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Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli C321.∆A. We found that defects in isoleucine biosynthesis and release factor activity, caused by mutations extant in all K-12 lineage strains, elicited profound fitness impairments in C321.∆A, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these and other C321.∆A-specific mutations, we engineered C321.∆A strains with doubling time reductions of 17% and 42% in rich and minimal medium, respectively, compared to ancestral C321. Strains with improved growth kinetics also demonstrated enhanced ribosomal non-standard amino acid incorporation capabilities. Proteomic analysis indicated that C321.∆A lacks the ability to regulate essential amino acid and nucleotide biosynthesis pathways, and that targeted mutation reversion restored regulatory capabilities. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other engineered microbes.

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During cancer pathogenesis, cell-generated mechanical stresses lead to dramatic alterations in the mechanical and organizational properties of the extracellular matrix (ECM). To date, contraction of the ECM is largely attributed to local mechanical stresses generated during cell invasion, but the impact of "elastocapillary" effects from surface tension on the tumor periphery has not been examined. Here, we embed glioblastoma cell spheroids within collagen gels, as a model of tumors within the ECM. We then modulate the surface tension of the spheroids, such that the spheroid contracts or expands. Surprisingly, in both cases, at the far-field, the ECM is contracted toward the spheroids prior to cellular migration from the spheroid into the ECM. Through computational simulation, we demonstrate that contraction of the ECM arises from a balance of spheroid surface tension, cell-ECM interactions, and time-dependent, poroelastic effects of the gel. This leads to the accumulation of ECM near the periphery of the spheroid and the contraction of the ECM without regard to the expansion or contraction of the spheroid. These results highlight the role of tissue-level surface stresses and fluid flow within the ECM in the regulation of cell-ECM interactions.

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The invasive potential of glioblastoma cells is attributed to large changes in pressure and volume, driven by diverse elements, including the cytoskeleton and ion cotransporters.  However, how the cell actuates changes in pressure and volume in confinement, and how these changes contribute to invasive motion is unclear. Here, we inhibited SPAK activity, with known impacts on the cytoskeleton and cotransporter activity and explored its role on the migration of glioblastoma cells in confining microchannels to model invasive spread through brain tissue. First, we found that confinement altered cell shape, inducing a transition in morphology that resembled droplet interactions with a capillary vessel, from "wetting" (more adherent) at low confinement, to "nonwetting" (less adherent) at high confinement. This transition was marked by a change from negative to positive pressure by the cells to the confining walls, and an increase in migration speed. Second, we found that the SPAK pathway impacted the migration speed in different ways dependent upon the extent of wetting. For nonwetting cells, SPAK inhibition increased cell-surface tension and cotransporter activity. By contrast, for wetting cells, it also reduced myosin II and YAP phosphorylation. In both cases, membrane-to-cortex attachment is dramatically reduced. Thus, our results suggest that SPAK inhibition differentially coordinates cotransporter and cytoskeleton-induced forces, to impact glioblastoma migration depending on the extent of confinement.

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Over the past two decades, synthetic biological systems have revolutionized the study of cellular physiology. The ability to site-specifically incorporate biologically relevant non-standard amino acids using orthogonal translation systems (OTSs) has proven particularly useful, providing unparalleled access to cellular mechanisms modulated by post-translational modifications, such as protein phosphorylation. However, despite significant advances in OTS design and function, the systems-level biology of OTS development and utilization remains underexplored. In this study, we employ a phosphoserine OTS (pSerOTS) as a model to systematically investigate global interactions between OTS components and the cellular environment, aiming to improve OTS performance. Based on this analysis, we design OTS variants to enhance orthogonality by minimizing host process interactions and reducing stress response activation. Our findings advance understanding of system-wide OTS:host interactions, enabling informed design practices that circumvent deleterious interactions with host physiology while improving OTS performance and stability. Furthermore, our study emphasizes the importance of establishing a pipeline for systematically profiling OTS:host interactions to enhance orthogonality and mitigate mechanisms underlying OTS-mediated host toxicity.

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Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. The identification of phosphorylation events through proteomic surveillance has dramatically outpaced our capacity for functional assignment using traditional strategies, which often require knowledge of the upstream kinase a priori. The development of phospho-amino-acid-specific orthogonal translation systems, evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs that enable co-translational insertion of a phospho-amino acids, has rapidly improved our ability to assess the physiological function of phosphorylation by providing kinase-independent methods of phosphoprotein production. Despite this utility, broad deployment has been hindered by technical limitations and an inability to reconstruct complex phopho-regulatory networks. Here, we address these challenges by optimizing genetically encoded phosphothreonine translation to characterize phospho-dependent kinase activation mechanisms and, subsequently, develop a multi-level protein interaction platform to directly assess the overlap of kinase and phospho-binding protein substrate networks with phosphosite-level resolution.

