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Biology subjects

Asara, J. M.

Publications and source records attributed to Asara, J. M..

At least 19 recordsLinked to original sources

Spatial isotope tracing maps compartmentalized nutrient use across the adult fruit fly

Drosophila melanogaster is a powerful model for studying systemic metabolism. Although metabolism is compartmentalized across tissues, prior work in adult Drosophila has mostly measured it in dissected tissues rather than in situ, limiting the spatial information that can be gained. Here, we combine whole-fly mass-spectrometry imaging with stable-isotope tracing and flux modeling to quantify metabolic activities across the adult fruit fly in a spatially resolved manner. Across 68 tracer-fed Oregon-R female flies and 187,709 pixels, we map 81 metabolites onto eight annotated regions, including muscle, fat body, gut, and nervous system. Glucose labeling and fitted relative fluxes indicate higher glycolytic and tricarboxylic acid cycle turnover in thoracic muscle than in abdominal fat body; proline labels the nervous system and eye; glutamine the abdominal reproductive system and gut. Alanine carries glucose-derived carbon through the hemolymph and transfers nitrogen into glutamate. In flies bearing yorkie-driven gut tumors, alanine contribution to malate rises in gut and abdomen, and reducing gut alanine uptake lowers tumor burden. Together, this work establishes a framework for quantitative spatial metabolic analysis, showing compartmentalized nutrient use across the adult fruit fly.

genetics↗

Newly identified COPD GWAS protein interactors reveal a potential disease network module

Background: Chronic obstructive pulmonary disease (COPD) is a complex, heterogeneous disease influenced by genetic and environmental factors. However, the interactions between COPD risk genes and their collective role in COPD susceptibility remain largely elusive. We hypothesize that the protein-protein interaction (PPI) network of genes associated with COPD risk can provide molecular insight into the mechanisms of COPD pathogenesis. Methods: We used affinity purification mass spectrometry (AP-MS) to detect protein-protein interactions of six well-established COPD GWAS gene products (AGER, FAM13A, FBXO38, HHIP, IREB2, MFAP2) in two relevant lung cell lines (IMR90, 16HBE). These genes are located in significant COPD GWAS regions, and their physiological role in COPD has been previously confirmed by functional studies. We analyzed the impact of the newly identified interactions in contextual, cell type-specific PPI networks built from the combination of publicly available PPI data (HUBRIS) and cell type-specific gene expression data. Results: Using AP-MS, we identified 482 unique interactors across 701 newly identified protein interactions with the six GWAS gene products in at least one of the cell lines (AGER: 122, FAM13A: 83, FBXO38: 71, HHIP: 108, IREB2: 8, MFAP2: 309). Subsequent network analysis incorporating these new PPIs with public PPI databases revealed: (1) the new interactions significantly reduce the network distance between known COPD GWAS gene products; (2) 5.2% of the newly identified interactors are differentially abundant between COPD cases and controls in lung proteomic data from the Lung Tissue Research Consortium (LTRC); and (3) 41 (14.3%) of the newly identified interactors were directly connected to more than one of the six COPD GWAS genes. We identified a tightly connected core network of COPD GWAS gene products and protein biomarkers with intersecting signals; this network consists of 8 proteins, including 5 of the 6 GWAS gene products (AGER, FAM13A, HHIP, FBXO38, and MFAP2) as well as CAVIN1, TGM2, and HSPA1A. Conclusions: By combining AP-MS experimental data, multiple types of "omics" data, and network analysis, we have constructed a disease network module for COPD. This module can be a foundation for understanding the collective influence of COPD GWAS gene products in disease pathogenesis.

systems biology↗

Epstein-Barr virus transformation creates a methionine-dependent ferroptosis vulnerability in B cells

