TIMP1 is an early biomarker for detection and prognosis of lung cancer.

BACKGROUND: Lung cancer remains the major cause of cancer-related deaths worldwide. Early stages of lung cancer are characterized by long asymptomatic periods that are ineffectively identified with the current screening programs. This deficiency represents a lost opportunity to improve the overall survival of patients. Serum biomarkers are among the most effective strategies for cancer screening and follow up. METHODS: Using bead-based multiplexing assays we screened plasma and tumours of the KrasG12D/+; Lkb1f/f (KL) mouse model of lung cancer for cytokines that could be used as biomarkers. We identified tissue inhibitor of metalloproteinase 1 (TIMP1) as an early biomarker and validated this finding in the plasma of lung cancer patients. We used immunohistochemistry (IHC), previously published single-cell RNA-seq and bulk RNA-seq data to assess the source and expression of TIMP1in the tumour. The prognostic value of TIMP1 was assessed using publicly available human proteomic and transcriptomic databases. RESULTS: We found that TIMP1 is a tumour-secreted protein with high sensitivity and specificity for aggressive cancer, even at early stages in mice. We showed that TIMP1 levels in the tumour and serum correlate with tumour burden and worse survival in mice. We validated this finding using clinical samples from our institution and publicly available human proteomic and transcriptomic databases. These data support the finding that high tumour expression of TIMP1 correlates with an unfavorable prognosis in lung cancer patients. CONCLUSION: TIMP1 is a suitable biomarker for lung cancer detection.

Coupling substrate-trapping with proximity-labeling to identify protein tyrosine phosphatase PTP1B signaling networks.

The ability to define functional interactions between enzymes and their substrates is crucial for understanding biological control mechanisms; however, such methods face challenges in the transient nature and low stoichiometry of enzyme-substrate interactions. Now, we have developed an optimized strategy that couples substrate-trapping mutagenesis to proximity-labeling mass spectrometry for quantitative analysis of protein complexes involving the protein tyrosine phosphatase PTP1B. This methodology represents a significant shift from classical schemes; it is capable of being performed at near-endogenous expression levels and increasing stoichiometry of target enrichment without a requirement for stimulation of supraphysiological tyrosine phosphorylation levels or maintenance of substrate complexes during lysis and enrichment procedures. Advantages of this new approach are illustrated through application to PTP1B interaction networks in models of HER2-positive and Herceptin-resistant breast cancer. We have demonstrated that inhibitors of PTP1B significantly reduced proliferation and viability in cell-based models of acquired and de novo Herceptin resistance in HER2-positive breast cancer. Using differential analysis, comparing substrate-trapping to wild-type PTP1B, we have identified multiple unreported protein targets of PTP1B with established links to HER2-induced signaling and provided internal validation of method specificity through overlap with previously identified substrate candidates. Overall, this versatile approach can be readily integrated with evolving proximity-labeling platforms (TurboID, BioID2, etc.), and is broadly applicable across all PTP family members for the identification of conditional substrate specificities and signaling nodes in models of human disease.

Prostate tumor-induced stromal reprogramming generates Tenascin C that promotes prostate cancer metastasis through YAP/TAZ inhibition.

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions that enhance PCa progression. How tumor-induced bone formation enhances PCa progression is not known. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition by tumor-secreted bone morphogenetic protein 4 (BMP4). Here, we show that EC-to-OSB transition leads to changes in the tumor microenvironment that increases the metastatic potential of PCa cells. We found that conditioned medium (CM) from EC-OSB hybrid cells increases the migration, invasion, and survival of PC3-mm2 and C4-2B4 PCa cells. Quantitative mass spectrometry (Isobaric Tags for Relative and Absolute Quantitation) identified Tenascin C (TNC) as one of the major proteins secreted from EC-OSB hybrid cells. TNC expression in tumor-induced OSBs was confirmed by immunohistochemistry of MDA PCa-118b xenograft and human bone metastasis specimens. Mechanistically, BMP4 increases TNC expression in EC-OSB cells through the Smad1-Notch/Hey1 pathway. How TNC promotes PCa metastasis was next interrogated by in vitro and in vivo studies. In vitro studies showed that a TNC-neutralizing antibody inhibits EC-OSB-CM-mediated PCa cell migration and survival. TNC knockdown decreased, while the addition of recombinant TNC or TNC overexpression increased migration and anchorage-independent growth of PC3 or C4-2b cells. When injected orthotopically, PC3-mm2-shTNC clones decreased metastasis to bone, while C4-2b-TNC-overexpressing cells increased metastasis to lymph nodes. TNC enhances PCa cell migration through α5ß1 integrin-mediated YAP/TAZ inhibition. These studies elucidate that tumor-induced stromal reprogramming generates TNC that enhances PCa metastasis and suggest that TNC may be a target for PCa therapy.

