RNA editing of microtubule-associated protein tau circular RNAs promotes their translation and tau tangle formation.

Aggregation of the microtubule-associated protein tau characterizes tauopathies, including Alzheimer's disease and frontotemporal lobar degeneration (FTLD-Tau). Gene expression regulation of tau is complex and incompletely understood. Here we report that the human tau gene (MAPT) generates two circular RNAs (circRNAs) through backsplicing of exon 12 to either exon 7 (12→7 circRNA) or exon 10 (12→10 circRNA). Both circRNAs lack stop codons. The 12→7 circRNA contains one start codon and is translated in a rolling circle, generating a protein consisting of multimers of the microtubule-binding repeats R1-R4. For the 12→10 circRNA, a start codon can be introduced by two FTLD-Tau mutations, generating a protein consisting of multimers of the microtubule-binding repeats R2-R4, suggesting that mutations causing FTLD may act in part through tau circRNAs. Adenosine to inosine RNA editing dramatically increases translation of circRNAs and, in the 12→10 circRNA, RNA editing generates a translational start codon by changing AUA to AUI. Circular tau proteins self-aggregate and promote aggregation of linear tau proteins. Our data indicate that adenosine to inosine RNA editing initiates translation of human circular tau RNAs, which may contribute to tauopathies.

A JAK/STAT-mediated inflammatory signaling cascade drives oncogenesis in AF10-rearranged AML.

Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.

Factor VIII exhibits chaperone-dependent and glucose-regulated reversible amyloid formation in the endoplasmic reticulum.

Hemophilia A, an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII), is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low-level secretion from mammalian host cells. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress, and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here, we identified intrinsic and extrinsic factors that cause FVIII to form aggregates. We show that FVIII forms amyloid-like fibrils within the ER lumen upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperone families and their cochaperones, immunoglobulin binding protein (BiP) and calnexin/calreticulin, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed ß-sheet polymerization, and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein aggregation in general and have implication for ongoing hemophilia A gene-therapy clinical trials.

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. METHODS: We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. RESULTS: EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. CONCLUSIONS: HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Overcoming the blood-brain barrier by Annexin A1-binding peptide to target brain tumours.

BACKGROUND: Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood-brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse. METHODS: (1) A dual-tumour model was generated by inoculating luciferase-expressing melanoma B16 cell line, B16-Luc, into the brain and under the skin of syngeneic C57BL/6 mice. IF7C(RR)-SN38 was injected intravenously daily at 7.0 µmoles/kg and growth of tumours was assessed by chemiluminescence using an IVIS imager. A similar dual-tumour model was generated with the C6-Luc line in immunocompromised SCID mice. (2) IF7C(RR)-SN38 formulated with 10% Solutol HS15 was injected intravenously daily at 2.5 µmoles/kg into two brain tumour mouse models: B16-Luc cells in C57BL/6 mice, and C6-Luc cells in nude mice. RESULTS: (1) Daily IF7C(RR)-SN38 injection suppressed tumour growth regardless of cell lines or mouse strains. (2) Daily injection of Solutol-formulated IF7C(RR)-SN38 led into complete disappearance of B16-Luc brain tumour in C57BL/6 mice, whereas this did not occur in C6-Luc in nude mice. CONCLUSIONS: IF7C(RR)-SN38 crosses the blood-brain barrier and suppresses growth of brain tumours in mouse models. Solutol HS15-formulated IF7C(RR)-SN38 may have promoted an antitumour immune response.

HDAC7 promotes cardiomyocyte proliferation by suppressing Myocyte Enhancer Factor 2.

Postnatal mammalian cardiomyocytes (CMs) rapidly lose proliferative capacity and exit the cell cycle and undergo further differentiation and maturation. Cell cycle activation has been a major strategy to stimulate postnatal CM proliferation, albeit achieving modest effects. One impediment is that postnatal CMs may need to undergo dedifferentiation before proliferation, if not simultaneously. Here, we report that overexpression of Hdac7 in neonatal mouse CMs results in significant CM dedifferentiation and proliferation. Mechanistically, we show that HDAC7-mediated CM proliferation is contingent on dedifferentiation, which is accomplished through suppressing MEF2. Hdac7 overexpression in CM shifts the chromatin state from binding MEF2, which favors the differentiation transcriptional program to AP-1, which favors the proliferative transcriptional program. Further, we found that HDAC7 interacts with minichromosome maintenance complex (MCM) components to initiate cell cycle progression. Our findings reveal that HDAC7 promotes CM proliferation by its dual action on CM dedifferentiation and proliferation, uncovering a potential new strategy for heart regeneration/repair.

