ACS Chemical Biology最新文献

N-Pyridinylthiophene carboxamide (compound 21) displays activity against peripheral nerve sheath cancer cells and mouse xenografts by an unknown mechanism. Through medicinal chemistry, we identified a more active derivative, compound 9, and found that only analogues with structures similar to nicotinamide retained activity. Genetic screens using compound 9 found that both NAMPT and NMNAT1, enzymes in the NAD salvage pathway, are necessary for activity. Compound 9 is metabolized by NAMPT and NMNAT1 into an adenine dinucleotide (AD) derivative in a cell-free system, cultured cells, and mice, and inhibition of this metabolism blocked compound activity. AD analogues derived from compound 9 inhibit IMPDH in vitro and cause cell death by inhibiting IMPDH in cells. These findings nominate these compounds as preclinical candidates for the development of tumor-activated IMPDH inhibitors to treat neuronal cancers.

Histone lysine acetylation (Kac) and crotonylation (Kcr) marks mediate the recruitment of YEATS domains to chromatin. In this way, YEATS domain-containing proteins such as AF9 participate in the regulation of DNA-templated processes. Our previous study showed that the replacement of Kac/Kcr by a 2-furancarbonyllysine (Kfu) residue led to greatly enhanced affinity toward the AF9 YEATS domain, rendering Kfu-containing peptides useful chemical tools to probe the AF9 YEATS–Kac/Kcr interactions. Here, we report the genetic incorporation of Kfu in Escherichia coli and mammalian cells through the amber codon suppression technology. We develop a Kfu-containing epitope tag, termed RAY-tag, which can robustly and selectively engage with the AF9 YEATS domain in vitro and in cellulo. We further demonstrate that the fusion of RAY-tag to different protein modules, including fluorescent proteins and DNA binding proteins, can facilitate the interrogation of the histone lysine acylation-mediated recruitment of the AF9 YEATS domain in different biological contexts.

Eliciting an antihapten antibody response to vaccination typically requires the use of constructs where multiple copies of the hapten are covalently attached to a larger carrier molecule. The carrier is required to elicit T cell help via presentation of peptide epitopes on major histocompatibility complex (MHC) class II molecules; as such, attachment to full-sized proteins, alone or in a complex, is generally used to account for the significant MHC diversity in humans. While such carrier-based vaccines have proven extremely successful, particularly in protecting against bacterial diseases, they can be challenging to manufacture, and repeated use can be compromised by pre-existing immunity against the carrier. One approach to reducing these complications is to recruit help from type I natural killer T (NKT) cells, which exhibit limited diversity in their antigen receptors and respond to glycolipid antigens presented by the highly conserved presenting molecule CD1d. Synthetic vaccines for universal use can, therefore, be prepared by conjugating haptens to an NKT cell agonist such as α-galactosylceramide (αGalCer, KRN7000). An additional advantage is that the quality of NKT cell help is sufficient to overcome the need for an extra immune adjuvant. However, while initial studies with αGalCer-hapten conjugate vaccines report strong and rapid antihapten antibody responses, they can fail to generate lasting memory. Here, we show that antibody responses to the hapten 4-hydoxy-3-nitrophenyl acetyl (NP) can be improved through additional attachment of a fusion peptide containing a promiscuous helper T cell epitope (Pan DR epitope, PADRE) that binds diverse MHC class II molecules. Such αGalCer-hapten-peptide tricomponent vaccines generate strong and sustained anti-NP antibody titers with increased hapten affinity compared to vaccines without the helper epitope. The tricomponent vaccine platform is therefore suitable for further exploration in the pursuit of efficacious antihapten immunotherapies.

Currently, the CRISPR-Cas9 system serves as a prevalent tool for genome editing and gene expression regulation. Its therapeutic application is limited by off-target effects that can affect genomic integrity through nonspecific, undesirable changes in the genome. Various strategies have been explored to mitigate the off-target effects. Many approaches focus on modifying components of the system, namely, Cas9 and guide RNAs, to enhance specificity. However, a common challenge is that methods aiming to increase specificity often result in a significant reduction in the editing efficiency. Here, we introduce a novel approach to modifying crRNA to balance CRISPR-Cas9 specificity and efficiency. Our approach involves incorporating nucleoside modifications, such as replacing ribo- to deoxyribonucleosides and backbone modifications, using phosphoryl guanidine groups, specifically 1,3-dimethylimidazolidin-2-ylidene phosphoramidate. In this case, within the first 10 nucleotides from the 5′ crRNA end, phosphodiester bonds are substituted with phosphoryl guanidine groups. We demonstrate that crRNAs containing a combination of deoxyribonucleosides and single or multiple phosphoryl guanidine groups facilitate the modulation of CRISPR-Cas9 system activity while improving its specificity in vitro.

