Protein degrons and degradation: Exploring substrate recognition and pathway selection in plants.

Proteome composition is dynamic and influenced by many internal and external cues, including developmental signals, light availability, or environmental stresses. Protein degradation, in synergy with protein biosynthesis, allows cells to respond to various stimuli and adapt by reshaping the proteome. Protein degradation mediates the final and irreversible disassembly of proteins, which is important for protein quality control and to eliminate misfolded or damaged proteins, as well as entire organelles. Consequently, it contributes to cell resilience by buffering against protein or organellar damage caused by stresses. Moreover, protein degradation plays important roles in cell signaling, as well as transcriptional and translational events. The intricate task of recognizing specific proteins for degradation is achieved by specialized systems that are tailored to the substrate's physicochemical properties and subcellular localization. These systems recognize diverse substrate cues collectively referred to as "degrons," which can assume a range of configurations. They are molecular surfaces recognized by E3 ligases of the ubiquitin-proteasome system but can also be considered as general features recognized by other degradation systems, including autophagy or even organellar proteases. Here we provide an overview of the newest developments in the field, delving into the intricate processes of protein recognition and elucidating the pathways through which they are recruited for degradation.

Molecular-Sieving Separation of Methanol/Benzene Azeotrope by a Flexible Metal-Organic Framework.

Separation of methanol/benzene azeotrope mixtures is very challenging not only by the conventional distillation technique but also by adsorbents. In this work, we design and synthesize a flexible Ca-based metal-organic framework MAF-58 consisting of cheap raw materials. MAF-58 shows selective methanol-induced pore-opening flexibility. Although the opened pores are large enough to accommodate benzene molecules, MAF-58 shows methanol/benzene molecular sieving with ultrahigh experimental selectivity, giving 5.1 mmol g-1 high-purity (99.99%+) methanol and 2.0 mmol g-1 high-purity (99.97%+) benzene in a single adsorption/desorption cycle. Computational simulations reveal that the preferentially adsorbed, coordinated methanol molecules act as the gating component to selectively block the diffusion of benzene, offering a new gating adsorption mechanism.

Human TDP1, APE1 and TREX1 repair 3'-DNA-peptide/protein cross-links arising from abasic sites in vitro.

Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3'-phospho-α,ß-unsaturated aldehyde (3'-PUA), to form unstable but long-lived Schiff base DNA-protein cross-links at 3'-DNA termini (3'-PUA-protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1's intrinsic redox capacity, yielding a stable 3'-PUA-PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3'-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3'-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3'-PUA-protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3'-PUA-histone DPCs, but not 3'-PUA-PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3'-PUA-protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.

Network Pharmacology Analysis of Liquid-Cultured Armillaria ostoyae Mycelial Metabolites and Their Molecular Mechanism of Action against Gastric Cancer.

Armillaria sp. are traditional edible medicinal mushrooms with various health functions; however, the relationship between their composition and efficacy has not yet been determined. Here, the ethanol extract of liquid-cultured Armillaria ostoyae mycelia (AOME), a pure wild Armillaria sp. strain, was analyzed using UHPLC-QTOF/MS, network pharmacology, and molecular docking techniques. The obtained extract affects various metabolic pathways, such as JAK/STAT and PI3K/AKT. The extract also contains important compounds such as 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide, isoliquiritigenin, and 7-hydroxycoumarin. Moreover, the extract targets key proteins, including EGFR, SCR, and IL6, to suppress the progression of gastric cancer, thereby synergistically inhibiting cancer development. The molecular docking analyses indicated that the main compounds stably bind to the target proteins. The final cell culture experimental data showed that the ethanol extract inhibited MGC-803 gastric cancer cells. In summary, our research revealed the beneficial components of AOME for treating gastric cancer and its associated molecular pathways. However, further research is needed to confirm its effectiveness and safety in gastric cancer patients.

In vitro eradication of abasic site-mediated DNA-peptide/protein cross-links by Escherichia coli long-patch base excision repair.

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA-protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5'-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5'-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2-36.1 kDa) DPCs is less efficient than that of an AP-peptide10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.

ECM1 is an essential factor for the determination of M1 macrophage polarization in IBD in response to LPS stimulation.