PSGL-1 attenuates early TCR signaling to suppress CD8+ T cell progenitor differentiation and elicit terminal CD8+ T cell exhaustion.

PSGL-1 (P-selectin glycoprotein-1) is a T cell-intrinsic checkpoint regulator of exhaustion with an unknown mechanism of action. Here, we show that PSGL-1 acts upstream of PD-1 and requires co-ligation with the T cell receptor (TCR) to attenuate activation of mouse and human CD8+ T cells and drive terminal T cell exhaustion. PSGL-1 directly restrains TCR signaling via Zap70 and maintains expression of the Zap70 inhibitor Sts-1. PSGL-1 deficiency empowers CD8+ T cells to respond to low-affinity TCR ligands and inhibit growth of PD-1-blockade-resistant melanoma by enabling tumor-infiltrating T cells to sustain an elevated metabolic gene signature supportive of increased glycolysis and glucose uptake to promote effector function. This outcome is coupled to an increased abundance of CD8+ T cell stem cell-like progenitors that maintain effector functions. Additionally, pharmacologic blockade of PSGL-1 curtails T cell exhaustion, indicating that PSGL-1 represents an immunotherapeutic target for PD-1-blockade-resistant tumors.

Proteomic analysis reveals dual requirement for Grb2 and PLCγ1 interactions for BCR-FGFR1-Driven 8p11 cell proliferation.

Translocation of Fibroblast Growth Factor Receptors (FGFRs) often leads to aberrant cell proliferation and cancer. The BCR-FGFR1 fusion protein, created by chromosomal translocation t(8;22)(p11;q11), contains Breakpoint Cluster Region (BCR) joined to Fibroblast Growth Factor Receptor 1 (FGFR1). BCR-FGFR1 represents a significant driver of 8p11 myeloproliferative syndrome, or stem cell leukemia/lymphoma, which progresses to acute myeloid leukemia or T-cell lymphoblastic leukemia/lymphoma. Mutations were introduced at Y177F, the binding site for adapter protein Grb2 within BCR; and at Y766F, the binding site for the membrane associated enzyme PLCγ1 within FGFR1. We examined anchorage-independent cell growth, overall cell proliferation using hematopoietic cells, and activation of downstream signaling pathways. BCR-FGFR1-induced changes in protein phosphorylation, binding partners, and signaling pathways were dissected using quantitative proteomics to interrogate the protein interactome, the phosphoproteome, and the interactome of BCR-FGFR1. The effects on BCR-FGFR1-stimulated cell proliferation were examined using the PLCγ1 inhibitor U73122, and the irreversible FGFR inhibitor futibatinib (TAS-120), both of which demonstrated efficacy. An absolute requirement is demonstrated for the dual binding partners Grb2 and PLCγ1 in BCR-FGFR1-driven cell proliferation, and new proteins such as ECSIT, USP15, GPR89, GAB1, and PTPN11 are identified as key effectors for hematopoietic transformation by BCR-FGFR1.

Vascular Proteome Responses Precede Organ Dysfunction in a Murine Model of Staphylococcus aureus Bacteremia.

Vascular dysfunction and organ failure are two distinct, albeit highly interconnected, clinical outcomes linked to morbidity and mortality in human sepsis. The mechanisms driving vascular and parenchymal damage are dynamic and display significant molecular cross talk between organs and tissues. Therefore, assessing their individual contribution to disease progression is technically challenging. Here, we hypothesize that dysregulated vascular responses predispose the organism to organ failure. To address this hypothesis, we have evaluated four major organs in a murine model of Staphylococcus aureus sepsis by combining in vivo labeling of the endothelial cell surface proteome, data-independent acquisition (DIA) mass spectrometry, and an integrative computational pipeline. The data reveal, with unprecedented depth and throughput, that a septic insult evokes organ-specific proteome responses that are highly compartmentalized, synchronously coordinated, and significantly correlated with the progression of the disease. These responses include abundant vascular shedding, dysregulation of the intrinsic pathway of coagulation, compartmentalization of the acute phase response, and abundant upregulation of glycocalyx components. Vascular cell surface proteome changes were also found to precede bacterial invasion and leukocyte infiltration into the organs, as well as to precede changes in various well-established cellular and biochemical correlates of systemic coagulopathy and tissue dysfunction. Importantly, our data suggest a potential role for the vascular proteome as a determinant of the susceptibility of the organs to undergo failure during sepsis. IMPORTANCE Sepsis is a life-threatening response to infection that results in immune dysregulation, vascular dysfunction, and organ failure. New methods are needed for the identification of diagnostic and therapeutic targets. Here, we took a systems-wide approach using data-independent acquisition (DIA) mass spectrometry to track the progression of bacterial sepsis in the vasculature leading to organ failure. Using a murine model of S. aureus sepsis, we were able to quantify thousands of proteins across the plasma and parenchymal and vascular compartments of multiple organs in a time-resolved fashion. We showcase the profound proteome remodeling triggered by sepsis over time and across these compartments. Importantly, many vascular proteome alterations precede changes in traditional correlates of organ dysfunction, opening a molecular window for the discovery of early markers of sepsis progression.