Epstein-Barr virus (EBV) causes over 200,000 cancers annually, including immunoblastic lymphomas in immunosuppressed hosts. Most transformed cells arrest, yet survive when deprived of the essential amino acid methionine. We instead find that EBV transformed lymphoblastoid cell lines (LCLs), which model the EBV latency III program-driven B-cell lymphoproliferative diseases of immunosuppressed hosts, rapidly die upon methionine restriction. Methionine restriction elevated LCL lipid reactive oxygen species and triggered ferroptosis. Whereas methionine restriction hypomethylates the EBV genome and triggers viral reactivation in latency I Burkitt cells by lowering the cellular methylation potential, the LCL latency III program instead redirected methionine toward redox defense, without altering the SAM/SAH ratio. Stable-isotope tracing revealed that latency III strongly induces transsulfuration, synthesizing cysteine de novo to support glutathione pools. The EBV oncoprotein LMP2A, which mimics B-cell receptor signaling, supported newly infected human B cell cystathionine-{beta}-synthase and cystathionine-{gamma}-lyase expression and methionine dependence, phenocopied by immunoglobulin crosslinking. In vivo, dietary methionine restriction impaired LCL xenograft outgrowth and depleted tumor cystine. Combined methioninase and cyst(e)inase administration blocked both cysteine sources, collapsed tumor glutathione levels, and triggered ferroptosis. Our results define methionine metabolism as a targetable ferroptosis vulnerability of EBV-transformed B cells. HighlightsO_LIMethionine restriction triggers EBV-transformed lymphoblastoid B cell ferroptosis C_LIO_LIEBV latency III induces transsulfuration to sustain LCL cysteine and glutathione C_LIO_LIMethioninase or dietary methionine restriction strongly impair LCL growth in vivo C_LIO_LIMethioninase plus cyst(e)inase collapses xenograft GSH levels and drives ferroptosis C_LI

biochemistry↗

Conserved principles of central carbon partitioning in Hippo-Yorkie-driven Drosophila gut tumors

Central carbon metabolism undergoes extensive remodeling in cancers, yet the extent to which the resulting network architectures and operating principles are conserved across species and oncogenic contexts in vivo remains unclear. Here, central carbon metabolism was evaluated in Hippo/Yki-driven Drosophila gut tumors, as Hippo-YAP/TAZ signaling links nutritional cues to metabolic state and contributes to epithelial tumorigenesis and therapy resistance. Using integrated steady-state metabolomics, transcriptomics and [U-13C6]glucose tracing, we defined how Hippo pathway activation reorganizes nutrient utilization and carbon flux in vivo and assessed how the resulting Yki-driven metabolic network aligns with mammalian cancer metabolism. Yki tumors exhibited a Warburg-like state with increased glycolytic throughput and enhanced conversion of glucose-derived carbon to lactate, accompanied by transcriptional upregulation of key glycolytic and lactate-production enzymes. Glucose carbon was also redirected into redox-supporting and anabolic nodes, including activation of the glycerol-3-phosphate shuttle and increased labeling of alanine and serine. Mitochondrial metabolism was reorganized into a non-canonical, segmented TCA network centered on -ketoglutarate, which accumulated and acted as a drain into glutamate/glutamine and 2-hydroxyglutarate rather than supporting complete oxidative turnover. Despite reduced abundance of pentose phosphate intermediates, non-oxidative PPP carbon rearrangements and ribose labeling were maintained, enabling robust glucose contribution to pyrimidine nucleotide pools, including strongly labeled dTTP. Together, these data establish a comprehensive map of Yki-driven central carbon partitioning in vivo and highlight conserved principles of tumor carbon allocation shared across oncogenic contexts and mammalian cancer metabolism.

cancer biology↗

Epstein-Barr Virus Latent Membrane Protein 1 Suppresses Ferroptosis via Pentose Phosphate Pathway and Glutathione Metabolism

