ACS Chemical Biology最新文献

Site-specific noncanonical amino acid (ncAA) mutagenesis in living cells has traditionally relied on heterologous, nonsense-suppressing aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that do not cross-react with their endogenous counterparts. Such heterologous pairs often perform suboptimally in a foreign host cell since they were not evolutionarily optimized to function in the foreign environment. This suboptimal performance restricts the number of ncAAs that can be simultaneously incorporated into a protein. Here, we show that the use of an endogenous aaRS/tRNA pair to drive ncAA incorporation can offer a potential solution to this limitation. To this end, we developed an engineered Escherichia coli strain (ATMY-C321), wherein the endogenous tyrosyl-tRNA synthetase (TyrRS)/tRNA pair has been functionally replaced with an archaeal counterpart, and the release factor 1 has been removed to eliminate competing termination at the UAG nonsense codons. The endogenous TyrRS/tRNA_CUA^Tyr pair exhibits remarkably efficient nonsense suppression in the resulting cell, relative to established orthogonal ncAA-incorporation systems in E. coli, allowing the incorporation of an ncAA at up to 10 contiguous sites in a reporter protein. Our work highlights the limitations of orthogonal translation systems using heterologous aaRS/tRNA pairs and offers a potential alternative involving the use of endogenous pairs.

Eliminating the core fucose from the N-glycans of the Fc antibody segment by pathway engineering or enzymatic methods has been shown to enhance the potency of therapeutic antibodies, especially in the context of antibody-dependent cytotoxicity (ADCC). However, there is a significant challenge due to the limited defucosylation efficiency of commercially available α-l-fucosidases. In this study, we report a unique α-l-fucosidase (PnfucA) from the bacterium Prevotella nigrescens that has a low sequence identity compared with all other known α-l-fucosidases and is highly reactive toward a core disaccharide substrate with fucose α(1,3)-, α (1,4)-and α(1,6)-linked to GlcNAc, and is less reactive toward the Fuc-α(1,2)-Gal on the terminal trisaccharide of the oligosaccharide Globo H (Bb3). The kinetic properties of the enzyme, such as its K_m and k_cat, were determined and the optimized expression of PnfucA gave a yield exceeding 30 mg/L. The recombinant enzyme retained its full activity even after being incubated for 6 h at 37 °C. Moreover, it retained 92 and 87% of its activity after freezing and freeze-drying treatments, respectively, for over 28 days. In a representative glycoengineering of adalimumab (Humira), PnfucA showed remarkable hydrolytic efficiency in cleaving the α(1,6)-linked core fucose from FucGlcNAc on the antibody with a quantitative yield. This enabled the seamless incorporation of biantennary sialylglycans by Endo-S2 D184 M in a one-pot fashion to yield adalimumab in a homogeneous afucosylated glycoform with an improved binding affinity toward Fcγ receptor IIIa.

Quenched activity-based probes (qABP) are invaluable tools to visualize aberrant protease activity. Unfortunately, most studies so far have only focused on cysteine proteases, and only a few studies describe the synthesis and use of serine protease qABPs. We recently used phosphinate ester electrophiles as a novel type of reactive group to construct ABPs for serine proteases. Here, we report on the construction of qABPs based on the phosphinate warhead, exemplified by probes for the neutrophil serine proteases. The most successful probes show sub-stoichiometric reaction with human neutrophil elastase, efficient fluorescence quenching, and rapid unquenching of fluorescence upon reaction with target proteases.

Glutaric Aciduria Type 1 (GA1) is a serious inborn error of metabolism with no pharmacological treatments. A novel strategy to treat this disease is to divert the toxic biochemical intermediates to less toxic or nontoxic metabolites. Here, we report a putative novel target, succinyl-CoA:glutarate-CoA transferase (SUGCT), which we hypothesize suppresses the GA1 metabolic phenotype through decreasing glutaryl-CoA and the derived 3-hydroxyglutaric acid. SUGCT is a type III CoA transferase that uses succinyl-CoA and glutaric acid as substrates. We report the structure of SUGCT, develop enzyme- and cell-based assays, and identify valsartan and losartan carboxylic acid as inhibitors of the enzyme in a high-throughput screen of FDA-approved compounds. The cocrystal structure of SUGCT with losartan carboxylic acid revealed a novel pocket in the active site and further validated the high-throughput screening approach. These results may form the basis for the future development of new pharmacological intervention to treat GA1.

tRNA modifications help maintain tRNA structure and facilitate translation and stress response. Found in all three kingdoms of life, m¹A tRNA modification occurs in the T loop of many tRNAs, stabilizes tertiary tRNA structure, and impacts translation. M¹A in the T loop is reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust its m¹A levels in cells. However, no prokaryotic tRNA demethylase enzyme has been identified that acts on endogenous RNA modifications. Using Streptomyces venezuelae as a model organism, we confirmed the presence and quantitative m¹A tRNA signatures using mass spectrometry and high-throughput tRNA sequencing. We identified two RNA demethylases that can remove m¹A in tRNA and validated the activity of a previously annotated tRNA m¹A writer. Using single-gene knockouts of these erasers and the m¹A writer, we found dynamic changes of m¹A levels in many tRNAs under stress conditions. Phenotypic characterization highlighted changes in their growth and altered antibiotic production. Our identification of the first prokaryotic tRNA demethylase enzyme paves the way for investigating new mechanisms of translational regulation in bacteria.

