Nature chemical biology最新文献

More than half of the ~20,000 protein-encoding human genes have paralogs. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to subsets of paralogous proteins. Here we explore whether such covalent compound–cysteine interactions can be used to discover ligandable pockets in paralogs lacking the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we substituted the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide–CCNE1-N112C interaction into in vitro NanoBRET (bioluminescence resonance energy transfer) and in cellulo activity-based protein profiling assays capable of identifying compounds that reversibly inhibit both the N112C mutant and wild-type CCNE1:CDK2 (cyclin-dependent kinase 2) complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings, thus, show how electrophile–cysteine interactions mapped by chemical proteomics can extend the understanding of protein ligandability beyond covalent chemistry.

By coupling rPAL to enzymatic glycan cleavage and sequential window acquisition of all theoretical mass spectra (SWATH-MS), the team found that acp³U was the most enriched linker (although other putative linker substrates were identified). Importantly, knocking out DTWD2, an enzyme responsible for installing acp³U in tRNAs, reduced glycoRNA levels. This study provides not only a useful tool for glycoRNA detection, but also the first direct evidence of a covalent linkage between a secretory N-glycan and RNA, paving the way for future functional studies.

Original reference: Cell https://doi.org/10.1016/j.cell.2024.07.044 (2024)

The formation of pathological protein aggregates occurs in many neurodegenerative diseases, such as the aggregation of the protein tau in patients with Alzheimer’s disease. Removing these aggregates offers a possible route to treat these protein misfolding diseases; however, targeting the aggregated form of a protein while sparing the monomeric form, which is required for normal cellular function, remains a challenge. Now, Benn, Cheng et al. have shown that fusing an E3 ligase-containing protein to a target-specific nanobody enables the construction of RING–nanobody (R-Nb) degraders that can target and selectively remove proteins that have assembled into protein aggregates.

The E3 ubiquitin ligase tripartite motif-containing protein 21 (TRIM21) induces degradation by the clustering and cross-activation of the RING domains of TRIM21. The team hypothesized that the structure of fibrillar aggregates may enable dense clustering of R-Nb degraders and therefore activation of the degradation pathway, whereas monomeric tau would not induce clustering of R-Nbs and therefore would not induce degradation. Tuning the affinity of the nanobody could also affect clustering and thereby provides another route to mediate protein degradation.

N-acetyltaurine is a metabolite whose levels fluctuate with diet and exercise and that undergoes hydrolysis to form taurine and acetate. However, the enzymes that facilitate this reaction were not known. In an effort to address this, Wei et al. used liquid chromatography–mass spectrometry-based activity guided analysis of mouse tissues, detecting high N-acetyltaurine hydrolysis activity in kidney and liver tissues, with reduced N-acetyltaurine levels and a corresponding increase in taurine. Fractionation of kidney cytosol fractions combined with size exclusion chromatography identified a peak of activity. Proteomic analysis revealed a series of candidates with exogenous expression of phosphotriesterase-related (PTER), an orphan metal-dependent hydrolase, sufficient to increase N-acetyltaurine hydrolytic activity in cells. Pter-deficient mice confirmed the loss of hydrolysis activity with targeted metabolomic analysis of N-acetylated amino acids showing only alterations in N-acetyltaurine levels with minimal changes in other N-acetyl amino acids. Previous work has shown a connection between PTER polymorphisms and early onset obesity, so the team examined Pter-deficient mice on a high-fat diet while supplemented with taurine or exposed to treadmill running. In both cases, the mice exhibited lower body weight and food intake with improved glucose homeostasis, suggesting a complex interplay between increased N-acetyltaurine, diet and exercise. The beneficial metabolic effects were attributed to the brainstem, where PTER was expressed and is known to regulate food intake. The addition of an antibody against a candidate brainstem regulator, glial cell-derived neurotrophic factor receptor alpha-like (GFRAL), blocked the effects of exogenous N-acetyltaurine. Although there remain open questions about the metabolic regulation between the brainstem and peripheral tissues and the identity of the enzymes required for N-acetyltaurine, the findings from Wei et al. offer the first step to understanding the metabolic and functional roles of N-acetyltaurine.

Original reference: Nature 633, 182–188 (2024)

Here, we report a modular multicellular system created by mixing and matching discrete engineered bacterial cells. This system can be designed to solve multiple computational decision problems. The modular system is based on a set of engineered bacteria that are modeled as an ‘artificial neurosynapse’ that, in a coculture, formed a single-layer artificial neural network-type architecture that can perform computational tasks. As a demonstration, we constructed devices that function as a full subtractor and a full adder. The system is also capable of solving problems such as determining if a number between 0 and 9 is a prime number and if a letter between A and L is a vowel. Finally, we built a system that determines the maximum number of pieces of a pie that can be made for a given number of straight cuts. This work may have importance in biocomputer technology development and multicellular synthetic biology.