Epstein-Barr virus (EBV) is associated with 200,000 cancers per year, including Burkitt lymphoma and post-transplant lymphomas. We previously reported that EBV latency oncogene programs dynamically remodel infected B cell metabolism and sensitivity to induction of ferroptosis, a programmed cell death pathway driven by lipid reactive oxygen species. However, much has remained unknown about how EBV remodels key redox defense pathways in support of infected B cell proliferation. Here, we identify EBV latent membrane protein 1 (LMP1), a key viral oncogene necessary for B cell immortalization and which mimics aspects of CD40 signaling, drives resistance to ferroptosis induction by erastin, a small molecule that blocks cystine uptake. LMP1 expression was sufficient to protect Burkitt cells from erastin ferroptosis induction. Mechanistically, signaling from the LMP1 TES2/CTAR2 region drove this phenotype, which was not shared by CD40 signaling, revealing that LMP1 evolved independent redox defense roles. Metabolomic analysis highlighted key LMP1 and TES2 signaling roles in support of antioxidant cysteine and glutathione levels. TES2 signaling supported cystine uptake, glutathione and NADPH pools in newly infected peripheral blood B cells. We identified PFKFB4, a host enzyme that shunts glucose into the pentose phosphate pathway to support NADPH production, as a major TES2 metabolic target. PFKFB4 knockdown increased EBV-transformed lymphoblastoid cell line lipid ROS levels, decreased glutathione and strongly sensitized them to ferroptosis induction by erastin treatment. PFKFB4 was also necessary for LMP1-mediated Burkitt B cell ferroptosis resistance. Collectively, these results identify PFKFB4 as a key host cell EBV metabolism remodeling target critical for infected B cell redox defense.

microbiology↗

Metabolic pathway competition sensitizes thioredoxin reductase-depleted Candida albicans to cell wall stress and antifungals

Candida albicans is the most common invasive human fungal pathogen. Each of the currently available 3 antifungal drug classes is limited by toxicities, drug interactions or inadequate penetration of critical tissues. We discovered a connection between C. albicans TOR and thioredoxin reductase, Trr1. Fungal and human thioredoxin reductases diverge fundamentally and are essential for repair of oxidatively damaged macromolecules, so they may be promising drug targets. We found that Trr1 is required for C. albicans growth at human body temperatures, hyphal growth regulation and management of oxidative and cell wall stress. Its depletion sensitizes C. albicans to AmphotericinB and to an echinocandin. TRR1-depleted cells have lower cell wall glucan content. Metabolomics experiments highlighted their perturbed pentose phosphate pathway (PPP). They have sharply elevated activity of glucose-6-phosphate dehydrogenase, the first, rate-limiting enzyme of the PPP. A key glycolytic, gluconeogenic and UDP-glucose biosynthetic enzyme each show decreased activity. UDP-glucose is the substrate of cell wall glucan-producing enzymes. We propose that Trr1-depleted cells accelerated glucose-6-phosphate flux into the PPP, driven by demand for NADPH reducing equivalents, diminishes glucose-6-phosphate availability for UDP-glucose production and hence for cell wall construction, weakening the wall. Strikingly dysregulated signaling pathways in Trr1-depleted cells contribute to their stress hypersensitivities.

microbiology↗

eIF2B Selectively Anchors and Activates Mutant KRAS

Much is known about how RAS oncoproteins regulate mRNA translation factors, but the reverse relationship, how translation factors influence RAS activity, has remained largely unexplored. At the plasma membrane (PM), Son of Sevenless (SOS) acts as the canonical guanine nucleotide exchange factor (GEF) for RAS proteins, yet mechanisms governing its specificity for individual RAS isoforms remain unknown. Here, we show that the translation initiation factor eIF2B, best known for its GEF function in translation initiation, forms a distinct complex with SOS and mutant KRAS at the PM, but not with other oncogenic RAS variants. Mechanistically, eIF2B acts as an allosteric regulator of SOS, selectively enhancing GDP-GTP exchange on mutant KRAS. This specificity arises from the translational activity of eIF2B, which upregulates glycosphingolipid (GSL) biosynthesis to remodel PM lipids and preferentially anchor mutant KRAS. Together, our results uncover an unexpected moonlighting function of eIF2B: acting both as a direct activator of SOS and as a regulator of GSL pathway that shapes the membrane landscape, both required for mutant KRAS activation. These insights redefine our understanding of eIF2B and mutant KRAS functions in cancer and have profound implications for KRAS-driven oncogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/686860v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f50934org.highwire.dtl.DTLVardef@2f55b9org.highwire.dtl.DTLVardef@1a51e39org.highwire.dtl.DTLVardef@1634aac_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIeIF2B interacts with mutant KRAS and SOS at the plasma membrane (PM). C_LIO_LIThe eIF2B:SOS complex promotes the GTP-bound active state of mutant KRAS. C_LIO_LIeIF2B enhances the translation of B4GALT5 mRNA, encoding a key enzyme of glycosphingolipid (GSL) biosynthesis. C_LIO_LIUpregulation of the GSL metabolites, ganglioside GM3 and sulfatide SM4, remodels PM lipid composition to facilitate eIF2B:SOS:KRAS complex formation and mutant KRAS nanoclustering. C_LIO_LIThrough its interaction with SOS and stimulation of GSL synthesis, eIF2B selectively activates mutant KRAS at the PM among RAS isoforms. C_LIO_LIeIF2B is required for the development of mKRAS-driven lung adenocarcinoma in mice. C_LIO_LIeIF2B is a marker of poor prognosis in mutant KRAS-driven cancers. C_LI