Inflammatory bowel disease (IBD) comprises chronic relapsing disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Here, we uncover a function of extracellular matrix protein 1 (ECM1) in promoting the pathogenesis of human and mouse IBD. ECM1 was highly expressed in macrophages, particularly tissue-infiltrated macrophages under inflammatory conditions, and ECM1 expression was significantly induced during IBD progression. The macrophage-specific knockout of ECM1 resulted in increased arginase 1 (ARG1) expression and impaired polarization into the M1 macrophage phenotype after lipopolysaccharide (LPS) treatment. A mechanistic study showed that ECM1 can regulate M1 macrophage polarization through the granulocyte-macrophage colony-stimulating factor/STAT5 signaling pathway. Pathological changes in mice with dextran sodium sulfate-induced IBD were alleviated by the specific knockout of the ECM1 gene in macrophages. Taken together, our findings show that ECM1 has an important function in promoting M1 macrophage polarization, which is critical for controlling inflammation and tissue repair in the intestine.

Water-Stable Metal Azolate Frameworks Showing Interesting Flexibilities for Highly Effective Bioethanol Dehydration.

The ethanol/water separation challenge highlights the adsorption capacity/selectivity trade-off problem. We show that the target guest can serve as a gating component of the host to block the undesired guest, giving molecular sieving effect for the adsorbent possessing large pores. Two hydrophilic/water-stable metal azolate frameworks were designed to compare the effects of gating and pore-opening flexibility. Large amounts (up to 28.7 mmol g-1 ) of ethanol with fuel-grade (99.5 %+) and even higher purities (99.9999 %+) can be produced in a single adsorption process from not only 95 : 5 but also 10 : 90 ethanol/water mixtures. More interestingly, the pore-opening adsorbent possessing large pore apertures showed not only high water adsorption capacity but also exceptionally high water/ethanol selectivity characteristic of molecular sieving. Computational simulations demonstrated the critical role of guest-anchoring aperture for the guest-dominated gating process.

Flexible Cuprous Triazolate Frameworks as Highly Stable and Efficient Electrocatalysts for CO2 Reduction with Tunable C2 H4 /CH4 Selectivity.

Cu-based metal-organic frameworks have attracted much attention for electrocatalytic CO2 reduction, but they are generally instable and difficult to control the product selectivity. We report flexible Cu(I) triazolate frameworks as efficient, stable, and tunable electrocatalysts for CO2 reduction to C2 H4 /CH4 . By changing the size of ligand side groups, the C2 H4 /CH4 selectivity ratio can be gradually tuned and inversed from 11.8 : 1 to 1 : 2.6, giving C2 H4 , CH4 , and hydrocarbon selectivities up to 51 %, 56 %, and 77 %, respectively. After long-term electrocatalysis, they can retain the structures/morphologies without formation of Cu-based inorganic species. Computational simulations showed that the coordination geometry of Cu(I) changed from triangular to tetrahedral to bind the reaction intermediates, and two adjacent Cu(I) cooperated for C-C coupling to form C2 H4 . Importantly, the ligand side groups controlled the catalyst flexibility by the steric hindrance mechanism, and the C2 H4 pathway is more sensitive than the CH4 one.

Proteasome-associated HECT-type ubiquitin ligase activity is required for plant immunity.

Regulated degradation of proteins by the 26S proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-26S proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. Here we show that specific members of the HECT domain-containing family of ubiquitin protein ligases (UPL) play important roles in proteasomal substrate processing during plant immunity. Mutations in UPL1, UPL3 and UPL5 significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of upl3 mutants indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. UPL3 was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-26S proteasome pathway components. In agreement, we demonstrate that UPL3 enabled proteasomes to form polyubiquitin chains, thereby regulating total cellular polyubiquitination levels. Taken together, our findings suggest that proteasome-associated ubiquitin ligase activity of UPL3 promotes proteasomal processivity and is indispensable for development of plant immunity.

Construction of Cationic Azahelicenes: Regioselective Three-Component Annulation Using In Situ Activation Strategy.

Described herein is a strategy to construct cationic azahelicenes through the three-component annulation reaction of isoquinoline, indole, and 1,2-dichloroethane (DCE), in which DCE serves as an in situ activating agent for C1-H activation of isoquinoline, a vinyl equivalent, and a solvent. This in situ activation annulation reaction features a facile one-step synthesis and complete regioselectivity. The complete regioselectivity of C1 over C3 for the isoquinoline ring paves a path to the helical structure in a highly ordered sequence. One of the synthesized ionic [5]azahelicenium fluorophores exhibits the potential to serve as a mitochondria-targeted biomarker with good photostability and low cytotoxicity.

