Biochemistry Biochemistry最新文献

Dipeptide repeat proteins (DPRs) are aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative diseases characterized by the cytoplasmic mislocalization and aggregation of RNA-binding proteins (RBPs). In particular, arginine (R)-rich DPRs (poly-GR and poly-PR) have been suggested to promiscuously interact with multiple cellular proteins and thereby exert high cytotoxicity. Components of the protein arginine methylation machinery have been identified as modulators of DPR toxicity and/or potential cellular interactors of R-rich DPRs; however, the molecular details and consequences of such an interaction are currently not well understood. Here, we demonstrate that several members of the family of protein arginine methyltransferases (PRMTs) can directly interact with R-rich DPRs in vitro and in the cytosol. In vitro, R-rich DPRs reduce solubility and promote phase separation of PRMT1, the main enzyme responsible for asymmetric arginine-dimethylation (ADMA) in mammalian cells, in a concentration- and length-dependent manner. Moreover, we demonstrate that poly-GR interferes more efficiently than poly-PR with PRMT1-mediated arginine methylation of RBPs such as hnRNPA3. We additionally show by two alternative approaches that poly-GR itself is a substrate for PRMT1-mediated arginine dimethylation. We propose that poly-GR may act as a direct competitor for arginine methylation of cellular PRMT1 targets, such as disease-linked RBPs.

Mitomycins make up a class of natural molecules produced by Streptomyces with strong antibacterial and antitumor activities. MitM is a key postmitosane modification enzyme involved in mitomycin biosynthesis in Streptomyces caespitosus. This protein was previously suggested to catalyze the aziridinium methylation of mitomycin A and the mitomycin intermediate 9a-demethyl-mitomycin A as an N-methyltransferase. The structural basis for MitM to recognize cofactor S-adenosyl-l-methionine (SAM) and substrate mitomycin A is unknown. Here, we determined the crystal structures of apo-MitM and MitM-mitomycin A-S-adenosylhomocysteine (SAH) ternary complexes with resolutions of 2.23 and 2.80 Å, respectively. We found that MitM adopts a class I SAM-dependent methyltransferase fold and forms a homodimer in solution. Conformational changes in a series of residues involved in the formation of active pockets assist MitM in binding SAH and mitomycin A. In particular, the ₂₈ALGAASLGE₃₆ loop changes most significantly. When mitomycin A binds, the bending direction of this loop is reversed, changing the entrance of the active site from open to closed. This study provides structural insights into MitM’s involvement in the postmitosane stage of mitomycin biosynthesis and provides a template for the engineering of methyltransferases.

In previous work, we reported on iridium(III) (Ir(III)) complex-peptide hybrids as amphiphilic conjugates (IPH-ACs) and triptycene-peptide hybrids as amphiphilic conjugates (TPH-ACs) and found that these hybrid compounds containing three cationic KK(K)GG peptide units through C₆–C₈ alkyl linkers induce paraptosis II, which is one of the nonapoptotic programmed cell death (PCD) types in Jurkat cells and different from previously reported paraptosis. The details of that study revealed that the paraptosis II induced by IPH-ACs (and TPH-ACs) proceeds via a membrane fusion or tethering of the endoplasmic reticulum (ER) and mitochondria, and Ca²⁺ transfer from the ER to mitochondria, which results in a loss of mitochondrial membrane potential (ΔΨ_m) in Jurkat cells. However, the detailed mechanistic studies of paraptosis II have been conducted only in Jurkat cells. In the present work, we decided to conduct mechanistic studies of paraptosis II in HeLa-S3 and A549 cells as well as in Jurkat cells to study the general mechanism of paraptosis II. Simultaneously, we designed and synthesized new TPH-ACs functionalized with peptides that contain cyclohexylalanine, which had been reported to enhance the localization of peptides to mitochondria. We found that TPH-ACs containing cyclohexylalanine promote paraptosis II processes in Jurkat, HeLa-S3 and A549 cells. The results of the experiments using fluorescence Ca²⁺ probes in mitochondria and cytosol, fluorescence staining agents of mitochondria and the ER, and inhibitors of paraptosis II suggest that TPH-ACs induce Ca²⁺ increase in mitochondria and the membrane fusion between the ER and mitochondria almost simultaneously, suggesting that our previous hypothesis on the mechanism of paraptosis II should be revised.

Active site lids are common features of enzymes and typically undergo conformational changes upon substrate binding to promote catalysis. Iodotyrosine deiodinase is no exception and contains a lid segment in all of its homologues from human to bacteria. The solution-state dynamics of the lid have now been characterized using ¹⁹F NMR spectroscopy with a CF₃-labeled enzyme and CF₃O-labeled ligands. From two-dimensional ¹⁹F–¹⁹F NMR exchange spectroscopy, interconversion rates between the free and bound states of a CF₃O-substituted tyrosine (45 ± 10 s^–1) and the protein label (40 ± 3 s^–1) are very similar and suggest a correlation between ligand binding and conformational reorganization of the lid. Both occur at rates that are ∼100-fold faster than turnover, and therefore these steps do not limit catalysis. A simple CF₃O-labeled phenol also binds to the active site and induces a conformational change in the lid segment that was not previously detectable by crystallography. Exchange rates of the ligand (130 ± 20 s^–1) and protein (98 ± 8 s^–1) in this example are faster than those above but remain self-consistent to affirm a correlation between ordering of the lid and binding of the ligand. Both ligands also protect the protein from limited proteolysis, as expected from their ability to stabilize a compact lid structure. However, the minimal turnover of simple phenol substrates indicates that such stabilization may be necessary but is not sufficient for efficient catalysis.