cancer biology↗

Arginine Metabolism Supports De Novo Pyrimidine Biosynthesis to Block DNA Damage and Maintain Epstein-Barr Virus Latency

Incompletely understood mechanisms serve to maintain Epstein-Barr virus (EBV) latency in most B-cell states, in which viral oncogene(s) are expressed but lytic antigens are repressed. Shortly after EBVs discovery and even before it was named, early pioneers Werne and Gertrude Henle identified that restriction of extracellular arginine de-represses EBV lytic antigens within Burkitt lymphoma tumor cells. However, for nearly 60 years, it has remained unknown how arginine metabolism supports EBV latency. To gain insights, we performed an amino acid restriction screen in Burkitt cell lines. This confirmed that arginine restriction was sufficient to trigger EBV reactivation in Burkitt B-cells and gastric carcinoma models. Arginine restriction strongly impaired de novo pyrimidine biosynthesis, and CRISPR or chemical genetic blockade of pyrimidine biosynthesis enzymes induced EBV immediate early and early lytic gene expression. However, arginine restriction blocked EBV lytic DNA replication and consequently also late gene expression, suggesting an abortive lytic cycle. Arginine restriction triggered DNA damage, which was an important driver of arginine restriction-driven EBV reactivation. Arginine restriction and DNA hypomethylation synergistically increased EBV reactivation. Together, our results highlight arginine and pyrimidine metabolism as potential targets for EBV lytic antigen induction therapy in B and epithelial cell contexts. ImportanceAltered metabolism is a hallmark of cancer, frequently increasing transformed cell dependence on extracellular amino acid supply. Despite current interest in EBV lytic antigen induction therapy, in which viral lytic reactivation sensitizes tumors to the highly cytotoxic effects of the antiviral ganciclovir, there has been no systemic study of extracellular amino acid that controls EBV latency. We identified that arginine uptake was important for the maintenance of EBV latency in both Burkitt lymphoma and gastric carcinoma contexts. Metabolic pathway analyses highlighted that arginine uptake and metabolism was required to supply pyrimidines. Disruption of arginine metabolism or de novo pyrimidine synthesis caused DNA damage. As arginine restriction was also found to cause Burkitt DNA hypermethylation, we provide evidence that the combination of arginine restriction and DNA hypomethylation by decitabine or by CRISPR approaches together induced EBV reactivation more highly than either alone, suggesting a therapeutic approach.

microbiology↗

Hepatic ceramide synthesis links systemic inflammation to organelle dysfunction in cancer

Paraneoplastic syndromes arise when tumor-derived cytokines reprogram distant organs. Although mediators such as Interleukin-6 have been implicated, how these signals impair host organ function remains incompletely defined. Here, we identify a cytokine-lipid axis that drives hepatic autophagy dysfunction. Specifically, in Drosophila, the gut tumor-derived interleukin-like cytokine Upd3 induces the expression of the triglyceride lipase CG5SCC, which we named "cancer-associated lipid mobilizer (calm)", and the ceramide synthase schlank in the fat body. This upregulation rewires fat body lipid metabolism, resulting in an autophagic-flux blockade. Genetic reduction of either CG5SCC or schlank restores organelle homeostasis and mitigates paraneoplastic phenotypes. This mechanism is conserved in mammals: in mice, IL-6 upregulates the lipoprotein lipase Lpl and ceramide synthases which in turn trigger a hepatic autophagy-flux blockade; in humans, hepatic LPL and ceramide synthases expression correlates with poorer survival in hepatocellular carcinoma. Our findings position hepatic lipid metabolism rewiring, especially ceramide synthesis as a critical, conserved node coupling systemic inflammation to organelle dysfunction, and suggest this pathway as a possible therapeutic entry point for cancer-associated liver disorders.