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Interactions between proteins from intracellular pathogens and host proteins in an infected cell are often mediated by post-translational modifications encoded in the host proteome. Identifying protein modifications, such as phosphorylation, that dictate these interactions remains a defining challenge in unraveling the molecular mechanisms of pathogenesis. We have developed a platform in engineered bacteria that displays over 110,000 phosphorylated human proteins coupled to a fluorescent reporter system capable of identifying the host-pathogen interactome of phosphoproteins (H-PIP). This resource broadly enables cell-type independent interrogation and discovery of proteins from intracellular pathogens capable of binding phosphorylated human proteins. As an example of the H-PIP platform, we generated a unique, high-resolution SARS-CoV-2 interaction network which expanded our knowledge of viral protein function and identified understudied areas of host pathology.

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Mass-spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground-truth phosphorylation positions. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides that is inexpensive, regenerable, and diverse, with precisely known positions of phosphorylation. We demonstrate possible uses of iSPI, including use as a phosphopeptide standard, a tool to evaluate and optimize phosphorylation-site localization algorithms, and a benchmark to compare performance across data analysis pipelines. We also present AScorePro, an updated version of the AScore algorithm specifically optimized for phosphorylation-site localization in higher energy fragmentation spectra, and the FLR viewer, a web tool for phosphorylation-site localization, to enable community use of the iSPI resource. iSPI and its associated data constitute a useful, multi-purpose resource for the phosphoproteomics community.

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Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.

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Bacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation (phenylalanyl-tRNA synthetase [PheRS]) were used to study the effects of quality control on antibiotic persistence. Upon amino acid limitation, elevated PheRS quality control led to significant decreases in aminoacylated tRNAPhe accumulation and increased antibiotic persistence. This increase in antibiotic persistence was most pronounced (65-fold higher) when the relA-encoded tRNA-dependent stringent response was inactivated. The increase in persistence with elevated quality control correlated with ∼2-fold increases in the levels of the RNase MazF and the NTPase MazG and a 3-fold reduction in cellular NTP pools. These data reveal a mechanism for persister formation independent of the stringent response where reduced translation capacity, as indicated by reduced levels of aminoacylated tRNA, is accompanied by active reduction of cellular NTP pools which in turn triggers antibiotic persistence. IMPORTANCE Bacterial antibiotic persistence is a transient physiological state wherein cells become dormant and thereby evade being killed by antibiotics. Once the antibiotic is removed, bacterial persisters are able to resuscitate and repopulate. It is thought that antibiotic bacterial persisters may cause reoccurring infections in the clinical setting. The molecular triggers and pathways that cause bacteria to enter into the persister state are not fully understood. Our results suggest that accumulation of deacylated tRNA is a trigger for antibiotic persistence independent of the RelA-dependent stringent response, a pathway thought to be required for persistence in many organisms. Overall, this provides a mechanism where changes in translation quality control in response to physiological cues can directly modulate bacterial persistence.

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ß-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

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Mechanisms have evolved to prevent errors in replication, transcription, and translation of genetic material, with translational errors occurring most frequently. Errors in protein synthesis can occur at two steps, during tRNA aminoacylation and ribosome decoding. Recent advances in protein mass spectrometry have indicated that previous reports of translational errors have potentially underestimated the frequency of these events, but also that the majority of translational errors occur during ribosomal decoding, suggesting that aminoacylation errors are evolutionarily less tolerated. Despite that interpretation, there is evidence that some aminoacylation errors may be regulated, and thus provide a benefit to the cell, while others are clearly detrimental. Here, we show that while it has been suggested that regulated Thr-to-Ser substitutions may be beneficial, there is a threshold beyond which these errors are detrimental. In contrast, we show that errors mediated by alanyl-tRNA synthetase (AlaRS) are not well tolerated and induce a global stress response that leads to gross perturbation of the Escherichia coli proteome, with potentially catastrophic effects on fitness and viability. Tolerance for Ala mistranslation appears to be much lower than with other translational errors, consistent with previous reports of multiple proofreading mechanisms targeting mischarged tRNAAla These results demonstrate the essential role of aminoacyl-tRNA proofreading in optimizing cellular fitness and suggest that any potentially beneficial effects of mistranslation may be confined to specific amino acid substitutions.IMPORTANCE Errors in protein synthesis have historically been assumed to be detrimental to the cell. While there are many reports that translational errors are consequential, there is a growing body of evidence that some mistranslation events may be tolerated or even beneficial. Using two models of mistranslation, we compare the direct phenotypic effects of these events in Escherichia coli This work provides insight into the threshold for tolerance of specific mistranslation events that were previously predicted to be broadly neutral to proteome integrity. Furthermore, these data reveal the effects of mistranslation beyond the general unfolded stress response, leading to global translational reprogramming.

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Cellular signaling and regulatory cascades often rely on post-translational modification of proteins, particularly phosphorylation, to quickly and effectively relay signals from a variety of inputs. Numerous kinases, the effectors of phosphorylation, and kinase networks have been implicated in human diseases. Until recently, an inability to produce high yields of physiologically phosphorylated proteins has proven to be a substantial barrier toward our understanding of many enzymatic processes. Orthogonal translation systems provide the means to overcome many of these limitations by enabling site-specific incorporation of phosphorylated amino acids into recombinantly expressed proteins. Site-by-site, combinatorial assessment of phosphorylation site function is unique to orthogonal translation system based approaches and offers unmatched precision in the study of PTM-enzymology, extending well beyond the scope of kinase biology.

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The aminoacylation status of the cellular tRNA pool regulates both general amino acid control (GAAC) and target of rapamycin (TOR) stress response pathways in yeast. Consequently, fidelity of translation at the level of aminoacyl-tRNA synthesis plays a central role in determining accuracy and sensitivity of stress responses. To investigate effects of translational quality control (QC) on cell physiology under stress conditions, phenotypic microarray analyses were used to identify changes in QC deficient cells. Nitrogen source growth assays showed QC deficient yeast grew differently compared to WT. The QC deficient strain was more tolerant to caffeine treatment than wild type through altered interactions with the TOR and GAAC pathways. Increased caffeine tolerance of the QC deficient strain was consistent with the observation that the activity of Gln3p, a transcription factor controlled by the TOR pathway, is decreased in the QC deficient strain compared to WT. GCN4 translation, which is typically repressed in the absence of nutritional stress, was enhanced in the QC deficient strain through TOR inhibition. QC did not impact cell cycle regulation; however, the chronological lifespan of QC deficient yeast strains decreased compared to wild type, likely due to translational errors and alteration of the TOR-associated regulon. These findings support the idea that changes in translational fidelity provide a mechanism of cellular adaptation by modulating TOR activity. This, in turn, supports a central role for aminoacyl-tRNA synthesis QC in the integrated stress response by maintaining the proper aa-tRNA pools necessary to coordinate the GAAC and TOR.

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Faithful translation of mRNA into the corresponding polypeptide is a complex multistep process, requiring accurate amino acid selection, transfer RNA (tRNA) charging and mRNA decoding on the ribosome. Key players in this process are aminoacyl-tRNA synthetases (aaRSs), which not only catalyse the attachment of cognate amino acids to their respective tRNAs, but also selectively hydrolyse incorrectly activated non-cognate amino acids and/or misaminoacylated tRNAs. This aaRS proofreading provides quality control checkpoints that exclude non-cognate amino acids during translation, and in so doing helps to prevent the formation of an aberrant proteome. However, despite the intrinsic need for high accuracy during translation, and the widespread evolutionary conservation of aaRS proofreading pathways, requirements for translation quality control vary depending on cellular physiology and changes in growth conditions, and translation errors are not always detrimental. Recent work has demonstrated that mistranslation can also be beneficial to cells, and some organisms have selected for a higher degree of mistranslation than others. The aims of this Review Article are to summarize the known mechanisms of protein translational fidelity and explore the diversity and impact of mistranslation events as a potentially beneficial response to environmental and cellular stress.

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Amino acid starvation activates the protein kinase Gcn2p, leading to changes in gene expression and translation. Gcn2p is activated by deacylated tRNA, which accumulates when tRNA aminoacylation is limited by lack of substrates or inhibition of synthesis. Pairing of amino acids and deacylated tRNAs is catalyzed by aminoacyl-tRNA synthetases, which use quality control pathways to maintain substrate specificity. Phenylalanyl-tRNA synthetase (PheRS) maintains specificity via an editing pathway that targets non-cognate Tyr-tRNAPhe. While the primary role of aaRS editing is to prevent misaminoacylation, we demonstrate editing of misaminoacylated tRNA is also required for detection of amino acid starvation by Gcn2p. Ablation of PheRS editing caused accumulation of Tyr-tRNAPhe (5%), but not deacylated tRNAPhe during amino acid starvation, limiting Gcn2p kinase activity and suppressing Gcn4p-dependent gene expression. While the PheRS-editing ablated strain grew 50% slower and displayed a 27-fold increase in the rate of mistranslation of Phe codons as Tyr compared to wild type, the increase in mistranslation was insufficient to activate an unfolded protein stress response. These findings show that during amino acid starvation a primary role of aaRS quality control is to help the cell mount an effective stress response, independent of the role of editing in maintaining translational accuracy.

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Ribosomal protein synthesis results in the genetically programmed incorporation of amino acids into a growing polypeptide chain. Faithful amino acid incorporation that accurately reflects the genetic code is critical to the structure and function of proteins as well as overall proteome integrity. Errors in protein synthesis are generally detrimental to cellular processes yet emerging evidence suggest that proteome diversity generated through mistranslation may be beneficial under certain conditions. Cumulative translational error rates have been determined at the organismal level, however codon specific error rates and the spectrum of misincorporation errors from system to system remain largely unexplored. In particular, until recently technical challenges have limited the ability to detect and quantify comparatively rare amino acid misincorporation events, which occur orders of magnitude less frequently than canonical amino acid incorporation events. We now describe a technique for the quantitative analysis of amino acid incorporation that provides the sensitivity necessary to detect mistranslation events during translation of a single codon at frequencies as low as 1 in 10,000 for all 20 proteinogenic amino acids, as well as non-proteinogenic and modified amino acids. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

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Amino acid misincorporation during protein synthesis occurs due to misacylation of tRNAs or defects in decoding at the ribosome. While misincorporation of amino acids has been observed in a variety of contexts, less work has been done to directly assess the extent to which specific tRNAs are misacylated in vivo, and the identity of the misacylated amino acid moiety. Here we describe tRNA isoacceptor specific aminoacylation profiling (ISAP), a method to identify and quantify the amino acids attached to a tRNA species in vivo. ISAP allows compilation of aminoacylation profiles for specific isoacceptors tRNAs. To demonstrate the efficacy and broad applicability of ISAP, tRNAPhe and tRNATyr species were isolated from total aminoacyl-tRNA extracted from both yeast and Escherichia coli. Isolated aminoacyl-tRNAs were washed until free of detectable unbound amino acid and subsequently deacylated. Free amino acids from the deacylated fraction were then identified and quantified by mass spectrometry. Using ISAP allowed quantification of the effects of quality control on the accumulation of misacylated tRNA species under different growth conditions.

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Mutations in mitochondrial aminoacyl-tRNA synthetases are an increasingly recognized cause of human diseases, often arising in individuals with compound heterozygous mutations and presenting with system-specific phenotypes, frequently neurologic. FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase (mtPheRS), perturbations of which have been reported in 6 cases of an infantile, lethal disease with refractory epilepsy and progressive myoclonus. Here the authors report the case of juvenile onset refractory epilepsy and progressive myoclonus with compound heterozygous FARS2 mutations. The authors describe the clinical course over 6 years of care at their institution and diagnostic studies including electroencephalogram (EEG), brain magnetic resonance imaging (MRI), serum and cerebrospinal fluid analyses, skeletal muscle biopsy histology, and autopsy gross and histologic findings, which include features shared with Alpers-Huttenlocher syndrome, Leigh syndrome, and a previously published case of FARS2 mutation associated infantile onset disease. The authors also present structure-guided analysis of the relevant mutations based on published mitochondrial phenylalanyl transfer RNA synthetase and related protein crystal structures as well as biochemical analysis of the corresponding recombinant mutant proteins.

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Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that 'cut and paste' certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell-wall polysaccharides (xyloglucan, mannans, mixed-linkage ß-glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero-trans-ß-glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early-diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero-transglycosylation: its preferred donor substrates (cellulose or mixed-linkage ß-glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose-xyloglucan and mixed-linkage ß-glucan-xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter-linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better-known xyloglucan-acting homo-transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable 'green' technology for industrially manipulating biomass.

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During mRNA decoding at the ribosome, deviations from stringent codon identity, or "mistranslation," are generally deleterious and infrequent. Observations of organisms that decode some codons ambiguously, and the discovery of a compensatory increase in mistranslation frequency to combat environmental stress have changed the way we view "errors" in decoding. Modern tools for the study of the frequency and phenotypic effects of mistranslation can provide quantitative and sensitive measurements of decoding errors that were previously inaccessible. Mistranslation with non-protein amino acids, in particular, is an enticing prospect for new drug therapies and the study of molecular evolution.

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