ChemBioChem最新文献

Staphylococcus aureus is an opportunistic pathogen that is persistently colonizing nearly 30% of the human population and can cause life-threatening infections. S. aureus secretes a variety of virulence factors, such as a set of extracellular serine protease-like proteins (Spls). Spls are expressed by most clinical isolates of S. aureus, but their pathophysiological substrates and role during infection are largely unknown. Pathogens use allelic variation of virulence factors to allow an adaption to different host cells and their defense mechanisms. The differences of these variants are marginally characterized so far. Here, we performed a biochemical characterization of selected allelic variants of the S. aureus SplA and SplB. Our data suggests different variants show differences in their stability, enzymatic activity, and substrate specificity. For the recently identified Spl target proteins RickULP and SseL, different cleavage patterns were observed, upon treatment with different Spl allelic variants. One SplB variant strongly differed in its substrate recognition at the P3/P4 position, more closely resembling SplE than SplB wildtype in its substrate selectivity. Our data provide valuable insights into the evolution of bacterial virulence factors and highlight the importance of including allelic variation of virulence factors to fully understand their role in host–pathogen interaction.

Coacervates formed through liquid–liquid phase separation represent a fundamental model for protocell and serve as a membraneless organelle in living cells, modulating various biological processes. During aging or under stress, protein misfolding and oligomerization trigger aberrant liquid-to-solid phase transition (LSPT), a process driven by multivalent interactions. These phase transitions disrupt cellular equilibrium, leading to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The regulatory strategies is summarize to enhance molecular interactions in coacervate LSPT into three categories, including physical stimulation, molecular modulation, and sequence regulation. This review aims to establish a conceptual framework for modulating coacervate LSPT and further explores potential clinical treatments for neurodegenerative diseases.

Numerous styrene monooxygenases (SMOs) have been identified and extensively applied in the asymmetric epoxidation of alkenes to prepare enantiopure epoxides, which are important intermediates in pharmaceuticals, agrochemicals, and fine chemicals. A current challenge is development of engineered SMOs capable of catalyzing a broad range of substrates with complementary enantioselectivity, thereby enabling the production of both (R)- and (S)-epoxides. Achieving this requires a deep understanding of the molecular basis underlying enantiocontrolling in SMO-catalyzed epoxidation. In this concept article, recent progress in elucidating the mechanisms of enantiocontrol by SMOs is summarized, with particular emphasis on the structural and mechanistic features that differentiate (R)- and (S)-selective SMOs. These insights not only expand fundamental knowledge of SMO catalysis but also provide a critical foundation for the rational design and protein engineering of highly efficient and versatile SMOs.

Protein ubiquitination is a pivotal posttranslational modification that regulates diverse biological processes depending on the type of ubiquitin chain linkage. Recently, ester-linked ubiquitin chains have been identified, yet their inherent hydrolytic instability has posed a significant challenge for biochemical investigations. In this study, a stable and isosteric amide analog of an ester-linked ubiquitin dimer, is chemically synthesized in which serine (Ser) at position 20 of the proximal ubiquitin is replaced with 2,3-diaminopropionic acid (Dap). The desired amide analog is synthesized using a convergent approach involving the sequential chemoselective ligation of three peptide fragments generated through Fmoc-based solid-phase peptide synthesis. Employing this chemically robust ubiquitin probe, a previously unrecognized interaction is uncovered between Ser20-linked ubiquitin chains and spliceosome-associated factors, notably ubiquitin-specific protease 39. These findings highlight the potential of the ester-to-amide bioisosteric strategy to unlock mechanistic insights into atypical ubiquitin modifications. The approach not only circumvents the intrinsic instability of ester-linked ubiquitin chains but also provides a broadly applicable framework for dissecting their biological roles, paving the way for future discoveries in ubiquitin signaling.

Heavily sulfated clusters of maltose are designed as mimetics of heparan sulfate (HS), exhibiting diverse biological activity properties of HS. Herein, the synthesis of three function-spacer-lipid (FSL) constructs containing mono, di, and tri sulfated maltoses is reported. FSLs are a class of synthetic glycolipids, the key features of which are simplicity of synthesis and capacity to integrate into cell membranes with sustained retention. A copper-catalyzed azide–alkyne cycloaddition click reaction is employed for the conjugation of the sulfated maltose entities with the respective spacer-lipid block because acylation with activated esters failed to provide the desired products. One FSL construct, featuring a single sulfated maltose unit conjugated to the lipid, demonstrated dose-dependent inhibition of complement activation in a cellular system, with maximal efficacy observed at 15 µg mL⁻¹. These findings highlight the potential of FSL-based HS mimetics for modulating immune responses.

The cover illustrates the first isolable cysteine sulfenyl iodide (Cys-SI), stabilized by a nanosized molecular cradle installed at the N-terminus of cysteine. This stabilization enabled direct structural and reactivity studies of the long-hypothesized intermediate in cysteine thiol oxidation. The Cys-SI exhibits high electrophilicity toward nucleophiles, including an indole ring, providing chemical evidence supporting mechanistic proposals for iodine mediated protein modification. More details can be found in the Research Article by Kei Goto and co-workers (DOI: 10.1002/cbic.202500619).

G-quadruplexes (G4s) are noncanonical nucleic acid structures with biological and therapeutic significance, and they are found in many aptamer sequences. Using c-kit 1 G4 DNA as a target, systematic evolution of ligands by exponential enrichment (SELEX) has been carried out using an RNA library, resulting in G4-rich aptamers. Herein, this article investigates the relationship between predicted G4-forming potential and aptamer enrichment by analyzing high-throughput SELEX libraries using three G4 prediction tools: G4NN, G4Hunter, and QGRS Mapper. While the tools demonstrate strong internal consistency and overlap in identifying G4-prone sequences, their predictions show limited concordance with experimental abundance and enrichment trends across SELEX rounds. Only a small fraction of sequences display both high G4 scores and consistent enrichment. Experimental validation using electrophoretic mobility shift assays confirmed that strong predicted G4-forming sequences often lack strong binding activity, whereas highly enriched aptamers with strong binding activities may show weaker G4 signatures computationally. These findings suggest that current G4-prediction tools alone are insufficient for aptamer candidate selection and highlight the need for integrative evaluation strategies that combine structural prediction with empirical performance data.

Dual cyclooxygenase-2/5-lipoxygenase (COX-2/5-LOX) inhibitors constitute safer alternatives to classical nonsteroidal anti-inflammatory drugs, widely used to effectively manage inflammation. In this article, molecular docking and molecular dynamics simulations guide the synthesis of novel thymol derivatives that interact with both COX-2 and 5-LOX active sites. Ligands are designed with the aim of improving thymol bioactivity, selectivity, stability, as well as pharmacokinetic properties. Therefore, –Br, –F, and –CF₃ inclusion on thymol is here evaluated, screening COX-2 and 5-LOX interactions with thymol (T), 4-fluorothymol (FT), 4-bromothymol (BT), isopropyl thymyl succinate (T1), 1,1,1,3,3,3-hexafluoroisopropyl thymyl succinate (T2), and 1′,1′,1′,3′,3′,3′-hexafluoroisopropyl 4-(4′’-thymyl)-4-oxobutanoate (T3). Molecular modeling reveals that the estimated ligands can establish favorable interactions with both COX-2 and 5-LOX active pockets, highlighting T1–T3 as the most promising compounds. In vitro assays identify T3 as the most active COX-2 inhibitor, while T2 results as the most effective ligand for 5-LOX. Interestingly, cavity analysis of the COX-2 entry site reveals that T3 insertion is favored over T1 and T2 due to its greater polarity, conferred by the presence of a free phenolic group (OH) able to establish H-bonds with surrounding residues.

A novel strategy based on genetic code expansion combined with click chemistry for the simultaneous visualization of distinct posttranslational modification (PTM) states of a single protein within living cells. As a model, it is focused on threonine 41 (T41) of β-catenin, a regulatory hotspot implicated in epithelial cancers and known to be phosphorylated, O-GlcNAcylated, or left unmodified. Using site-specific incorporation of the unnatural phenylselenocysteine, a β-catenin-EGFP fusion protein is engineered allowing selective installation of a S-GlcNAc moiety via oxidative elimination and thiol-Michael ligation. Additional β-catenin variants, phosphomimetic T41E-mCherry and wild-type-blue fluorescent protein fusions, are produced to represent other PTM states. All constructs are successfully introduced into Hep3B and HeLa cells by lipofection or TAT-mediated transduction. Fluorescence microscopy revealed distinct subcellular localization profiles for each PTM form. Notably, the S-GlcNAcylated β-catenin exhibited enhanced resistance to proteasomal degradation, consistent with known roles of O-GlcNAcylation in protein stability. This approach provides a versatile platform to functionally probe PTMs in a comparative, cell-based context.

Phosphoribosyl pyrophosphate (PRPP) functions as a central metabolic intermediate, supplying ribose-5-phosphate moieties for the biosynthesis of nucleotides, certain amino acids, and a range of essential cofactors. In this study, a thermostable phosphoribosyl pyrophosphate synthetase (PfPRS) was identified from the hyper thermophilic archaeon Pyrolobus fumarii 1A, a hyper thermophilic archaeon that grows optimally at 90–113 °C. The prs gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Peak catalytic activity of PfPRS was observed at approximately pH 7.5 and 55 °C and retained over 85% of its activity after 2 h of incubation across pH 4.0–10.5. PfPRS exhibited high thermal stability. The enzyme exhibited half-lives of 12 h at 90 °C, 5 h at 95 °C, and 3 h at 100 °C. Among the nucleotides tested as diphosphate donors, PfPRS showed a strong preference for ATP, whereas ADP served as an effective inhibitor. Kinetic analysis revealed K_m values of 35 µM for R5P and 46 µM for ATP, with turnover rates (k_cat) of 71 s⁻¹ and 56 s⁻¹. PfPRS was co-immobilized with polyphosphate kinase 2 (DrPPK2) from Deinococcus radiodurans using a cross-linked enzyme aggregate (CLEA) system to enable ATP regeneration and to explore the feasibility of using PfPRS for PRPP biosynthesis.