genetics↗

Lysosome-dependent nutrient scavenging underliesstress adaptation during epithelial-to-mesenchymaltransition

Metastatic cancer cells invade tissue, overcome nutrient stress, and survive transit to distant sites. Many of the mechanisms that support these processes are incompatible with proliferation. This study defines cellular transition states in breast epithelial cells undergoing epithelial-mesenchymal transition (EMT) driven by ERK2 and TGF-{beta} signaling. EMT triggers robust endolysosomal system upregulation and metabolic adaptations that balance proliferative and invasive states. Surprisingly, invasive cells rely on scavenging via lysosomes and macropinocytosis to acquire amino acids, rather than plasma membrane transport, even in nutrient-rich conditions. Macropinocytosis increases intracellular amino acid storage, promoting survival during amino acid deprivation. This metabolic shift depends on c-MYC downregulation, an early EMT event. Reintroducing c-MYC suppresses the metabolic switch, endolysosomal induction, macropinocytosis, and the proliferation-to-migration transition. These findings reveal how cells dynamically balance proliferation and invasion, offering insights into transition states difficult to capture in models of breast cancer metastasis.

cell biology↗

Renal Coenzyme A (CoA) Production Fuels Stem Cell Proliferation and Tumor Growth

Coenzyme A (CoA), derived from Vitamin B5 (VB5; also called pantothenate), is essential for lipid metabolism, energy production, and cell proliferation. While the intracellular functions of CoA are well-characterized, much less is known about its tissue-specific regulation and systemic physiological roles. Here, using Drosophila melanogaster, we uncover a gut-renal circuit in which dietary VB5 fuels CoA biosynthesis specifically in the Malpighian tubules (MTs, the fly kidney), non-autonomously impacting gut homeostasis. We show that, in the MTs, Myc boosts renal CoA production by directly upregulating the pantothenate kinase Fbl (human PANK1-3 ortholog) and downregulating CG5828, which we characterize as the functional ortholog of the metabolite phosphatase and CoA synthesis suppressor PANK4 (dPANK4). Elevated CoA biosynthesis enhances the mevalonate-isoprenoid pathway activity in the gut, promoting intestinal stem cell proliferation. We further demonstrate that renal CoA production is required for gut tumor growth in a fly model. Consistently, MYC and genes within the CoA-isoprenoid axis display strong association with clinical outcomes in human cancers. Together, our findings establish that Myc-driven CoA metabolism generates an inter-organ signal that couples VB5 availability to stem cell control and tumor growth, and identify the CoA-isoprenoid axis as a targetable metabolic vulnerability in cancer.

genetics↗

Multi-omic mapping of Drosophila protein secretomes reveals tissue-specific origins and inter-organ trafficking

Secreted proteins regulate many aspects of animal biology and are attractive targets for biomarkers and therapeutics. However, comprehensively identifying the "secretome", along with their tissues of origin, remains extremely challenging. To address this, we employed multiple omics methods to define a tissue-secretome map of 535 blood plasma proteins derived from specific cell-types and organs in Drosophila melanogaster. This map was enabled by methodological improvements including a collection of transgenic flies to label endogenous secreted proteins in 10 major tissue types, large-scale blood isolation, whole animal snRNA-seq, and a collection of 40 knock-in strains. Using this map, we discover surprising findings about circulating proteins: most originate from specific tissues including unusual sources (e.g. glia), many are uncharacterized, and some are shed ectodomains of transmembrane proteins. In addition, in vivo experiments revealed circulating proteins with remarkably tissue-specific expression, as well as proteins that are deposited in a different tissue from where they are synthesized, suggesting potential inter-organ functions. Our secretome map will serve as a resource to investigate blood protein function, discover novel tissue-tissue communication signals, and mine for homologues of human biomarkers.

