we found that ducks were seropositive for anti- AIV-NP antibodies for around twice as long as for H5-specific antibodies. The population was H5 seronegative to the latter approximately six months after the initial H5N1 incursion. In late February 2023, H5N1 clade 2.3.4.4b viruses were again detected in the duck population as a result of a second incursion into Newfoundland from Eurasia, which resulted in a second population-wide infection event. We observed a clear relationship of increasing antibody levels with decreasing viral RNA loads that allowed for interpretation of the course of infection and immune response in infected individuals and applied these findings to two cases of resampled ducks to infer infection history. Our study highlights the significance of applying both AIV surveillance and seroprevalence monitoring to provide a better understanding of AIV dynamics in wild populations, which may be crucial following the arrival of 2.3.4.4b H5Nx subtypes to assess the threats they pose to both wild and domestic animals, and to humans.

microbiology↗

bioRxiv2024

Copper drives remodeling of metabolic state and progression of clear cell renal cell carcinoma

Copper (Cu) is an essential trace element required for mitochondrial respiration. Late-stage clear cell renal cell carcinoma (ccRCC) accumulates Cu and allocates it to mitochondrial cytochrome c oxidase. We show that Cu drives coordinated metabolic remodeling of bioenergy, biosynthesis and redox homeostasis, promoting tumor growth and progression of ccRCC. Specifically, Cu induces TCA cycle-dependent oxidation of glucose and its utilization for glutathione biosynthesis to protect against H2O2 generated during mitochondrial respiration, therefore coordinating bioenergy production with redox protection. scRNA-seq determined that ccRCC progression involves increased expression of subunits of respiratory complexes, genes in glutathione and Cu metabolism, and NRF2 targets, alongside a decrease in HIF activity, a hallmark of ccRCC. Spatial transcriptomics identified that proliferating cancer cells are embedded in clusters of cells with oxidative metabolism supporting effects of metabolic states on ccRCC progression. Our work establishes novel vulnerabilities with potential for therapeutic interventions in ccRCC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/575895v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@14e1b89org.highwire.dtl.DTLVardef@f1c83forg.highwire.dtl.DTLVardef@191aff8org.highwire.dtl.DTLVardef@1b7ccd1_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAccumulation of copper is associated with progression and relapse of ccRCC and drives tumor growth. C_LIO_LICu accumulation and allocation to cytochrome c oxidase (CuCOX) remodels metabolism coupling energy production and nucleotide biosynthesis with maintenance of redox homeostasis. C_LIO_LICu induces oxidative phosphorylation via alterations in the mitochondrial proteome and lipidome necessary for the formation of the respiratory supercomplexes. C_LIO_LICu stimulates glutathione biosynthesis and glutathione derived specifically from glucose is necessary for survival of CuHi cells. Biosynthesis of glucose-derived glutathione requires activity of glutamyl pyruvate transaminase 2, entry of glucose-derived pyruvate to mitochondria via alanine, and the glutamate exporter, SLC25A22. Glutathione derived from glucose maintains redox homeostasis in Cu-treated cells, reducing Cu-H2O2 Fenton-like reaction mediated cell death. C_LIO_LIProgression of human ccRCC is associated with gene expression signature characterized by induction of ETC/OxPhos/GSH/Cu-related genes and decrease in HIF/glycolytic genes in subpopulations of cancer cells. Enhanced, concordant expression of genes related to ETC/OxPhos, GSH, and Cu characterizes metabolically active subpopulations of ccRCC cells in regions adjacent to proliferative subpopulations of ccRCC cells, implicating oxidative metabolism in supporting tumor growth. C_LI

cancer biology↗