Jesse Rinehart

The genetic code is conserved across all domains of life, yet exceptions have revealed variations in codon assignments and associated translation factors^1,2,3. Inspired by this natural malleability, synthetic approaches have demonstrated whole-genome replacement of synonymous codons to construct genomically recoded organisms (GROs)^4,5 with alternative genetic codes. However, no efforts have fully leveraged translation factor plasticity and codon degeneracy to compress translation function to a single codon and assess the possibility of a non-degenerate code. Here we describe construction and characterization of Ochre, a GRO that fully compresses a translational function into a single codon. We replaced 1,195 TGA stop codons with the synonymous TAA in ∆TAG Escherichia coli C321.∆A⁴. We then engineered release factor 2 (RF2) and tRNA^Trp to mitigate native UGA recognition, translationally isolating four codons for non-degenerate functions. Ochre thus utilizes UAA as the sole stop codon, with UGG encoding tryptophan and UAG and UGA reassigned for multi-site incorporation of two distinct non-standard amino acids into single proteins with more than 99% accuracy. Ochre fully compresses degenerate stop codons into a single codon and represents an important step toward a 64-codon non-degenerate code that will enable precise production of multi-functional synthetic proteins with unnatural encoded chemistries and broad utility in biotechnology and biotherapeutics.

Na ⁺ –K ⁺ –Cl ⁻ cotransporters functions as an anion importers, regulating trans-epithelial chloride secretion, cell volume, and renal salt reabsorption. Loop diuretics, including furosemide, bumetanide, and torsemide, antagonize both NKCC1 and NKCC2, and are first-line medicines for the treatment of edema and hypertension. NKCC1 activation by the molecular crowding sensing WNK kinases is critical if cells are to combat shrinkage during hypertonic stress; however, how phosphorylation accelerates NKCC1 ion transport remains unclear. Here, we present co-structures of phospho-activated NKCC1 bound with furosemide, bumetanide, or torsemide showing that furosemide and bumetanide utilize a carboxyl group to coordinate and co-occlude a K ⁺ , whereas torsemide encroaches and expels the K ⁺ from the site. We also found that an amino-terminal segment of NKCC1, once phosphorylated, interacts with the carboxyl-terminal domain, and together, they engage with intracellular ion exit and appear to be poised to facilitate rapid ion translocation. Together, these findings enhance our understanding of NKCC-mediated epithelial ion transport and the molecular mechanisms of its inhibition by loop diuretics.

The Na⁺-Cl⁻ cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated renal salt retention and have been cornerstones for treating hypertension and edema since the 1950s. Here we determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they fit into an orthosteric site and occlude the NCC ion translocation pathway. Aberrant NCC activation by the WNKs-SPAK kinase cascade underlies Familial Hyperkalemic Hypertension, but it remains unknown whether/how phosphorylation transforms the NCC structure to accelerate ion translocation. We show that an intracellular amino-terminal motif of NCC, once phosphorylated, associates with the carboxyl-terminal domain, and together, they interact with the transmembrane domain. These interactions suggest a phosphorylation-dependent allosteric network that directly influences NCC ion translocation.

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.

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.

Fitness landscapes are models of the sequence space of a genetic element that map how each sequence corresponds to its activity and can be used to guide laboratory evolution. The ribosome is a macromolecular machine that is essential for protein synthesis in all organisms. Because of the prevalence of dominant lethal mutations, a comprehensive fitness landscape of the ribosomal peptidyl transfer center (PTC) has not yet been attained. Here, we develop a method to functionally map an orthogonal tethered ribosome (oRiboT), which permits complete mutagenesis of nucleotides located in the PTC and the resulting epistatic interactions. We found that most nucleotides studied showed flexibility to mutation, and identified epistatic interactions between them, which compensate for deleterious mutations. This work provides a basis for a deeper understanding of ribosome function and malleability and could be used to inform design of engineered ribosomes with applications to synthesize next-generation biomaterials and therapeutics.

Metabolic reprogramming is a hallmark of malignant transformation, and loss of isozyme diversity (LID) contributes to this process. Isozymes are distinct proteins that catalyze the same enzymatic reaction but can have different kinetic characteristics, subcellular localization, and tissue specificity. Cancer-dominant isozymes that catalyze rate-limiting reactions in critical metabolic processes represent potential therapeutic targets. Here, we examined the isozyme expression patterns of 1,319 enzymatic reactions in 14 cancer types and their matching normal tissues using The Cancer Genome Atlas mRNA expression data to identify isozymes that become cancer-dominant. Of the reactions analyzed, 357 demonstrated LID in at least one cancer type. Assessment of the expression patterns in over 600 cell lines in the Cancer Cell Line Encyclopedia showed that these reactions reflect cellular changes instead of differences in tissue composition; 50% of the LID-affected isozymes showed cancer-dominant expression in the corresponding cell lines. The functional importance of the cancer-dominant isozymes was assessed in genome-wide CRISPR and RNAi loss-of-function screens: 17% were critical for cell proliferation, indicating their potential as therapeutic targets. Lists of prioritized novel metabolic targets were developed for 14 cancer types; the most broadly shared and functionally validated target was acetyl-CoA carboxylase 1 (ACC1). Small molecule inhibition of ACC reduced breast cancer viability in vitro and suppressed tumor growth in cell line– and patient-derived xenografts in vivo. Evaluation of the effects of drug treatment revealed significant metabolic and transcriptional perturbations. Overall, this systematic analysis of isozyme expression patterns elucidates an important aspect of cancer metabolic plasticity and reveals putative metabolic vulnerabilities.Significance:This study exploits the loss of metabolic isozyme diversity common in cancer and reveals a rich pool of potential therapeutic targets that will allow the repurposing of existing inhibitors for anticancer therapy.See related commentary by Kehinde and Parker, p. 1695

Supplementary Table from Comprehensive Analysis of Metabolic Isozyme Targets in Cancer