genomics↗

Succinate dehydrogenase activity supports de novo purine synthesis

The de novo purine synthesis pathway is fundamental for nucleic acid production and cellular energetics, yet the role of mitochondrial metabolism in modulating this process remains underexplored. In many cancers, metabolic reprogramming supports rapid proliferation and survival, but the specific contributions of the tricarboxylic acid (TCA) cycle enzymes to nucleotide biosynthesis are not fully understood. Here, we demonstrate that the TCA cycle enzyme succinate dehydrogenase (SDH) is essential for maintaining optimal de novo purine synthesis in normal and cancer cells. Genetic or pharmacological inhibition of SDH markedly attenuates purine synthesis, leading to a significant reduction in cell proliferation. Mechanistically, SDH inhibition causes an accumulation of succinate, which directly impairs the purine biosynthetic pathway. In response, cancer cells compensate by upregulating the purine salvage pathway, a metabolic adaptation that represents a potential therapeutic vulnerability. Notably, co-inhibition of SDH and the purine salvage pathway induces pronounced antiproliferative and antitumoral effects in preclinical models. These findings not only reveal a signaling role for mitochondrial succinate in regulating nucleotide metabolism but also provide a promising therapeutic strategy for targeting metabolic dependencies in cancer.

biochemistry↗

The epigenetic factor Zrf1 regulates intestinal stem cell proliferation during midgut regeneration

Stem cells are essential for tissue maintenance and regeneration, balancing self-renewal and differentiation to support homeostasis and repair. Through an RNAi screen in the Drosophila midgut, we identified the epigenetic factor Zrf1 as a critical regulator of intestinal stem cell (ISC) proliferation. Functional analyses reveal that Zrf1 integrates inputs from multiple signaling pathways and interacts with components of the RNA-induced silencing complex (RISC). Zrf1 mutants exhibit elevated expression of transposable elements (TEs) and chromatin disruption, highlighting a broader role in genome stability. Single-nuclei RNA sequencing (snRNA-seq) further demonstrated the influence of Zrf1 on chromatin organization and TE repression, particularly within stem cell progenitors. Our findings suggest that Zrf1 is potentially a key chromatin regulator necessary for maintaining stem cell proliferation and genome integrity, enhancing our understanding of the molecular controls underlying stem cell function and chromatin dynamics.

genetics↗

Epstein-Barr Virus Latent Membrane Protein 1 Subverts IMPDH pathways to drive B-cell oncometabolism

Epstein-Barr virus (EBV) is associated with multiple types of cancers, many of which express the key viral oncoprotein Latent Membrane Protein 1 (LMP1). LMP1 is the only EBV-encoded protein whose expression is sufficient to transform both epithelial and B-cells. Although metabolism reprogramming is a cancer hallmark, much remains to be learned about how LMP1 alters lymphocyte oncometabolism. To gain insights into key B-cell metabolic pathways subverted by LMP1, we performed systematic metabolomic analyses on B cells with conditional LMP1 expression. This approach highlighted that LMP highly induces de novo purine biosynthesis, with xanthosine-5-P (XMP) as one of the most highly LMP1-upregulated metabolites. Consequently, IMPDH inhibition by mycophenolic acid (MPA) triggered apoptosis of LMP1-expressing EBV-transformed lymphoblastoid cell lines (LCL), a key model for EBV-driven immunoblastic lymphomas. Whereas MPA instead caused growth arrest of Burkitt lymphoma cells with the EBV latency I program, conditional LMP1 expression triggered their apoptosis. Although both IMPDH isozymes are expressed in LCLs, only IMPDH2 was critical for LCL survival, whereas both contributed to proliferation of Burkitt cells with the EBV latency I program. Both LMP1 C-terminal cytoplasmic tail domains critical for primary human B-cell transformation were important for XMP production, and each contributed to LMP1-driven Burkitt cell sensitivity to MPA. MPA also de-repressed EBV lytic antigens including LMP1 in latency I Burkitt cells, highlighting crosstalk between the purine biosynthesis pathway and the EBV epigenome. These results suggest novel oncometabolism-based therapeutic approaches to LMP1-driven lymphomas. IMPORTANCEAltered metabolism is a hallmark of cancer, yet much remains to be learned about how EBV rewires host cell metabolism to support multiple malignancies. While the oncogene LMP1 is the only EBV-encoded gene that is sufficient to transform murine B-cells and rodent fibroblasts, knowledge has remained incomplete about how LMP1 alters host cell oncometabolism to aberrantly drive infected B-cell growth and survival. Likewise, it has remained unknown whether LMP1 expression creates metabolic vulnerabilities that can be targeted by small molecule approaches to trigger EBV-transformed B-cell programmed cell death. We therefore used metabolomic profiling to define how LMP1 signaling remodels the B-cell metabolome. We found that LMP1 upregulated purine nucleotide biosynthesis, likely to meet increased demand. Consequently, LMP1 expression sensitized Burkitt B-cells to growth arrest upon inosine monophosphate dehydrogenase blockade. Thus, while LMP1 itself may not be a therapeutic target, its signaling induces dependence on downstream druggable host cell nucleotide metabolism enzymes, suggesting rational therapeutic approaches.

