1,721,026 research outputs found
The big picture of chromatin biology by cryo-EM
No full textModifications of chromatin structure are one of the key mechanisms regulating epigenetic gene expression. Proteins involved in chromatin modification mainly function as large multi-subunit complexes, and each component in the complex contributes to the function and activity of the complex. However, little is known about the structures of whole complexes and the mechanisms by which the chromatin modifying complexes function, the functional roles of each component in the complexes, and how the complexes recognize the central unit of chromatin, the nucleosome. This lack of information is partially due to the lack of structural information for whole complexes. Recent advances in cryo-EM have begun to reveal the structures of whole chromatin modifying complexes that enable us to understand the big picture of chromatin biology. In this review, we discuss the recent discoveries related to the mechanisms of chromatin modifying complexes.
Caf1 regulates the histone methyltransferase activity of Ash1 by sensing unmodified histone H3
No full textAbstract Histone modifications are one of the many key mechanisms that regulate gene expression. Ash1 is a histone H3K36 methyltransferase and is involved in gene activation. Ash1 forms a large complex with Mrg15 and Caf1/p55/Nurf55/RbAp48 (AMC complex). The Ash1 subunit alone exhibits very low activity due to the autoinhibition, and the binding of Mrg15 releases the autoinhibition. Caf1 is a scaffolding protein commonly found in several chromatin modifying complexes and has two histone binding pockets: one for H3 and the other for H4. Caf1 has the ability to sense unmodified histone H3K4 residues using the H3 binding pocket. However, the role of Caf1 in the AMC complex has not been investigated. Here, we dissected the interaction among the AMC complex subunits, revealing that Caf1 uses the histone H4 binding pocket to interact with Ash1 near the histone binding module cluster. Furthermore, we showed that H3K4 methylation inhibits AMC HMTase activity via Caf1 sensing unmodified histone H3K4 to regulate the activity in an internucleosomal manner, suggesting that crosstalk between H3K4 and H3K36 methylation. Our work revealed a delicate mechanism by which the AMC histone H3K36 methyltransferase complex is regulated
The crystal structure of Capicua HMG‐box domain complexed with the ETV5‐DNA and its implications for Capicua‐mediated cancers
No full textCapicua (CIC) is a transcriptional repressor and functions downstream of the receptor tyrosine kinase (RTK) signaling pathway. Somatic mutations found in the HMG-box DNA binding domain in CIC have been implicated in several cancers such as oligodendroglioma, oligoastrocytoma, and adenocarcinoma. However, the molecular basis of the DNA binding of CIC and the effect of the somatic mutations found in cancers on DNA binding have not been investigated. Here, we report the crystal structure of the HMG-box domain of CIC complexed with its target DNA, the promoter of Ets Translocation Variant 5 (ETV5). The structure shows that the HMG-box domain has an L-shaped structure and recognizes the minor groove leading to DNA bending. Our structure combined with an electrophoretic mobility shift assay (EMSA) revealed that cancer-associated mutations in the HMG-box domain abrogate the interaction with DNA. These results provide the molecular insight into the DNA binding of CIC and reveal the effects of carcinogenic mutations on DNA binding.
Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage
No full textFacile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)(6)(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37 degrees C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4 h, 2 h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues.
Structural basis of histone H4 recognition by p55
No full textp55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. Here, we present a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a beta-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones
A pH-Responsive Virus-Like Particle as a Protein Cage for a Targeted Delivery
No full textA stimuli-responsive protein self-assembly offers promising utility as a protein nanocage for biotechnological and medical applications. Herein, the development of a virus-like particle (VLP) that undergoes a transition between assembly and disassembly under a neutral and acidic pH, respectively, for a targeted delivery is reported. The structure of the bacteriophage P22 coat protein is used for the computational design of coat subunits that self-assemble into a pH-responsive VLP. Subunit designs are generated through iterative computational cycles of histidine substitutions and evaluation of the interaction energies among the subunits under an acidic and neutral pH. The top subunit designs are tested and one that is assembled into a VLP showing the highest pH-dependent structural transition is selected. The cryo-EM structure of the VLP is determined, and the structural basis of a pH-triggered disassembly is delineated. The utility of the designed VLP is exemplified through the targeted delivery of a cytotoxic protein cargo into tumor cells in a pH-dependent manner. These results provide strategies for the development of self-assembling protein architectures with new functionality for diverse applications. A virus-like particle (VLP) showing pH-responsive assembly and disassembly is developed for targeted drug delivery through the computational design of bacteriophage P22 coat proteins. The underlying mechanism for the pH responsiveness of the designed VLP is demonstrated by determining its cryo-EM structure. The utility of the VLP is exemplified by the pH-dependent delivery of a tumor-targeting cytotoxic protein.
Dynamic Anchoring of the 3 '-End of the Guide Strand Controls the Target Dissociation of Argonaute-Guide Complex
No full textArgonaute (Ago) is the catalytic core of small RNA-based gene regulation. Despite plenty of mechanistic studies on Ago, the dynamical aspects and the mechanistic determinants of target mRNA binding and dissociation of Ago guide strand remain unclear. Here, by using single-molecule fluorescence resonance energy transfer (FRET) assays and Thermus thermophilus Ago (TtAgo), we reveal that the 3'-end of the guide strand dynamically anchors at and releases from the PAZ domain of Ago, and that the 3'-end anchoring of the guide strand greatly accelerates the target dissociation by destabilizing the guide-target duplex. Our results indicate that the target binding/dissociation of Ago-guide is executed through the dynamic interplays among Ago, guide, and target
Hydrophobic Residues near the Bilin Chromophore-Binding Pocket Modulate Spectral Tuning of Insert-Cys Subfamily Cyanobacteriochromes
No full textCyanobacteriochromes (CBCRs) are a subfamily of phytochrome photoreceptors found exclusively in photosynthetic cyanobacteria. Four CBCRs containing a second Cys in the insert region (insert-Cys) have been identified from the nonheterocystous cyanobacterium Microcoleus B353 (Mbr3854g4 and Mbl3738g2) and the nitrogen fixing, heterocystous cyanobacterium Nostoc punctiforme (NpF2164g3 and NpR1597g2). These insert-Cys CBCRs can sense light in the near-UV to orange range, but key residues responsible for tuning their colour sensitivity have not been reported. In the present study, near-UV/Green (UG) photosensors Mbr3854g4 (UG1) and Mbl3738g2 (UG2) were chosen for further spectroscopic analysis of their spectral sensitivity and tuning. Consistent with most dual-Cys CBCRs, both UGs formed a second thioether linkage to the phycocyanobilin (PCB) chromophore via the insert-Cys. This bond is subject to breakage and relinkage during forward and reverse photoconversions. Variations in residues equivalent to Phe that are in close contact with the PCB chromophore D-ring in canonical red/green CBCRs are responsible for tuning the light absorption peaks of both dark and photoproducts. This is the first time these key residues that govern light absorption in insert-Cys family CBCRs have been identified and characterised.
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