Sequential release of interacting proteins and Ub-modifying enzymes by disulfide heterotypic ubiquitin reagents.

Heterotypic ubiquitin (Ub) chains have emerged as fundamental components in a wide range of cellular processes. The integrative identification of Ub-interacting proteins (readers) and Ub-modifying enzymes (writers and erasers) that selectively recognize and regulate heterotypic ubiquitination may provide crucial insights into these processes. In this study, we employed the bifunctional molecule-assisted (CAET) strategy to develop a type of disulfide bond-activated heterotypic Ub reagents, which allowed to enrich heterotypic Ub-interacting proteins and modifying enzymes simultaneously. The sequential release of readers which are non-covalently bound and writers or erasers which are covalently conjugated by using urea and reductant, respectively, combined with label-free quantitative (LFQ) MS indicated that these heterotypic Ub reagents would facilitate future investigations into functional roles played by heterotypic Ub chains.

Effects of Combined Therapy of Olanzapine and Samidorphan on Safety and Metabolic Parameters in Schizophrenia Patients: A Meta-Analysis.

Purpose: This meta-analysis intended to evaluate the safety and metabolic effects of the combination of olanzapine (OLZ) and samidorphan (SAM) in the treatment of schizophrenia (SCZ) patients. Patients and Methods: We searched for the English and Chinese databases for randomized controlled trials (RCTs) on the OLZ combined with SAM for SCZ. The English databases included PubMed, Web of Science, EMbase, and Cochrane Library, however, Chinese databases included Chinese Biology Medicine (CBM), VIP, Wanfang, and China National Knowledge Infrastructure (CNKI). All database searches were due by May 31, 2023. Using Review Manager 5.4 software, a meta-analysis was conducted following a literature review and data extraction. Results: This study included five RCTs involving 1781 patients. Regarding safety, the meta-analysis revealed that the probability of weight gain was reduced in the OLZ and SAM group than in the OLZ group (RR = 0.83, 95% CI (0.69, 0.99), P < 0.05). Statistically, the incidence of severe adverse safety events, dry mouth, headache, drowsiness, death, and suicidal perception events was insignificant (P > 0.05); in terms of metabolism, compared with the OLZ group, the OLZ plus SAM group reduced total cholesterol (TC) levels (MD = -3.58, 95% CI (-6.81, -0.34), P < 0.05). However, it had no significant effect on metabolic indices, including low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides, glucose, and insulin index (P > 0.05). Conclusion: In patients with SCZ, treatment with the combination of OLZ and SAM decreased the incidence of weight gain adverse events and TC levels; nevertheless, it did not affect other adverse events or metabolic parameters. These findings provide clinicians with evidence-based guidance and support for drug selection. However, it is crucial to confirm these findings through further high-quality research.

A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system.

Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.

Thioester-Assisted Sortase-A-Mediated Ligation.

Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a C-terminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N- or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.

Structural insights into Ubr1-mediated N-degron polyubiquitination.

The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.

Chemical Synthesis of Activity-Based E2-Ubiquitin Probes for the Structural Analysis of E3 Ligase-Catalyzed Transthiolation.

Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.

Precise synchronization of hyperthermia-chemotherapy: photothermally induced on-demand release from injectable hydrogels of gold nanocages.

Though a therapeutic sequence plays a key role in tumor therapy, little attention has been paid to its influence on multimodal combined therapy. Herein, we developed gold nanocages (GNC@PNA-hls) decorated with two kinds of temperature sensitive p(N-isopropyl-acrylamide-acrylic acid) copolymers (PNA-hs and PNA-ls) for precise antitumor coordination of thermo-chemotherapy. Doxorubicin-loaded GNC@PNA-hls (Dox-GNC@PNA-hls) showed a steady photothermally induced on-demand release under multiple near-infrared (NIR) irradiations. In vitro evaluations indicated that concurrent thermo-chemotherapy treatments (Dox - L) showed the best antitumor effect, compared with the sequence of either doxorubicin treatment followed by NIR radiation (Dox + L) or NIR radiation followed by doxorubicin treatment (L + Dox). The in vivo antitumor efficacy also indicated that the tumor volume was totally suppressed (ca. 0.14 cm3) by the treatment of Dox-GNC@PNA-hls with NIR radiation for 14 days. These results indicated that Dox-GNC@PNA-hls could achieve precise synchronization between hyperthermia and chemotherapy, and effectively enhance their antitumor efficacy.