Multicomponent Reactions of Pyridines To Give Ring-Fused Pyridiniums: In Situ Activation Strategy Using 1,2-Dichloroethane as a Vinyl Equivalent.

Reported herein is a rhodium(III)-catalyzed three-component annulation reaction of simple pyridines, alkynes, and 1,2-dichloroethane (DCE), affording a streamlined pathway to diverse ring-fused pyridiniums. DCE not only serves as a vinyl equivalent but also as an in situ activating agent for pyridine C2-H activation. A cationic five-membered rhodacycle complex has been isolated and proposed as a possible intermediate. This strategy can be extended to other N-containing heteroarenes for the synthesis of multiring-fused pyridiniums. These multicomponent reactions exhibit excellent regioselectivity for 1,3-diynes, paving a path to the cascade cyclization of 3-fluoropyridine or N-methylpyridin-3-amine with 1,3-diynes for the construction of brand-new tricyclic-fused pyrano- or hydropyridoquinolizinium salts. These ionic fluorophores have been investigated as potential biomarkers.

An Ultrastable Metal Azolate Framework with Binding Pockets for Optimal Carbon Dioxide Capture.

In the evolution of metal-organic frameworks (MOFs) for carbon capture, a lasting challenge is to strike a balance between high uptake capacity/selectivity and low energy cost for regeneration. Meanwhile, these man-made materials have to survive from practical demands such as stability under harsh conditions and feasibility of scale-up synthesis. Reported here is a new MOF, Zn(imPim) (aka. MAF-stu-1), with an imidazole derivative ligand, featuring binding pockets that can accommodate CO2 molecules in a fit-like-a-glove manner. Such a high degree of shape complementarity allows direct observation of the loaded CO2 in the pockets, and warrants its optimal carbon capture performances exceeding the best-performing MOFs nowadays. Coupled with the record thermal (up to 680 °C) and chemical stability, as well as rapid large-scale production, both encoded in the material design, Zn(imPim) represents a most competitive candidate to tackle the immediate problems of carbon dioxide capture.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 2 controls floral organ development and plant fertility by activating ASYMMETRIC LEAVES 2 in Arabidopsis thaliana.

A network of genes is coordinately expressed to ensure proper development of floral organs and fruits, which are essential for generating new offspring in flowering plants. In Arabidopsis thaliana, microRNA156 (miR156) plays a role in regulating the development of flowers and siliques by targeting members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family. Despite the important roles of the miR156/SPL network, our understanding of its downstream genes that are involved in floral organ and silique growth is still incomplete. Here, we report that the miR156/SPL2 regulatory pathway regulates pollen production, fertility rate, and the elongation of floral organs, including petals, sepals, and siliques in Arabidopsis. Transgenic plants exhibiting both overexpression of miR156 and dominant-negative alleles of SPL2 had reduced ASYMMETRIC LEAVES 2 (AS2) transcript levels in their siliques. Furthermore, their fertility phenotype was similar to that of the AS2 loss-of-function mutant. We also demonstrate that the SPL2 protein binds to the 5'UTR of the AS2 gene in vivo, indicating that AS2 is directly regulated by SPL2. Our results suggest that the miR156/SPL2 pathway affects floral organs, silique development and plant fertility, as well as directly regulates AS2 expression.

Ectopic expression of miR156 represses nodulation and causes morphological and developmental changes in Lotus japonicus.

The effects of microRNA156 overexpression on general plant architecture, branching, flowering time and nodulation were investigated in the model legume, Lotus japonicus. We cloned an miR156 homolog, LjmiR156a, from L. japonicus, and investigated its SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes and its biological function at enhancing vegetative biomass yield, extending flowering time, and its impact on nodulation. Thirteen potential targets for LjmiR156 were identified in vitro and their expression profiles were determined in aerial and underground parts of mature plants, including genes coding for eight SPLs, one WD-40, one RNA-directed DNA polymerase, two transport proteins, and one histidine-phosphotransfer protein. Two SPL and one WD-40 cleavage targets for LjmiR156-TC70253, AU089191, and TC57859-were identified. Transgenic plants with ectopic expression of LjmiR156a showed enhanced branching, dramatically delayed flowering, underdeveloped roots, and reduced nodulation. We also examined the transcript levels of key genes involved in nodule organogenesis and infection thread formation to determine the role of miR156 in regulating symbiosis. Overexpression of LjmiR156a led to repression of several nodulation genes during the early stages of root development such as three ENOD genes, SymPK, POLLUX, CYCLOPS, Cerberus, and Nsp1, and the stimulation of NFR1. Our results show that miR156 regulates vegetative biomass yield, flowering time and nodulation by silencing downstream target SPLs and other genes, suggesting that the miR156 regulatory network could be modified in forage legumes (such as alfalfa and trefoils) and in leafy vegetables (like lettuce and spinach) to positively impact economically valuable crop species.

Designing dynamic coordination bonds in polar hybrid crystals for a high-temperature ferroelastic transition.

Ferroelastic materials have gained widespread attention as promising candidates for mechanical switches, shape memory, and information processing. Their phase-transition mechanisms usually originate from conventional order-disorder and/or displacive types, while those involving dynamic coordination bonds are still scarce. Herein, based on a strategic molecular design of organic cations, we report three new polar hybrid crystals with a generic formula of AA'RbBiCl6 (A = A' = Me3SO+ for 1; A = Me3SO+ and A' = Me4N+ for 2; A = A' = Me3NNH2+ for 3). Their A-site cations link to the [RbBiCl6]n2n- inorganic framework with lon topology through Rb-O/N coordination bonds, while their significantly different interactions between A'-site cations and inorganic frameworks provide distinct phase-transition behaviour. In detail, the strongly coordinative A'-site Me3SO+ cations prevent 1 from a structural phase transition, while coordinatively free A'-site Me4N+ cations trigger a conventional order-disorder ferroelastic transition at 247 K in 2, accompanied by a latent heat of 0.63 J g-1 and a usual "high → low" second-harmonic-generation (SHG) switch. Interestingly, the A'-site Me3NNH2+ cations in 3 reveal unusual dynamic coordination bonds, driving a high-temperature ferroelastic transition at 369 K with a large latent heat of 18.34 J g-1 and an unusual "low → high" SHG-switching behaviour. This work provides an effective molecular assembly strategy to establish dynamic coordination bonds in a new type of host-guest model and opens an avenue for designing advanced ferroelastic multifunctional materials.

Celastrol inhibits oligodendrocyte and neuron ferroptosis to promote spinal cord injury recovery.

BACKGROUND: Spinal cord injury (SCI) is a traumatic injury to the central nervous system and can cause lipid peroxidation in the spinal cord. Ferroptosis, an iron-dependent programmed cell death, plays a key role in the pathophysiology progression of SCI. Celastrol, a widely used antioxidant drug, has potential therapeutic value for nervous system. PURPOSE: To investigate whether celastrol can be a reliable candidate for ferroptosis inhibitor and the molecular mechanism of celastrol in repairing SCI by inhibiting ferroptosis. METHODS: First, a rat SCI model was constructed, and the recovery of motor function was observed after treatment with celastrol. The regulatory effect of celastrol on ferroptosis pathway Nrf2-xCT-GPX4 was detected by Western blot and immunofluorescence. Finally, the ferroptosis model of neurons and oligodendrocytes was constructed in vitro to further verify the mechanism of inhibiting ferroptosis by celastrol. RESULTS: Our results demonstrated that celastrol promoted the recovery of spinal cord tissue and motor function in SCI rats. Further in vitro and in vivo studies showed that celastrol significantly inhibited ferroptosis in neurons and oligodendrocytes and reduced the accumulation of ROS. Finally, we found that celastrol could inhibit ferroptosis by up-regulating the Nrf2-xCT-GPX4 axis to repair SCI. CONCLUSION: Celastrol effectively inhibits ferroptosis after SCI by upregulating the Nrf2-xCT-GPX4 axis, reducing the production of lipid ROS, protecting the survival of neurons and oligodendrocytes, and improving the functional recovery.

ROS-responsive nanoparticle delivery of ferroptosis inhibitor prodrug to facilitate mesenchymal stem cell-mediated spinal cord injury repair.

Spinal cord injury (SCI) is a traumatic condition that results in impaired motor and sensory function. Ferroptosis is one of the main causes of neural cell death and loss of neurological function in the spinal cord, and ferroptosis inhibitors are effective in reducing inflammation and repairing SCI. Although human umbilical cord mesenchymal stem cells (Huc-MSCs) can ameliorate inflammatory microenvironments and promote neural regeneration in SCI, their efficacy is greatly limited by the local microenvironment after SCI. Therefore, in this study, we constructed a drug-release nanoparticle system with synergistic Huc-MSCs and ferroptosis inhibitor, in which we anchored Huc-MSCs by a Tz-A6 peptide based on the CD44-targeting sequence, and combined with the reactive oxygen species (ROS)-responsive drug nanocarrier mPEG-b-Lys-BECI-TCO at the other end for SCI repair. Meanwhile, we also modified the classic ferroptosis inhibitor Ferrostatin-1 (Fer-1) and synthesized a new prodrug Feborastatin-1 (Feb-1). The results showed that this treatment regimen significantly inhibited the ferroptosis and inflammatory response after SCI, and promoted the recovery of neurological function in rats with SCI. This study developed a combination therapy for the treatment of SCI and also provides a new strategy for the construction of a drug-coordinated cell therapy system.

Analysis of Proteasome-Associated Ubiquitin Ligase Activity.

The ubiquitin-proteasome system (UPS) is the predominant protein degradation machinery in eukaryotic cells. It is highly conserved among eukaryotes and essential for their survival. Through regulated proteolysis the UPS plays a key role in a myriad of cellular functions, including developmental and stress signaling, cell differentiation, and cell death. Attachment of a ubiquitin chain to a substrate can trigger its recruitment to the proteasome for proteolysis. To efficiently degrade substrates, however, the proteasome employs HECT-type ubiquitin ligases that can further remodel ubiquitin chains of proteasome-captured substrates. It is thought that this remodeling process is necessary to maintain substrate affinity for the proteasome and to completely translocate the substrate into the 20S proteolytic barrel. Here, we describe a protocol for purifying proteasomes and their associated accessory proteins and provide a practical way to detect proteasome-associated E3 ligase activity. This assay is reliable and efficient for assessing the ability of proteasomes to form ubiquitin conjugates and is applicable to a wide range of eukaryotic species.

Exploration of the effect and potential mechanism of quercetin in repairing spinal cord injury based on network pharmacology and in vivo experimental verification.

Spinal cord injury (SCI) is a highly complex neurological disease, but there is no effective repair method. Quercetin is a flavonol drug and has a variety of biological activities, such as scavenging oxygen free radicals in the body to resist oxidation, inhibiting inflammation, and so on. In this study, quercetin was firstly demonstrated to reduce tissue damage, promote neuron survival and repair motor function after SCI in rats through in vivo experiments. Then, 293 potential targets of quercetin repair for SCI were predicted by network pharmacology. GO analysis revealed that the biological processes of potential targets focused mainly on signal transduction, negative regulation of the apoptotic process, protein phosphorylation, drug response, and so on. Similarly, KEGG analysis suggested that these potential targets were involved in cell growth regulation, differentiation, apoptosis, and a few metabolic pathways. PPI network analysis predicted that the key genes were EP300, CREBBP, SRC, HSP90AA1, TP53, PIK3R1, EGFR, ESR1, and CBL. Further, the molecular docking showed that quercetin binds well with these proteins. Finally, RT-qPCR and Western blotting experiments verified that quercetin downregulated the expression levels of PIK3R1 and EGFR. It is suggested that quercetin can repair SCI in rats through PI3K-AKT signaling pathway and EGFR/MAPK pathway, which may provide a new theoretical basis for the repair of spinal cord injury.

The discovery of the new mechanism: Celastrol improves spinal cord injury by increasing cAMP through VIP-ADCYAP1R1-GNAS pathway.

Spinal cord injury (SCI) is a debilitating condition that results in significant impairment of motor function and sensation. Despite the ongoing efforts to develop effective treatments, there are currently very limited options available for patients with SCI. Celastrol, a natural anti-inflammatory compound extracted from Tripterygium wilfordii, has been shown to exhibit anti-inflammatory and anti-apoptotic properties. In this study, we aimed to explore the therapeutic potential of celastrol for SCI and elucidate the underlying molecular mechanisms involved. We found that local tissue often experiences a significant decrease in cAMP content and occurrs apoptosis after SCI. However, the treatment of celastrol could promote the production of cAMP by up-regulating the VIP-ADCYAP1R1-GNAS pathway. This could effectively inhibit the phosphorylation of JNK and prevent apoptosis, ultimately improving the exercise ability after SCI. Together, our results reveal celastrol may be a promising therapeutic agent for the treatment of SCI.