The main biopolymers in nature are oligonucleotides and polypeptides. However, naturally occurring peptide–nucleobase hybrids are rare. Here we report the characterization of the founding member of a class of peptide–nucleobase hybrid natural products with a pyrimidone motif from a widely distributed ribosomally synthesized and post-translationally modified (RiPP) biosynthetic pathway. This pathway features two steps where a heteromeric RRE–YcaO–dehydrogenase complex catalyzes the formation of a six-membered pyrimidone ring from an asparagine residue on the precursor peptide, and an acyl esterase selectively recognizes this moiety to cleave the C-terminal follower peptide. Mechanistic studies reveal that the pyrimidone formation occurs in a substrate-assisted catalysis manner, requiring a His residue in the precursor to activate asparagine for heterocyclization. Our study expands the chemotypes of RiPP natural products and the catalytic scope of YcaO enzymes. This discovery opens avenues to create artificial biohybrid molecules that resemble both peptide and nucleobase, a modality of growing interest.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species including food crops, such as tomato, potato and eggplant. Unlike true alkaloids, nitrogen is introduced at a late stage of SGA biosynthesis through an unknown transamination reaction. Here, we reveal the mechanism by which GLYCOALKALOID METABOLISM12 (GAME12) directs the biosynthesis of nitrogen-containing steroidal alkaloid aglycone in Solanum. We report that GAME12, a neofunctionalized γ-aminobutyric acid (GABA) transaminase, undergoes changes in both active site specificity and subcellular localization to switch from its renown and generic activity in core metabolism to function in a specialized metabolic pathway. Moreover, overexpression of GAME12 alone in engineered S. nigrum leaves is sufficient for de novo production of nitrogen-containing SGAs. Our results highlight how hijacking a core metabolism GABA shunt enzyme is crucial in numerous Solanum species for incorporating a nitrogen to a steroidal-specialized metabolite backbone and form defensive alkaloids.

Superoxide anion is thought to be a natural by-product with strong oxidizing ability in all living organisms and was recently found to accumulate in plant meristems to maintain stem cells in the shoot and undifferentiated meristematic cells in the root. Here we show that the DNA demethylase repressor of silencing 1 (ROS1) is one of the direct targets of superoxide in stem cells. The Fe–S clusters in ROS1 are oxidized by superoxide to activate its DNA glycosylase/lyase activity. We demonstrate that superoxide extensively participates in the establishment of active DNA demethylation in the Arabidopsis genome and that ARABIDOPSIS RESPONSE REGULATOR 12 acts downstream of ROS1-mediated superoxide signaling to maintain stem cell fate. Our results provide a mechanistic framework for superoxide control of the stem cell niche and demonstrate how redox and DNA demethylation interact to define stem cell fate in plants.

Synthetic biology aims to modify cellular behaviors by implementing genetic circuits that respond to changes in cell state. Integrating genetic biosensors into endogenous gene coding sequences using clustered regularly interspaced short palindromic repeats and Cas9 enables interrogation of gene expression dynamics in the appropriate chromosomal context. However, embedding a biosensor into a gene coding sequence may unpredictably alter endogenous gene regulation. To address this challenge, we developed an approach to integrate genetic biosensors into endogenous genes without modifying their coding sequence by inserting into their terminator region single-guide RNAs that activate downstream circuits. Sensor dosage responses can be fine-tuned and predicted through a mathematical model. We engineered a cell stress sensor and actuator in CHO-K1 cells that conditionally activates antiapoptotic protein BCL-2 through a downstream circuit, thereby increasing cell survival under stress conditions. Our gene sensor and actuator platform has potential use for a wide range of applications that include biomanufacturing, cell fate control and cell-based therapeutics.

Darwinian evolution has given rise to all the enzymes that enable life on Earth. Mimicking natural selection, scientists have learned to tailor these biocatalysts through recursive cycles of mutation, selection and amplification, often relying on screening large protein libraries to productively modulate the complex interplay between protein structure, dynamics and function. Here we show that by removing destabilizing mutations at the library design stage and taking advantage of recent advances in gene synthesis, we can accelerate the evolution of a computationally designed enzyme. In only five rounds of evolution, we generated a Kemp eliminase—an enzymatic model system for proton transfer from carbon—that accelerates the proton abstraction step >10⁸-fold over the uncatalyzed reaction. Recombining the resulting variant with a previously evolved Kemp eliminase HG3.17, which exhibits similar activity but differs by 29 substitutions, allowed us to chart the topography of the designer enzyme’s fitness landscape, highlighting that a given protein scaffold can accommodate several, equally viable solutions to a specific catalytic problem.