Carrier proteins (CPs) play a fundamental role in the biosynthesis of fatty acids, polyketides, and non-ribosomal peptides, encompassing many medicinally and pharmacologically relevant compounds. Current approaches to analyze novel carrier-protein-dependent synthetic pathways are hampered by a lack of activity-based assays for natural product biosynthesis. To fill this gap, we turned to 3-methoxychromones, highly solvatochromic fluorescent molecules whose emission intensity and wavelength are heavily dependent on their immediate molecular environment. We have developed a solvatochromic carrier-protein-targeting probe which is able to selectively fluoresce when bound to a target carrier protein. Additionally, the probe displays distinct responses upon CP binding in carrier-protein-dependent synthases. This discerning approach demonstrates the design of solvatochromic fluorophores with the ability to identify biosynthetically active CP-enzyme interactions.

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is important for nociception and inflammatory pain and is activated by a variety of nociceptive stimuli─including lipids such as capsaicin (CAP) and endocannabinoids. TRPV1's role in physiological systems is often studied by activating it with externally perfused ligands; however, this approach is plagued by poor spatiotemporal resolution. Lipid agonists are insoluble in physiological buffers and can permeate membranes to accumulate nonselectively inside cells, where they can have off-target effects. To increase the spatiotemporal precision with which we can activate lipids on cells and tissues, we previously developed optically cleavable targeted (OCT) ligands, which use protein tags (SNAP-tags) to localize a photocaged ligand on a target cellular membrane. After enrichment, the active ligand is released on a flash of light to activate nearby receptors. In our previous work, we developed an OCT-ligand to control a cannabinoid-sensitive GPCR. Here, we expand the scope of OCT-ligand technology to target TRPV1 ion channels. We synthesize a probe, OCT-CAP, that tethers to membrane-bound SNAP-tags and releases a TRPV1 agonist when triggered by UV-A irradiation. Using Ca²⁺ imaging and electrophysiology in HEK293T cells expressing TRPV1, we demonstrate that OCT-CAP uncaging activates TRPV1 with superior spatiotemporal precision when compared to standard diffusible ligands or photocages. This study is the first example of an OCT-ligand designed to manipulate an ion-channel target. We anticipate that these tools will find many applications in controlling lipid signaling pathways in various cells and tissues.

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

The precise spatial and temporal orchestration of gene expression is crucial for the ontogeny of an organism and is mainly governed by transcription factors (TFs). The mechanism of recognition of cognate sites amid millions of base pairs in the genome by TFs is still incompletely understood. In this study, we focus on DNA sequence composition, shape, and flexibility preferences of 28 quintessential TFs from Drosophila melanogaster that are critical to development and body patterning mechanisms. Our study finds that TFs exhibit distinct predilections for DNA shape, flexibility, and sequence compositions in the proximity of transcription factor binding sites (TFBSs). Notably, certain zinc finger proteins prefer GC-rich areas with less negative propeller twist, while homeodomains mainly seek AT-rich regions with a more negative propeller twist at their sites. Intriguingly, while numerous cofactors share similar binding site preferences and bind closer to each other in the genome, some cofactors that have different preferences bind farther apart. These findings shed light on TF DNA recognition and provide novel insights into possible cofactor binding and transcriptional regulation mechanisms.

Peptide-bile acid hybrids offer promising drug candidates due to enhanced pharmacological properties, such as improved protease resistance and oral bioavailability. However, it remains unknown whether bile acids can be incorporated into peptide chains by the ribosome to produce a peptide-bile acid hybrid macrocyclic peptide library for target-based de novo screening. In this study, we achieved the ribosomal incorporation of lithocholic acid (LCA)-d-tyrosine into peptide chains. This led to the construction of a peptide-LCA hybrid macrocyclic peptide library, which enabled the identification of peptides TP-2C-4L3 (targeting Trop2) and EP-2C-4L5 (targeting EphA2) with strong binding affinities. Notably, LCA was found to directly participate in binding to EphA2 and confer on the peptides improved stability and resistance to proteases. Cell staining experiments confirmed the high specificity of the peptides for targeting Trop2 and EphA2. This study highlights the benefits of LCA in peptides and paves the way for de novo discovery of stable peptide-LCA hybrid drugs.