ACS Chemical Biology最新文献

Bacterial peptidoglycan, the essential cell surface polymer that protects bacterial integrity, also serves as the molecular pattern recognized by the host's innate immune system. Although the minimal motifs of bacterial peptidoglycan fragments (PGNs) that activate mammalian NOD1 and NOD2 sensors are well-known and often represented by small canonical ligands, the immunostimulatory effects of natural PGNs, which are structurally more complex and potentially can simultaneously activate both the NOD1 and NOD2 signaling pathways in hosts, have not been comprehensively investigated. In particular, many bacteria incorporate additional structural modifications in peptidoglycans to evade host immune surveillance, resulting in diverse structural variations among natural PGNs that may influence their biological effects in hosts. The focus of this study is on the amidation status of γ-d-glutamic acid and meso-diaminopimelic acid (mDAP) at the second and third positions of stem peptides in peptidoglycan, which represent key structural features that vary across different bacterial species. With four synthetic mDAP-containing disaccharide PGNs of different amidation states, we systematically investigated their structure-activity relationship in stimulating host innate immune responses in vitro. Our findings revealed that the amidation of disaccharide PGNs has distinct effects on NOD1 and NOD2 induction, along with their differential immunostimulatory activities in macrophage cells. Additionally, we found that, like the canonical NOD2 ligand, natural PGNs confer immune tolerance to LPS, and amidation states do not affect this outcome. Overall, our work highlights the potential immunological implications of these differentially amidated mDAP-type disaccharide PGNs in host-microbe crosstalk.

Although methods for Cys-specific bioconjugation and functionalization of proteins have been developed and widely utilized in biomolecule engineering and therapeutic development, reagents for this purpose are generally designed to accomplish bioconjugation only. Consequently, additional clickable groups must be attached to these reagents to accomplish functionalization. Herein, we describe a new, simple, dual-performing bioconjugation-functionalization reagent, VMeTz, which possesses an electron-withdrawing tetrazine (Tz) substituted vinyl (V) moiety to serve as both a Michael receptor for selective conjugation with Cys and a site for click with TCO derivatives to introduce functionality. Critically, VMeTz contains a methyl group that prevents the formation of multiple Tz-containing Cys-adducts. Reactions of VMeTz with Cys-containing peptides and proteins both in vitro and in live cells produce single stable Michael adducts with high selectivity. Moreover, the Cys-VMeTz peptide and protein conjugates undergo facile click reactions with TCO-functionalized reagents for labeling and protein profiling. Furthermore, VMeTz selectively activates and delivers the TCO-caged toxic substances Dox and PROTAC ARV-771 to cancer cells to produce therapeutic effects that are comparable to those of the parent drugs. Collectively, the studies demonstrate that VMeTz is a useful reagent for therapeutically significant Cys-specific protein bioconjugation and functionalization.

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties. Aromatic polyketides are produced by type II polyketide synthase (PKS) systems, each minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (holo-ACP). Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro. In cases where the KS-CLF can be accessed via E. coli heterologous expression, often the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli, which allowed us to study this minimal type II PKS in vitro. Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.

Hyperammonemia is characterized by the accumulation of ammonia within the bloodstream upon liver injury. Left untreated, hyperammonemia contributes to conditions such as hepatic encephalopathy that have high rates of patient morbidity and mortality. Previous studies have identified gut bacterial urease, an enzyme that converts urea into ammonia, as a major contributor to systemic ammonia levels. Here, we demonstrate use of benurestat, a clinical candidate used against ureolytic organisms in encrusted uropathy, to inhibit urease activity in gut bacteria. Benurestat inhibits ammonia production by urease-encoding gut bacteria and is effective against individual microbes and complex gut microbiota. When administered to conventional mice with liver injury induced by thioacetamide exposure, benurestat reduced gut and serum ammonia levels and rescued 100% of mice from lethal acute liver injury. Overall, this study provides an important proof-of-concept for modulating host ammonia levels and microbiota-driven risks for hyperammonemia with gut microbiota-targeted small-molecule inhibitors.

Fibroblast growth factor 2 (FGF2) is a multipotent growth factor and signaling protein that exhibits broad functions across multiple cell types. These functions are often initiated by binding to growth factor receptors and fine-tuned by glycosaminoglycan (GAG)-modified proteins called proteoglycans. The various outputs of FGF2 signaling and functions arise from a dynamic and cell type-specific set of binding partners. However, the interactome of FGF2 has yet to be comprehensively determined. Moreover, the identity of the proteoglycan proteins carrying GAG chains is often overlooked and remains unknown in most cell contexts. Here, we perform peroxidase-catalyzed live cell proximity labeling using an engineered APEX2-FGF2 fusion protein to map the interactome of FGF2. Across two cell lines with established and distinct FGF2-driven functions, we greatly expand upon the known FGF2 interactome, identifying >600 new putative FGF2 interactors. Notably, our results demonstrate a key role for the GAG binding capacity of FGF2 in modulating its interactome.

MicroRNAs (miRNAs) play a significant role in tumor progression, and regulating miRNA expression with small molecules may offer a new approach to cancer therapy. Among them, miRNA-20b has been found to be dysregulated in several cancers, including nonsmall cell lung cancer (NSCLC). Herein, an in silico high-throughput computer screen was conducted to identify small molecules that downregulate miR-20b using the three-dimensional structure of the Dicer binding site on pre-miR-20b. Among 1058 small molecule compounds, Methotrexate (MTX), was discovered to be a potential miR-20b-specific inhibitor, which has been found to suppress miR-20b by specifically blocking Dicer processing in p53 wild-type A549 NSCLC cells but not in H1299 cells with p53 depletion. MTX effectively inhibited the proliferation, survival, migration, and invasion of A549 cells in a dose-dependent manner. Furthermore, the treatment of MTX up-regulated the expression of miR-20b target genes PTEN, STAT3, and HIF1α. Notably, MTX also significantly inhibited tumor growth in a mouse xenograft tumor model of NSCLC, with no observed tissue toxicity. Our findings indicate that MTX may have a novel role as an established drug in p53 wild-type NSCLC tumor therapy by down-regulating miR-20b expression. These findings are expected to provide preclinical evidence for miR-20b-targeting NSCLC therapeutic strategies.

Cell-cell interactions are fundamental in biology for maintaining physiological conditions with direct contact being the most straightforward mode of interaction. Recent advancements have led to the development of various chemical tools for detecting or identifying these interactions. However, the use of exogenous cues, such as toxic reagents, bulky probes, and light irradiation, can disrupt normal cell physiology. For example, the toxicity of hydrogen peroxide (H₂O₂) limits the applications of peroxidases in the proximity labeling field. In this study, we aimed to address this limitation by demonstrating that membrane-localized horseradish peroxidase (HRP-TM) efficiently utilizes endogenously generated extracellular H₂O₂. By harnessing endogenous H₂O₂, we observed that HRP-TM-expressing cells can effectively label contacting cells without the need for exogenous H₂O₂ treatment. Furthermore, we confirmed that HRP-TM labels proximal cells in an interaction-dependent manner. These findings offer a novel approach for studying cell-cell interactions under more physiological conditions without the confounding effects of exogenous stimuli. Our study contributes to elucidating cell-cell interaction networks in various model organisms, providing valuable insights into the dynamic interplay between cells in their native network.

Chronic kidney fibrosis poses a significant global health challenge with effective therapeutic strategies remaining elusive. While cell–extracellular matrix (ECM) interactions are known to drive fibrosis progression, the specific role of focal adhesions (FAs) in kidney fibrosis is not fully understood. In this study, we investigated the role of FAs in kidney tubular epithelial cell fibrosis by employing precise nanogold patterning to modulate integrin distribution. We demonstrate that increasing ligand spacing disrupts integrin clustering, thereby inhibiting FA formation and attenuating fibrosis. Importantly, enhanced FA activity is associated with kidney fibrosis in both human disease specimens and murine models. Mechanistically, FAs regulate fibrosis through mechanotransduction pathways, and our in vivo experiments show that suppressing mechanotransduction significantly mitigates kidney fibrosis in mice. These findings highlight the potential of targeting FAs as a therapeutic strategy, offering new insights into clinical intervention in kidney fibrosis.

The APOBEC3 family of polynucleotide cytidine deaminases has diverse roles as viral restriction factors and oncogenic mutators. These enzymes convert cytidine to uridine in single-stranded (ss)DNA, inducing genomic mutations that promote drug resistance and tumor heterogeneity. Of the seven human APOBEC3 members, APOBEC3A (A3A) and APOBEC3B (A3B) are most implicated in driving pro-tumorigenic mutations. How these enzymes engage and selectively deaminate ssDNA over RNA is not well understood. We previously conducted molecular dynamics (MD) simulations that support the role of sugar conformation as a key molecular determinant in nucleic acid recognition by A3B. We hypothesize that A3A and A3B selectively deaminate substrates in the 2'-endo (DNA) conformation and show reduced activity for 3'-endo (RNA) conformation substrates. Consequently, we have characterized A3A- and A3B-binding and deaminase activity with chimeric oligonucleotides containing cytidine analogues that promote either the 2'-endo or 3'-endo conformation. Using fluorescence polarization and gel-based deamination assays, we determined that sugar conformation preferentially impacts the ability of these enzymes to deaminate substrates and less so binding to substrates. Using MD simulations, we identify specific active site interactions that promote selectivity based on the 2'-endo conformation. These findings help inform the biological functions of A3A and A3B in providing antiviral innate immunity and pathogenic functions in cancer.

The regulation of reactive oxygen species (ROS) such as superoxide (SO) and nitric oxide (NO) is crucial in biology, influencing metabolism and signaling pathways. Imbalances in these species lead to oxidative stress and various diseases. Traditional methods for measuring SO and NO face challenges in terms of sensitivity and specificity, particularly in complex biological matrices. This report introduces bioluminescent probes that leverage the intrinsic sensitivity of bioluminescence for direct and selective detection of SO and NO. These probes release analogs of d-luciferin upon reaction with their target ROS. Following addition of luciferase, luminescence is generated proportional to the amount of accumulated luciferin, allowing for quantitation of SO or NO. Both probes exhibit high specificity, confirmed through cell-free assays and cell-based studies in macrophages, demonstrating their utility in measuring cellular SO and NO production. These assays offer a robust, high-throughput platform for studying ROS, providing direct insights into oxidative stress-related mechanisms.