RAD18-catalysed formation of ubiquitination intermediate mimic of proliferating cell nuclear antigen PCNA.

The 2-((2-chloroethyl)amino)ethane-1-thiol (CAET)-based chemical trapping strategy is a practical tool for mechanistic studies of E3-catalysed ubiquitination. However, the construction of ubiquitination intermediate mimics (E2-Ub-substrate conjugates) via CAET has been limited to peptides, while its application to folded protein substrates remains unexplored. Here, we report that disulfide bond formation between E2-Ub (RAD6A-Ub) and the folded protein substrate PCNA (proliferating cell nuclear antigen) occurs upon the addition of the PCNA-associated E3 ligase RAD18.

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally Defined K63-Polyubiquitylated Linker Histone H1.

The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). In this study, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.

Synovial sarcoma X breakpoint 1 protein uses a cryptic groove to selectively recognize H2AK119Ub nucleosomes.

The cancer-specific fusion oncoprotein SS18-SSX1 disturbs chromatin accessibility by hijacking the BAF complex from the promoters and enhancers to the Polycomb-repressed chromatin regions. This process relies on the selective recognition of H2AK119Ub nucleosomes by synovial sarcoma X breakpoint 1 (SSX1). However, the mechanism underlying the selective recognition of H2AK119Ub nucleosomes by SSX1 in the absence of ubiquitin (Ub)-binding capacity remains unknown.

Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A.

Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B.

Flexural Behavior of Damaged Hollow RC Box Girders Repaired with Prestressed CFRP.

In recent years, numerous studies have explored the benefits of utilizing prestressed carbon fiber-reinforced polymer (CFRP) for strengthening concrete structures. However, research on the reinforcement of prestressed CFRP on full-scale hollow RC box girders, particularly damaged bridges, remains limited. In this study, both experiments and finite element analysis (FEA) were performed to investigate the flexural behavior of full-scale hollow RC box girders with varying degrees of damage, which were strengthened using CFRP with different levels of prestress.

Chemical Synthesis of Post-Translationally Modified H2AX Reveals Redundancy in Interplay between Histone Phosphorylation, Ubiquitination, and Methylation on the Binding of 53BP1 with Nucleosomes.

The chemical synthesis of homogeneously modified histones is a powerful approach to quantitatively decipher how post-translational modifications (PTMs) modulate epigenetic events. Herein, we describe the expedient syntheses of a selection of phosphorylated and ubiquitinated H2AX proteins in a strategy integrating expressed protein hydrazinolysis and auxiliary-mediated protein ligation. These modified H2AX proteins were then used to discover that although H2AXS139 phosphorylation can enhance the binding of the DNA damage repair factor 53BP1 to either an unmodified nucleosome or that bearing a single H2AXK15ub or H4K20me2 modification, it augments 53BP1's binding only weakly to nucleosomes bearing both H2AXK15ub and H4K20me2.

Met1-specific motifs conserved in OTUB subfamily of green plants enable rice OTUB1 to hydrolyse Met1 ubiquitin chains.

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive.

Flexural Behavior of Full-Scale Damaged Hollow RC Beams Strengthened with Prestressed SCFRP Plate under Four-Point Bending.

The advantages of using prestressed carbon fiber reinforced polymer (CFRP) for strengthening and retrofitting structures have been reported in recent years. In this regard, most of the studies on prestressed CFRP technique have been carried out in the laboratory test with small-scale and no damage (reinforced concrete) RC beam. However, the real structures that need to be retrofitted in service are often degraded or damaged due to early cracking.

H2B Lys34 Ubiquitination Induces Nucleosome Distortion to Stimulate Dot1L Activity.

Ubiquitination-dependent histone crosstalk plays critical roles in chromatin-associated processes and is highly associated with human diseases. Mechanism studies of the crosstalk have been of the central focus. Here our study on the crosstalk between H2BK34ub and Dot1L-catalyzed H3K79me suggests a novel mechanism of ubiquitination-induced nucleosome distortion to stimulate the activity of an enzyme.

The Bre1/Rad6 machinery: writing the central histone ubiquitin mark on H2B and beyond.

Mono-ubiquitination on H2B (H2Bub1) is an evolutionarily conserved histone post-translational modification implicated in various important physiological processes including DNA replication, transcription activation, and DNA damage repair. The Bre1/Rad6 ubiquitination machinery is currently considered to be the sole writer of H2Bub1, but the mechanistic basis by which it operates is unclear. Recently, the RING-type E3 ligase Bre1 was proposed to associate with the E2 enzyme Rad6 through a novel interaction between Bre1 RBD (Rad6 binding domain) and Rad6; and the RING domain of Bre1 that is responsible for the nucleosomal acidic patch binding also interacts with Rad6 to stimulate its catalytic activity.

Mechanical Performance of Steel Slag Concrete under Biaxial Compression.

The mechanical performance of steel slag concrete (SSC) under biaxial compression is investigated by a servo-controlled static-dynamic true triaxial machine (TAWZ-5000/3000). Three replacement ratios of steel slags and four kinds of stress ratio (0.25:1, 0.

Experimental Investigation on the Impact Resistance of Carbon Fibers Reinforced Coral Concrete.

In this study, the impact resistance of coral concrete with different carbon fiber (CF) dosages subjected to drop-weight impact test was investigated. For this purpose, three concrete strength grades (C20, C30, C40) and six CF dosages (0.0%, 0.