microbiology↗

Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved post-translational modification that facilitates rapid and reversible modulation of enzyme activity, localization or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in non-tumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the epidermal growth factor receptor (EGFR) inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (PKM, PGAM1) and two novel (GSTP1, GLUD1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference (CRISPRi) system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous re-expression of wild-type, phosphomutant or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance.

biochemistry↗

Identification of high sugar diet-induced dysregulated metabolic pathways in muscle using tissue-specific metabolic models in Drosophila

Individual tissues perform highly specialized metabolic functions to maintain whole-body metabolic homeostasis. Although Drosophila serves as a powerful model for studying human metabolic diseases, modeling tissue-specific metabolism has been limited in this organism. To address this gap, we reconstruct 32 tissue-specific genome-scale metabolic models (GEMs) by integrating a curated Drosophila metabolic network with pseudo-bulk single-nuclei transcriptomics data, revealing distinct metabolic network structures and subsystem coverage across tissues. We validate enriched pathways identified through tissue-specific GEMs, particularly in muscle and fat body, using metabolomics and pathway analysis. Moreover, to demonstrate the utility in disease modeling, we apply muscle-GEM to investigate high sugar diet (HSD)-induced metabolic dysregulation. Constraint-based semi-quantitative flux and sensitivity analyses identify altered NAD(H)-dependent reactions and distributed control of glycolytic flux, including GAPDH. This prediction is further validated through in vivo 13C-glucose isotope tracing study. Notably, decreased glycolytic flux, including GAPDH, is linked to increased redox modifications. Finally, our pathway-level flux analyses identify dysregulation in fructose metabolism. Together, this work establishes a quantitative framework for tissue-specific metabolic modeling in Drosophila, demonstrating its utility for identifying dysregulated reactions and pathways in muscle in response to HSD.

systems biology↗

HHIP protein interactions in lung cells provide insight into COPD pathogenesis

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. The primary causes of COPD are environmental, including cigarette smoking; however, genetic susceptibility also contributes to COPD risk. Genome-Wide Association Studies (GWASes) have revealed more than 80 genetic loci associated with COPD, leading to the identification of multiple COPD GWAS genes. However, the biological relationships between the identified COPD susceptibility genes are largely unknown. Genes associated with a complex disease are often in close network proximity, i.e. their protein products often interact directly with each other and/or similar proteins. In this study, we use affinity purification mass spectrometry (AP-MS) to identify protein interactions with HHIP, a well-established COPD GWAS gene which is part of the sonic hedgehog pathway, in two disease-relevant lung cell lines (IMR90 and 16HBE). To better understand the network neighborhood of HHIP, its proximity to the protein products of other COPD GWAS genes, and its functional role in COPD pathogenesis, we create HUBRIS, a protein-protein interaction network compiled from 8 publicly available databases. We identified both common and cell type-specific protein-protein interactors of HHIP. We find that our newly identified interactions shorten the network distance between HHIP and the protein products of several COPD GWAS genes, including DSP, MFAP2, TET2, and FBLN5. These new shorter paths include proteins that are encoded by genes involved in extracellular matrix and tissue organization. We found and validated interactions to proteins that provide new insights into COPD pathobiology, including CAVIN1 (IMR90) and TP53 (16HBE). The newly discovered HHIP interactions with CAVIN1 and TP53 implicate HHIP in response to oxidative stress.

genetics↗