COG4 mutation in Saul-Wilson syndrome selectively affects secretion of proteins involved in chondrogenesis in chondrocyte-like cells.

Saul-Wilson syndrome is a rare skeletal dysplasia caused by a heterozygous mutation in COG4 (p.G516R). Our previous study showed that this mutation affected glycosylation of proteoglycans and disturbed chondrocyte elongation and intercalation in zebrafish embryos expressing the COG4p.G516R variant. How this mutation causes chondrocyte deficiencies remain unsolved. To analyze a disease-relevant cell type, COG4p.G516R variant was generated by CRISPR knock-in technique in the chondrosarcoma cell line SW1353 to study chondrocyte differentiation and protein secretion. COG4p.G516R cells display impaired protein trafficking and altered COG complex size, similar to SWS-derived fibroblasts. Both SW1353 and HEK293T cells carrying COG4p.G516R showed very modest, cell-type dependent changes in N-glycans. Using 3D culture methods, we found that cells carrying the COG4p.G516R variant made smaller spheroids and had increased apoptosis, indicating impaired in vitro chondrogenesis. Adding WT cells or their conditioned medium reduced cell death and increased spheroid sizes of COG4p.G516R mutant cells, suggesting a deficiency in secreted matrix components. Mass spectrometry-based secretome analysis showed selectively impaired protein secretion, including MMP13 and IGFBP7 which are involved in chondrogenesis and osteogenesis. We verified reduced expression of chondrogenic differentiation markers, MMP13 and COL10A1 and delayed response to BMP2 in COG4p.G516R mutant cells. Collectively, our results show that the Saul-Wilson syndrome COG4p.G516R variant selectively affects the secretion of multiple proteins, especially in chondrocyte-like cells which could further cause pleiotropic defects including hampering long bone growth in SWS individuals.

Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I.

SMIP004-7 is a small molecule inhibitor of mitochondrial respiration with selective in vivo anti-cancer activity through an as-yet unknown molecular target. We demonstrate here that SMIP004-7 targets drug-resistant cancer cells with stem-like features by inhibiting mitochondrial respiration complex I (NADH:ubiquinone oxidoreductase, complex I [CI]). Instead of affecting the quinone-binding site targeted by most CI inhibitors, SMIP004-7 and its cytochrome P450-dependent activated metabolite(s) have an uncompetitive mechanism of inhibition involving a distinct N-terminal region of catalytic subunit NDUFS2 that leads to rapid disassembly of CI. SMIP004-7 and an improved chemical analog selectively engage NDUFS2 in vivo to inhibit the growth of triple-negative breast cancer transplants, a response mediated at least in part by boosting CD4+ and CD8+ T cell-mediated immune surveillance. Thus, SMIP004-7 defines an emerging class of ubiquinone uncompetitive CI inhibitors for cell autonomous and microenvironmental metabolic targeting of mitochondrial respiration in cancer.

Transcription recycling assays identify PAF1 as a driver for RNA Pol II recycling.

RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states.

SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells.

Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle. ONE-SENTENCE SUMMARY: SARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.

Unbiased Profiling of the Human Proinsulin Biosynthetic Interaction Network Reveals a Role for Peroxiredoxin 4 in Proinsulin Folding.

The ß-cell protein synthetic machinery is dedicated to the production of mature insulin, which requires the proper folding and trafficking of its precursor, proinsulin. The complete network of proteins that mediate proinsulin folding and advancement through the secretory pathway, however, remains poorly defined. Here we used affinity purification and mass spectrometry to identify, for the first time, the proinsulin biosynthetic interaction network in human islets. Stringent analysis established a central node of proinsulin interactions with endoplasmic reticulum (ER) folding factors, including chaperones and oxidoreductases, that is remarkably conserved in both sexes and across three ethnicities. The ER-localized peroxiredoxin PRDX4 was identified as a prominent proinsulin-interacting protein. In ß-cells, gene silencing of PRDX4 rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress, while exogenous PRDX4 improved proinsulin folding. Moreover, proinsulin misfolding induced by oxidative stress or high glucose was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. Notably, islets from patients with type 2 diabetes (T2D) exhibited significantly higher levels of sulfonylated PRDX4 than islets from healthy individuals. In conclusion, we have generated the first reference map of the human proinsulin interactome to identify critical factors controlling insulin biosynthesis, ß-cell function, and T2D.