Tryptophan, commonly regarded as buried within the interior cores of proteins to maintain secondary structures, is now being recognized for its significant contributions to protein functionality. However, investigating functional tryptophan-involved interactions across the proteome and manipulating these interactions in live cells are considerable challenges. In this In Focus article, we summarize emerging advances in the field, describing innovative chemistries that leverage distinctive biochemical properties of the indole moiety for targeting and functionally manipulating tryptophan interactions.

N-Methylated amino acids are constituents of natural bioactive peptides and proteins. N^α-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing N^α-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded N^α-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of N^α-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.

Methylglyoxal (MGO) is an electrophilic α-oxoaldehyde generated endogenously through metabolism of carbohydrates and exogenously due to autoxidation of sugars, degradation of lipids, and fermentation during food and drink processing. MGO can react with nucleophilic sites within proteins and DNA to form covalent adducts. MGO-induced advanced glycation end-products such as protein and DNA adducts are thought to be involved in oxidative stress, inflammation, diabetes, cancer, renal failure, and neurodegenerative diseases. Additionally, MGO has been hypothesized to form toxic DNA–protein cross-links (DPC), but the identities of proteins participating in such cross-linking in cells have not been determined. In the present work, we quantified DPC formation in human cells exposed to MGO and identified proteins trapped on DNA upon MGO exposure using mass spectrometry-based proteomics. A total of 265 proteins were found to participate in MGO-derived DPC formation including gene products engaged in telomere organization, nucleosome assembly, and gene expression. In vitro experiments confirmed DPC formation between DNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as histone proteins H3.1 and H4. Collectively, our study provides the first evidence for MGO-mediated DNA–protein cross-linking in living cells, prompting future studies regarding the relevance of these toxic lesions in cancer, diabetes, and other diseases linked to elevated MGO levels.

Copper is an essential trace element that participates in many biological processes through its unique redox cycling between cuprous (Cu⁺) and cupric (Cu²⁺) oxidation states. To elucidate the biological functions of copper, chemical biology tools that enable selective visualization and detection of copper ions and proteins in copper-rich environments are required. Herein, we describe the design of Cu⁺-responsive reagents based on a conditional protein labeling strategy. Upon binding Cu⁺, the probes generated quinone methide via oxidative bond cleavage, which allowed covalent labeling of surrounding proteins with high Cu⁺ selectivity. Using gel- and imaging-based analyses, the best-performing probe successfully detected changes in the concentration of labile Cu⁺ in living cells. Moreover, conditional proteomics analysis suggested intramitochondrial Cu⁺ accumulation in cells undergoing cuproptosis. Our results highlight the power of Cu⁺-responsive protein labeling in providing insights into the molecular mechanisms of Cu⁺ metabolism and homeostasis.

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5′ cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.

Triceptides are a class of ribosomally synthesized and post-translationally modified peptides defined by an aromatic C(sp²) to Cβ(sp³) bond. The Gly-rich repeat family of triceptide maturases (TIGR04261) are paired with precursor peptides (TIGR04260) containing a Gly-rich core peptide. These maturases are prevalent in cyanobacteria and catalyze cyclophane formation on multiple Ω1-X2-X3 motifs (Ω1 = Trp and Phe) of the Gly-rich precursor peptide. The topology of the individual rings has not been completely elucidated, and the promiscuity of these enzymes is not known. In this study, we characterized all the cyclophane rings formed by the triceptide maturase OscB and show the ring topology is uniform with respect to the substitution at Trp-C7 and the atropisomerism (planar chirality). Additionally, the enzyme OscB demonstrated substrate promiscuity on Gly-rich precursors and can accommodate a diverse array of engineered sequences. These findings highlight the versatility and implications for using OscB as a biocatalyst for producing polycyclophane-containing peptides for biotechnological applications.