PHAROH lncRNA regulates Myc translation in hepatocellular carcinoma via sequestering TIAR.

Hepatocellular carcinoma, the most common type of liver malignancy, is one of the most lethal forms of cancer. We identified a long non-coding RNA, Gm19705, that is overexpressed in hepatocellular carcinoma and mouse embryonic stem cells. We named this RNA Pluripotency and Hepatocyte Associated RNA Overexpressed in HCC, or PHAROH. Depletion of PHAROH impacts cell proliferation and migration, which can be rescued by ectopic expression of PHAROH. RNA-seq analysis of PHAROH knockouts revealed that a large number of genes with decreased expression contain a Myc motif in their promoter. MYC is decreased in knockout cells at the protein level, but not the mRNA level. RNA-antisense pulldown identified nucleolysin TIAR, a translational repressor, to bind to a 71-nt hairpin within PHAROH, sequestration of which increases MYC translation. In summary, our data suggest that PHAROH regulates MYC translation by sequestering TIAR and as such represents a potentially exciting diagnostic or therapeutic target in hepatocellular carcinoma.

Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

MaTAR25 lncRNA regulates the Tensin1 gene to impact breast cancer progression.

Misregulation of long non-coding RNA (lncRNA) genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 (MaTAR25), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B (PURB), and associating with a major downstream target gene Tensin1 (Tns1) to regulate its expression in trans. The Tns1 protein product is a critical component of focal adhesions linking signaling between the extracellular matrix and the actin cytoskeleton. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. We identify LINC01271 as the human ortholog of MaTAR25, and importantly, increased expression of LINC01271 is associated with poor patient prognosis and metastasis. Our findings demonstrate that LINC01271 represents a potential therapeutic target to alter breast cancer progression.

Mito-oncology agent: fermented extract suppresses the Warburg effect, restores oxidative mitochondrial activity, and inhibits in vivo tumor growth.

Mitochondrial dysfunction and significant changes in metabolic pathways accompany cancer development and are responsible for maintaining the tumor microenvironment. Normal mitochondria can trigger intrinsic apoptosis by releasing cytochrome c into the cytosol. The survival of malignant cells highly depends on the suppression of this function. We validated that A250, a highly purified fraction of fermented wheat germ extract (FWGE), increases the carbon flux into the mitochondria, the expression of key elements of the Krebs cycle and oxidative phosphorylation (OXPHOS). The increased respiratory chain activity is related to the mitochondria's ability to release cytochrome c into the cytosol, which triggers the apoptotic cascade. The 68% tumor growth inhibitory effect observed in the murine melanoma study is related to this effect, as proteomic analysis validated similar changes in mitochondrial protein levels in the isolated tumor tissue samples. Blood count data indicated that this effect was not accompanied by general toxicity. This study is significant, as it shows that a highly concentrated form of FWGE is an effective agent that increases normal mitochondrial functionality. The lack of hepatotoxic and general toxic effects makes A250 an excellent candidate targeting mitochondria function in cancer therapy.

Tryp-N: A Thermostable Protease for the Production of N-terminal Argininyl and Lysinyl Peptides.

Bottom-up proteomics is a mainstay in protein identification and analysis. These studies typically employ proteolytic treatment of biological samples to generate suitably sized peptides for tandem mass spectrometric (MS) analysis. In MS, fragmentation of peptides is largely driven by charge localization. Consequently, peptides with basic centers exclusively on their N-termini produce mainly b-ions. Thus, it was long ago realized that proteases that yield such peptides would be valuable proteomic tools for achieving simplified peptide fragmentation patterns and peptide assignment. Work by several groups has identified such proteases, however, structural analysis of these suggested that enzymatic optimization was possible. We therefore endeavored to find enzymes that could provide enhanced activity and versatility while maintaining specificity. Using these previously described proteases as informatic search templates, we discovered and then characterized a thermophilic metalloprotease with N-terminal specificity for arginine and lysine. This enzyme, dubbed Tryp-N, affords many advantages including improved thermostability, solvent and detergent tolerance, and rapid digestion time.

HYPERsol: High-Quality Data from Archival FFPE Tissue for Clinical Proteomics.

Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing an invaluable resource for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present high-yield protein extraction and recovery by direct solubilization (HYPERsol), an optimized workflow using ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936). When applied to archival samples, HYPERsol resulted in high-quality data from FFPE specimens in storage for up to 17 years, and may enable the discovery of new immunohistochemical markers.

Regulation of PTP1B activation through disruption of redox-complex formation.

We have identified a molecular interaction between the reversibly oxidized form of protein tyrosine phosphatase 1B (PTP1B) and 14-3-3ζ that regulates PTP1B activity. Destabilizing the transient interaction between 14-3-3ζ and PTP1B prevented PTP1B inactivation by reactive oxygen species and decreased epidermal growth factor receptor phosphorylation. Our data suggest that destabilizing the interaction between 14-3-3ζ and the reversibly oxidized and inactive form of PTP1B may establish a path to PTP1B activation in cells.

The glycan CA19-9 promotes pancreatitis and pancreatic cancer in mice.

Glycosylation alterations are indicative of tissue inflammation and neoplasia, but whether these alterations contribute to disease pathogenesis is largely unknown. To study the role of glycan changes in pancreatic disease, we inducibly expressed human fucosyltransferase 3 and ß1,3-galactosyltransferase 5 in mice, reconstituting the glycan sialyl-Lewisa, also known as carbohydrate antigen 19-9 (CA19-9). Notably, CA19-9 expression in mice resulted in rapid and severe pancreatitis with hyperactivation of epidermal growth factor receptor (EGFR) signaling. Mechanistically, CA19-9 modification of the matricellular protein fibulin-3 increased its interaction with EGFR, and blockade of fibulin-3, EGFR ligands, or CA19-9 prevented EGFR hyperactivation in organoids. CA19-9-mediated pancreatitis was reversible and could be suppressed with CA19-9 antibodies. CA19-9 also cooperated with the KrasG12D oncogene to produce aggressive pancreatic cancer. These findings implicate CA19-9 in the etiology of pancreatitis and pancreatic cancer and nominate CA19-9 as a therapeutic target.

Muller's Ratchet and Ribosome Degeneration in the Obligate Intracellular Parasites Microsporidia.

Microsporidia are fungi-like parasites that have the smallest known eukaryotic genome, and for that reason they are used as a model to study the phenomenon of genome decay in parasitic forms of life. Similar to other intracellular parasites that reproduce asexually in an environment with alleviated natural selection, Microsporidia experience continuous genome decay that is driven by Muller's ratchet-an evolutionary process of irreversible accumulation of deleterious mutations that lead to gene loss and the miniaturization of cellular components. Particularly, Microsporidia have remarkably small ribosomes in which the rRNA is reduced to the minimal enzymatic core. In this study, we analyzed microsporidian ribosomes to study an apparent impact of Muller's ratchet on structure of RNA and protein molecules in parasitic forms of life. Through mass spectrometry of microsporidian proteome and analysis of microsporidian genomes, we found that massive rRNA reduction in microsporidian ribosomes appears to annihilate the binding sites for ribosomal proteins eL8, eL27, and eS31, suggesting that these proteins are no longer bound to the ribosome in microsporidian species. We then provided an evidence that protein eS31 is retained in Microsporidia due to its non-ribosomal function in ubiquitin biogenesis. Our study illustrates that, while Microsporidia carry the same set of ribosomal proteins as non-parasitic eukaryotes, some ribosomal proteins are no longer participating in protein synthesis in Microsporidia and they are preserved from genome decay by having extra-ribosomal functions. More generally, our study shows that many components of parasitic cells, which are identified by automated annotation of pathogenic genomes, may lack part of their biological functions due to continuous genome decay.

DPM-1001 decreased copper levels and ameliorated deficits in a mouse model of Wilson's disease.

The levels of copper, which is an essential element in living organisms, are under tight homeostatic control. Inactivating mutations in ATP7B, a P-type Cu-ATPase that functions in copper excretion, promote aberrant accumulation of the metal, primarily the in liver and brain. This condition underlies Wilson's disease, a severe autosomal recessive disorder characterized by profound hepatic and neurological deficits. Current treatment regimens rely on the use of broad specificity metal chelators as "decoppering" agents; however, there are side effects that limit their effectiveness. Here, we present the characterization of DPM-1001 {methyl 4-[7-hydroxy-10,13-dimethyl-3-({4-[(pyridin-2-ylmethyl)amino]butyl}amino)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl] pentanoate} as a potent and highly selective chelator of copper that is orally bioavailable. Treatment of cell models, including fibroblasts derived from Wilson's disease patients, eliminated adverse effects associated with copper accumulation. Furthermore, treatment of the toxic milk mouse model of Wilson's disease with DPM-1001 lowered the levels of copper in the liver and brain, removing excess copper by excretion in the feces while ameliorating symptoms associated with the disease. These data suggest that it may be worthwhile to investigate DPM-1001 further as a new therapeutic agent for the treatment of Wilson's disease, with potential for application in other indications associated with elevated copper, including cancer and neurodegenerative diseases.

Error-prone protein synthesis in parasites with the smallest eukaryotic genome.

Microsporidia are parasitic fungi-like organisms that invade the interior of living cells and cause chronic disorders in a broad range of animals, including humans. These pathogens have the tiniest known genomes among eukaryotic species, for which they serve as a model for exploring the phenomenon of genome reduction in obligate intracellular parasites. Here we report a case study to show an apparent effect of overall genome reduction on the primary structure and activity of aminoacyl-tRNA synthetases, indispensable cellular proteins required for protein synthesis. We find that most microsporidian synthetases lack regulatory and eukaryote-specific appended domains and have a high degree of sequence variability in tRNA-binding and catalytic domains. In one synthetase, LeuRS, an apparent sequence degeneration annihilates the editing domain, a catalytic center responsible for the accurate selection of leucine for protein synthesis. Unlike accurate LeuRS synthetases from other eukaryotic species, microsporidian LeuRS is error-prone: apart from leucine, it occasionally uses its near-cognate substrates, such as norvaline, isoleucine, valine, and methionine. Mass spectrometry analysis of the microsporidium Vavraia culicis proteome reveals that nearly 6% of leucine residues are erroneously replaced by other amino acids. This remarkably high frequency of mistranslation is not limited to leucine codons and appears to be a general property of protein synthesis in microsporidian parasites. Taken together, our findings reveal that the microsporidian protein synthesis machinery is editing-deficient, and that the proteome of microsporidian parasites is more diverse than would be anticipated based on their genome sequences.

Mitochondrial Complex I Inhibitors Expose a Vulnerability for Selective Killing of Pten-Null Cells.

A hallmark of advanced prostate cancer (PC) is the concomitant loss of PTEN and p53 function. To selectively eliminate such cells, we screened cytotoxic compounds on Pten-/-;Trp53-/- fibroblasts and their Pten-WT reference. Highly selective killing of Pten-null cells can be achieved by deguelin, a natural insecticide. Deguelin eliminates Pten-deficient cells through inhibition of mitochondrial complex I (CI). Five hundred-fold higher drug doses are needed to obtain the same killing of Pten-WT cells, even though deguelin blocks their electron transport chain equally well. Selectivity arises because mitochondria of Pten-null cells consume ATP through complex V, instead of producing it. The resulting glucose dependency can be exploited to selectively kill Pten-null cells with clinically relevant CI inhibitors, especially if they are lipophilic. In vivo, deguelin suppressed disease in our genetically engineered mouse model for metastatic PC. Our data thus introduce a vulnerability for highly selective targeting of incurable PC with inhibitors of CI.

Proteomic profile of cystic fibrosis sputum cells in adults chronically infected with Pseudomonas aeruginosa.

Lung disease is the main cause of morbidity and mortality in cystic fibrosis (CF), and involves chronic infection and perturbed immune responses. Tissue damage is mediated mostly by extracellular proteases, but other cellular proteins may also contribute to damage through their effect on cell activities and/or release into sputum fluid by means of active secretion or cell death.We employed MudPIT (multidimensional protein identification technology) to identify sputum cellular proteins with consistently altered abundance in adults with CF, chronically infected with Pseudomonas aeruginosa, compared with healthy controls. Ingenuity Pathway Analysis, Gene Ontology, protein abundance and correlation with lung function were used to infer their potential clinical significance.Differentially abundant proteins relate to Rho family small GTPase activity, immune cell movement/activation, generation of reactive oxygen species, and dysregulation of cell death and proliferation. Compositional breakdown identified high abundance of proteins previously associated with neutrophil extracellular traps. Furthermore, negative correlations with lung function were detected for 17 proteins, many of which have previously been associated with lung injury.These findings expand our current understanding of the mechanisms driving CF lung disease and identify sputum cellular proteins with potential for use as indicators of disease status/prognosis, stratification determinants for treatment prescription or therapeutic targets.

NRF2 Promotes Tumor Maintenance by Modulating mRNA Translation in Pancreatic Cancer.

Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nfe2l2/Nrf2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.

Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.

BACKGROUND: In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. METHODS: To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. RESULTS: Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14. CONCLUSIONS: Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.