Proteomic atlas of organ vasculopathies triggered by Staphylococcus aureus sepsis.

Sepsis is a life-threatening condition triggered by a dysregulated host response to microbial infection resulting in vascular dysfunction, organ failure and death. Here we provide a semi-quantitative atlas of the murine vascular cell-surface proteome at the organ level, and how it changes during sepsis. Using in vivo chemical labeling and high-resolution mass spectrometry, we demonstrate the presence of a vascular proteome that is perfusable and shared across multiple organs. This proteome is enriched in membrane-anchored proteins, including multiple regulators of endothelial barrier functions and innate immunity. Further, we automated our workflows and applied them to a murine model of methicillin-resistant Staphylococcus aureus (MRSA) sepsis to unravel changes during systemic inflammatory responses. We provide an organ-specific atlas of both systemic and local changes of the vascular proteome triggered by sepsis. Collectively, the data indicates that MRSA-sepsis triggers extensive proteome remodeling of the vascular cell surfaces, in a tissue-specific manner.

Integrative proteomics and pharmacogenomics analysis of methylphenidate treatment response.

Transcriptomics and candidate gene/protein expression studies have indicated several biological processes modulated by methylphenidate (MPH), widely used in attention-deficit/hyperactivity disorder (ADHD) treatment. However, the lack of a differential proteomic profiling of MPH treatment limits the understanding of the most relevant mechanisms by which MPH exerts its pharmacological effects at the molecular level. Therefore, our aim is to investigate the MPH-induced proteomic alterations using an experimental design integrated with a pharmacogenomic analysis in a translational perspective. Proteomic analysis was performed using the cortices of Wistar-Kyoto rats, which were treated by gavage with MPH (2 mg/kg) or saline for two weeks (n = 6/group). After functional enrichment analysis of the differentially expressed proteins (DEP) in rats, the significant biological pathways were tested for association with MPH response in adults with ADHD (n = 189) using genome-wide data. Following MPH treatment in rats, 98 DEPs were found (P < 0.05 and FC < -1.0 or > 1.0). The functional enrichment analysis of the DEPs revealed 18 significant biological pathways (gene-sets) modulated by MPH, including some with recognized biological plausibility, such as those related to synaptic transmission. The pharmacogenomic analysis in the clinical sample evaluating these pathways revealed nominal associations for gene-sets related to neurotransmitter release and GABA transmission. Our results, which integrate proteomics and pharmacogenomics, revealed putative molecular effects of MPH on several biological processes, including oxidative stress, cellular respiration, and metabolism, and extended the results involving synaptic transmission pathways to a clinical sample. These findings shed light on the molecular signatures of MPH effects and possible biological sources of treatment response variability.

A Proteomic Variant Approach (ProVarA) for Personalized Medicine of Inherited and Somatic Disease.

The advent of precision medicine for genetic diseases has been hampered by the large number of variants that cause familial and somatic disease, a complexity that is further confounded by the impact of genetic modifiers. To begin to understand differences in onset, progression and therapeutic response that exist among disease-causing variants, we present the proteomic variant approach (ProVarA), a proteomic method that integrates mass spectrometry with genomic tools to dissect the etiology of disease. To illustrate its value, we examined the impact of variation in cystic fibrosis (CF), where 2025 disease-associated mutations in the CF transmembrane conductance regulator (CFTR) gene have been annotated and where individual genotypes exhibit phenotypic heterogeneity and response to therapeutic intervention. A comparative analysis of variant-specific proteomics allows us to identify a number of protein interactions contributing to the basic defects associated with F508del- and G551D-CFTR, two of the most common disease-associated variants in the patient population. We demonstrate that a number of these causal interactions are significantly altered in response to treatment with Vx809 and Vx770, small-molecule therapeutics that respectively target the F508del and G551D variants. ProVarA represents the first comparative proteomic analysis among multiple disease-causing mutations, thereby providing a methodological approach that provides a significant advancement to existing proteomic efforts in understanding the impact of variation in CF disease. We posit that the implementation of ProVarA for any familial or somatic mutation will provide a substantial increase in the knowledge base needed to implement a precision medicine-based approach for clinical management of disease.

A Transcript-Specific eIF3 Complex Mediates Global Translational Control of Energy Metabolism.

The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears to be conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer.