ChemBioChem最新文献

The ubiquitin (Ub) ligase E6AP, encoded by the UBE3A gene, has been causally associated with human diseases including cervical cancer and Angelman syndrome, a neurodevelopmental disorder. Yet, our knowledge about disease-relevant substrates of E6AP is still limited, presumably because at least some of these interactions are rather transient, a phenomenon observed for many enzyme-substrate interactions. Here, we introduce a novel approach to trap such potential transient interactions by combining a stable E6AP-Ub conjugate mimicking the active state of this enzyme with photo-crosslinking (PCL) followed by affinity enrichment coupled to mass spectrometry (AE-MS). To enable PCL, we equipped Ub with diazirine moieties at distinct positions. We validated our PCL assisted AE-MS approach by identification of known (e. g. PSMD4, UCHL5) and potential new (e. g. MSH2) substrates of E6AP. Our findings suggest that PCL assisted AE-MS is indeed suited to identify substrates of E6AP, thereby providing insights into E6AP-associated pathologies, and, potentially, of other enzymes of the Ub-conjugating system.

Reactive oxygen species (ROS) play crucial roles in both cell signaling and defense mechanisms. Hypochlorous acid (HOCl), a strong oxidant, aids the immune response by damaging pathogens. In this study, we developed two pyridinium-based fluorophores PSSM and PSSE for selective hypochlorite detection. Out of these two fluorescent probes, PSSM shows a strong turn-on emission via a photoinduced electron transfer (PeT) mechanism, excellent mitochondrial localization, and rapid response to HOCl with high selectivity among reactive oxygen species by achieving a detection limit of 2.41 μM. It successfully detects both exogenous and endogenous HOCl in live cells, enabling the study of HOCl's role at the organelle level. Structural analysis of PSSM via thioether oxidation confirmed by HPLC, NMR and HRMS further supports its specificity. Confocal imaging and flow cytometry studies further highlights its utility in investigating oxidative stress, positioning this fluorophore as a valuable tool for monitoring HOCl imbalances in biological systems.

In the last decade the important role of small non-coding RNAs such as micro RNAs (miRs) in gene regulation in healthy and disease states became more and more evident. The miR-200-family of miRs has been shown to play a critical role in many diseases such as cancer and neurodegenerative disorders and could be potentially important for diagnosis and treatment. However, the size of miRs of about ~21-23 nt provide challenges for their investigation. Here we report the conversion and optimization of the Broccoli fluorescent light-up RNA-aptamer into a specific sensor for miR-200c using a strand-displacement design principle. This aptamer can differentiate miR-200c from its family members whose sequence differ by more than one nucleotide. By adding this in vitro transcribed aptamer to RNA extracts from human cells, we can detect miR-200c in vitro in a plate reader assay.

The α-helix is an abundant and functionally important element of protein secondary structure, which has motivated intensive efforts toward chemical strategies to stabilize helical folds. One such method is the incorporation of non-canonical backbone composition through an additional methyl substituent at the C_α atom. Examples of monomers include the achiral 2-aminoisobutyric acid (Aib) with geminal dimethyl substitution and chiral analogues with one methyl and one non-methyl substituent. While Aib and chiral C_α-Me residues are both established helix promoting moieties, their comparative ability in this regard has not been quantitatively investigated. Addressing this gap would help to inform the use of these building blocks in the construction of peptide and protein mimetics as well as provide fundamental insights into consequences of backbone methylation on folding. Here, we report a quantitative comparison of the impacts of Aib and chiral αMe residues on the high-resolution folded structure and folding thermodynamics of a small helical protein. These results reveal a synergistic stabilizing effect arising from the presence of C_α methylation in conjunction with a C_α stereocenter.

Pancreatic ductal adenocarcinoma (PDAC) is marked by significant desmoplastic reactions, or the accumulation of excessive extracellular matrices. PDAC stroma has abnormally high stiffness, which alters cancer cell behaviors and creates a barrier for effective drug delivery. Unfortunately, clinical trials using a combination of chemotherapy and matrix-degrading enzyme have led to disappointing results, as the degradation of stromal tissue likely accelerated the dissemination of cancer cells. High matrix stiffness has been shown to activate cancer-associated fibroblasts (CAFs), increasing their interaction with pancreatic cancer cells (PCCs) through promoting proliferation, migration, and resistance to chemotherapy. With the advance of biomaterials science and engineering, it is now possible to design chemically defined matrices to understand the role of stiffness in activating pancreatic CAFs and how this may alter cancer cell migration. Here, we developed a norbornene-based click hydrogel system with independently tunable stiffness and cell adhesive ligand to evaluate stiffness-induced activation of CAFs and migration of PCCs. Our results show that matrix stiffness did not alter matrix deposition from CAFs but affected nuclear localization of Yes-associated protein (YAP). Our results also verify the role of CAFs on promoting PCC migration and an elevated substrate stiffness further increased PCC motility.

Membrane proteins play a crucial role in a variety of biological processes and are key targets for pharmaceutical development. Structural studies of membrane proteins provide molecular insights into the mechanisms of these processes and are essential for effective drug discovery. Historically, these studies have relied on solubilization of the target protein using detergents, but conventional detergents often fail to maintain the stability of challenging membrane proteins. To address this issue, there is a need to develop novel detergents with enhanced protein stabilization properties. In this study, we synthesized unsymmetric variants of recently reported tris(hydroxymethyl)aminomethane(TRIS)-linker-bearing triazine-based triglucosides (TTGs) by incorporating two different alkyl chains (long and short) into the detergent structure. When tested with model membrane proteins, including a G protein-coupled receptor, TTG-8,12 demonstrated superior efficacy in stabilizing membrane proteins compared to the original TTGs and the gold standard detergents DDM/LMNG. These results suggest that detergent unsymmetry is an important concept for improving detergent performance and unsymmetric detergents such as TTG-8,12 hold significant potential for advancing membrane protein structural studies.

DNA double crossover (DX) motifs including DAE (double crossover, antiparallel, even spacing) and DAO (double crossover, antiparallel, odd spacing) are well-known monolayered DNA building blocks for construction of 2D DNA arrays and tubes in nanoscale and microscale. Compared to the 3D architectures of DNA origami and single-stranded DNA bricks to build nanoscale 3D bundles, tessellations, gears, castles, etc., designs of double- and multi-layers of DX motifs for 3D architectures are still limited. Herein, we report two types of double-layered tiles derived from DAE motifs with single-stranded circular 42- and 64-nt oligonucleotides as scaffold strands. Further tiling of the tiles generated planar 3D crystalline domains and curved tubes, correspondingly. Finally, we applied the chiral index theory to derive the unit tube parameters of six E-tiling (inter-tile distance of even spacing) tubes and analyzed the causation of difference between these tubes.

Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein Cas9 system has been widely used for genome editing. However, the editing or cleavage specificity of CRISPR Cas9 remains a major concern due to the off-target effects. The existing approaches to control or modulate CRISPR Cas9 cleavage include engineering Cas9 protein and development of anti-CRISPR proteins. There are also attempts on direct modification of sgRNA, for example, structural modification via truncation or hairpin design, or chemical modification on sgRNA such as partially replacing RNA with DNA. The above-mentioned strategies rely on extensive protein engineering and direct chemical or structural modification of sgRNA. In this study, we proposed an indirect method to modulate CRISPR Cas9 cleavage without modification on Cas9 protein or sgRNA. An oligonucleotide was used to form an RNA-DNA hybrid structure with the sgRNA spacer, creating steric hindrance during the Cas9 mediated DNA cleavage process. We first introduced a simple and robust method to assemble the oligo-ribonucleoprotein (oligo-RNP). Next, the cleavage efficiency of the assembled oligo-RNP was examined using different oligo lengths in vitro. Lastly, we showed that the oligo-RNP directly delivered into cells could also modulate Cas9 activity inside cells using three model gene targets with reduced off-target effects.

Marine natural products show a large variety of unique chemical structures and potent biological activities. Elucidating the target molecule and the mechanism of action is an essential and challenging step in drug development starting with a natural product. Odoamide, a member of aurilide-family isolated from Okinawan marine cyanobacterium, has been known to exhibit highly potent cytotoxicity against various cancer cell lines. In this study, we investigated the target protein and the cytotoxic mechanism of odoamide. Compared to existing anticancer agents, odoamide showed a unique fingerprint in the JFCR39 cancer cell panel and a characteristic pattern in gene expression profiling. Affinity chromatography utilizing a biologically active odoamide probe identified ATPase Na⁺/K⁺ transporting subunit alpha 1 (ATP1A1) as a specific binding protein. Additionally, cells resistant to odoamide were found to have mutations at Gly98 and Gly99 of the ATP1A1 protein. The apparently attenuated cytotoxic and apoptotic activities of odoamide in odoamide-resistant cells suggests that the induction of these activities by odoamide is critically dependent on its interaction with ATP1A1. We conclude that odoamide induces apoptotic cell death by targeting ATP1A1, and we discuss the impact of affinity-based target identification for natural products and the potential of ATP1A1 inhibitors for drug discovery.

In this study, we have successfully synthesized bis (cholesterol-dibenzo-18-crown-6-ether)-pillar[5]arene compound 1 through a click reaction, which could spontaneously insert into lipid bilayers to form ion channel due to the membrane anchor cholesterol group and show significant transport activity of K⁺ superior to Na⁺, with a permeability ratio of K⁺/Na⁺ equal to 4.58. Compound 1 two crown ether modules act as selective filters similar to natural K⁺ channel, which are determined to 1 : 2 binding stoichiometry to K⁺ by Job's plot and NMR titration. This structurally unambiguously unimolecule artificial channel provides ideas for constructing highly K⁺/Na⁺ selective molecular filters.