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) ¹H–¹⁵N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed β7-β8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (K_d = 3–8 μM). Inhibitor 6c displayed tighter binding (K_d ∼ 0.3 μM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.

The intricate regulation of gene expression is fundamental to the biological complexity of higher organisms, and is primarily governed by transcriptional and post-transcriptional mechanisms. The 3′-untranslated region (3′UTR) of mRNA is rich in cis-regulatory elements like G-quadruplexes (G4s), and plays a crucial role in post-transcriptional regulation. G4s have emerged as significant gene regulators, impacting mRNA stability, translation, and localization. In this study, we investigate the role of a robust parallel G4 structure situated within the 3′UTR of CCN1 mRNA in post-transcriptional regulation. This G4 structure is proximal to the stop codon of human CCN1, and evolutionarily conserved. We elucidated its interaction with the insulin-like growth factor 2 binding protein 1 (IGF2BP1), a noncanonical RNA N6-methyladenosine (m6A) modification reader, revealing a novel interplay between RNA modifications and G-quadruplex structures. Knockdown experiments and mutagenesis studies demonstrate that IGF2BP1 binds specifically to the G4 structure, modulating CCN1 mRNA stability. Additionally, we unveil the role of IGF2BP1’s RNA recognition motifs in G4 recognition, highlighting this enthalpically driven interaction. Our findings offer fresh perspectives on the complex mechanisms of post-transcriptional gene regulation mediated by G4 RNA secondary structures.

Berberine bridge enzyme-like oxidases are often involved in natural product biosynthesis and are seen as essential enzymes for the generation of intricate pharmacophores. These oxidases have the ability to transfer a hydride atom to the FAD cofactor, which enables complex substrate modifications and rearrangements including (intramolecular) cyclizations, carbon–carbon bond formations, and nucleophilic additions. Despite the diverse range of activities, the mechanistic details of these reactions often remain incompletely understood. In this Review, we delve into the complexity that BBE-like oxidases from bacteria, fungal, and plant origins exhibit by providing an overview of the shared catalytic features and emphasizing the different reactivities. We propose four generalized modes of action by which BBE-like oxidases enable the synthesis of natural products, ranging from the classic alcohol oxidation reactions to less common amine and amide oxidation reactions. Exploring the mechanisms utilized by nature to produce its vast array of natural products is a subject of considerable interest and can lead to the discovery of unique biochemical activities.

Melanin biosynthesis in different organisms is performed by a tyrosinase action. Excessive enzyme activity and pigment accumulation result in different diseases and disorders including skin cancers, blemishes, and darkening. In fruits and vegetables, it causes unwanted browning of these products and reduces their appearance quality and economic value. Inhibiting enzyme activity and finding novel powerful and safe inhibitors are highly important in agriculture, food, medical, and pharmaceutical industries. In this regard, in the present study, some novel synthetic pyridine-based compounds including 2,6-bis (tosyloxymethyl) pyridine (compound 3), 2,6-bis (butylthiomethyl) pyridine (compound 4), and 2,6-bis (phenylthiomethyl) pyridine (compound 5) were synthesized for the first time, and their inhibitory potencies were assessed on mushroom tyrosinase diphenolase activity. The results showed that while all tested compounds significantly decreased the enzyme activity, compounds 4 and 5 had the highest inhibitory effects (respectively, 80 and 89% inhibition with the IC₅₀ values of 17.0 and 9.0 μmol L^–1), and the inhibition mechanism was mixed-type for both compounds. Ligand-binding studies were carried out by fluorescence quenching and molecular docking methods to investigate the enzyme–compound interactions. Fluorescence quenching results revealed that the compounds can form nonfluorescent complexes with the enzyme and result in quenching of its intrinsic emission by the static process. Molecular docking analyses predicted the binding positions and the amino acid residues involved in the interactions. These compounds appear to be suitable candidates for more studies on tyrosinase inhibition.

Collagen hydrolysis, catalyzed by Zn(II)-dependent matrix metalloproteinases (MMPs), is a critical physiological process. Despite previous computational investigations into the catalytic mechanisms of MMP-mediated collagenolysis, a significant knowledge gap in understanding remains regarding the influence of conformational sampling and entropic contributions at physiological temperature on enzymatic collagenolysis. In our comprehensive multilevel computational study, employing quantum mechanics/molecular mechanics (QM/MM) metadynamics (MetD) simulations, we aimed to bridge this gap and provide valuable insights into the catalytic mechanism of MMP-1. Specifically, we compared the full enzyme-substrate complex in solution, clusters in solution, and gas-phase to elucidate insights into MMP-1-catalyzed collagenolysis. Our findings reveal significant differences in the catalytic mechanism when considering thermal effects and the dynamic evolution of the system, contrasting with conventional static potential energy surface QM/MM reaction path studies. Notably, we observed a significant stabilization of the critical tetrahedral intermediate, attributed to contributions from conformational flexibility and entropy. Moreover, we found that protonation of the scissile bond nitrogen occurs via proton transfer from a Zn(II)-coordinated hydroxide rather than from a solvent water molecule. Following C-N bond cleavage, the C-terminus remains coordinated to the catalytic Zn(II), while the N-terminus forms a hydrogen bond with a solvent water molecule. Subsequently, the release of the C-terminus is facilitated by the coordination of a water molecule. Our study underscores the pivotal role of protein conformational dynamics at physiological temperature in stabilizing the transition state of the rate-limiting step and key intermediates, compared to the corresponding reaction in solution. These fundamental insights into the mechanism of collagen degradation provide valuable guidance for the development of MMP-1-specific inhibitors.