The Mevalonate Pathway Is a Druggable Target for Vaccine Adjuvant Discovery.

Motivated by the clinical observation that interruption of the mevalonate pathway stimulates immune responses, we hypothesized that this pathway may function as a druggable target for vaccine adjuvant discovery. We found that lipophilic statin drugs and rationally designed bisphosphonates that target three distinct enzymes in the mevalonate pathway have potent adjuvant activities in mice and cynomolgus monkeys. These inhibitors function independently of conventional "danger sensing." Instead, they inhibit the geranylgeranylation of small GTPases, including Rab5 in antigen-presenting cells, resulting in arrested endosomal maturation, prolonged antigen retention, enhanced antigen presentation, and T cell activation. Additionally, inhibiting the mevalonate pathway enhances antigen-specific anti-tumor immunity, inducing both Th1 and cytolytic T cell responses. As demonstrated in multiple mouse cancer models, the mevalonate pathway inhibitors are robust for cancer vaccinations and synergize with anti-PD-1 antibodies. Our research thus defines the mevalonate pathway as a druggable target for vaccine adjuvants and cancer immunotherapies.

A Type I-F Anti-CRISPR Protein Inhibits the CRISPR-Cas Surveillance Complex by ADP-Ribosylation.

CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

Thermally Stable and Chemically Recyclable Poly(ketal-ester)s Regulated by Floor Temperature.

Chemical recycling to monomers (CRM) offers a promising closed-loop approach to transition from current linear plastic economy toward a more sustainable circular paradigm. Typically, this approach has focused on modulating the ceiling temperature (Tc) of monomers. Despite considerable advancements, polymers with low Tc often face challenges such as inadequate thermal stability, exemplified by poly(γ-butyrolactone) (PGBL) with a decomposition temperature of ∼200 °C. In contrast, floor temperature (Tf)-regulated polymers, particularly those synthesized via the ring-opening polymerization (ROP) of macrolactones, inherently exhibit enhanced thermodynamic stability as the temperature increases. However, the development of those Tf regulated chemically recyclable polymers remains relatively underexplored. In this context, by judicious design and efficient synthesis of a biobased macrocyclic diester monomer (HOD), we developed a type of Tf -regulated closed-loop chemically recyclable poly(ketal-ester) (PHOD). First, the entropy-driven ROP of HOD generated high-molar mass PHOD with exceptional thermal stability with a Td,5% reaching up to 353 °C. Notably, it maintains a high Td,5% of 345 °C even without removing the polymerization catalyst. This contrasts markedly with PGBL, which spontaneously depolymerizes back to the monomer above its Tc in the presence of catalyst. Second, PHOD displays outstanding closed-loop chemical recyclability at room temperature within just 1 min with tBuOK. Finally, copolymerization of pentadecanolide (PDL) with HOD generated high-performance copolymers (PHOD-co-PPDL) with tunable mechanical properties and chemical recyclability of both components.

Fumarate suppresses B-cell activation and function through direct inactivation of LYN.

Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.

Novel primers to identify a wider diversity of butyrate-producing bacteria.

Butyrate-producing bacteria are a functionally important part of the intestinal tract flora, and the resulting butyric acid is essential for maintaining host intestinal health, regulating the immune system, and influencing energy metabolism. However, butyrate-producing bacteria have not been defined as a coherent phylogenetic group. They are primarily identified using primers for key genes in the butyrate-producing pathway, and their use has been limited to the Bacillota and Bacteroidetes phyla. To overcome this limitation, we developed functional gene primers able to identify butyrate-producing bacteria through the butyrate kinase gene, which encodes the enzyme involved in the final step of the butyrate-producing pathway. Genomes extracted from human and rat feces were used to amplify the target genes through PCR. The obtained sequences were analyzed using BLASTX to construct a developmental tree using the MEGA software. The newly designed butyrate kinase gene primers allowed to recognize a wider diversity of butyrate-producing bacteria than that recognized using currently available primers. Specifically, butyrate-producing bacteria from the Synergistota and Spirochaetota phyla were identified for the first time using these primers. Thus, the developed primers provide a more accurate method for researchers and doctors to identify potential butyrate-producing bacteria and deepen our understanding of butyrate-producing bacterial species.

Discovery of a first-in-class CDK2 selective degrader for AML differentiation therapy.

The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.

Photocaging of Activity-Based Ubiquitin Probes via a C-Terminal Backbone Modification Strategy.

Photocaged, activity-based ubiquitin (Ub) probes (Ub-ABPs) have been developed for the time-resolved probing of deubiquitinating enzyme (DUB) activities, but many Ub-ABPs are still challenging to photocage because their warheads (e.g. propargylamide (PA) or dehydroalanine (Dha)) are difficult to temporally block and activate. Here, we describe a new C-terminal backbone modification strategy for the construction of photocaged Ub-ABPs in which a light-sensitive group is placed at the backbone amide bond of the Ub Gly75. This strategy enabled the facile generation of cell-permeable photocaged Ub-PA and Dha probes that could be activated to capture DUBs after photo-irradiation, and were used to profile DUBs in cells under specially designed conditions (e.g. in cells experiencing oxidative stress) or DUBs with isopeptide linkage selectivity. This backbone modification strategy is anticipated to provide a general solution for the development of photocaged Ub ABPs bearing any warheads for DUB profiling.

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.

Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells.

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones.

Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz's auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5-6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BKC34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.

Efficient Semi-Synthesis of Atypical Ubiquitin Chains and Ubiquitin-Based Probes Forged by Thioether Isopeptide Bonds.

The development of powerful and general methods to acquire ubiquitin (Ub) chains has prompted the deciphering of Ub-mediated processes. Herein, the cysteine-aminoethylation assisted chemical ubiquitination (CAACU) strategy is extended and improved to enable the efficient semi-synthesis of atypical Ub chain analogues and Ub-based probes. Combining the Cys aminoethylation and the auxiliary-mediated protein ligation, several linkage- and length-defined atypical Ub chains including di-Ubs, K27C-linked tri-Ub, K11/K48C-branched tri-Ub, and even the SUMOlated Ub are successfully prepared from recombinantly expressed starting materials at about a 9-20 mg L-1 expression level. In addition, the utility of this strategy is demonstrated with the synthesis of a novel non-hydrolyzable di-Ub PA probe, which may provide a new useful tool for the mechanistic studies of deubiquitinase (DUB) recognition.

Discovery and characterization of a novel C-terminal peptide carboxyl methyltransferase in a lassomycin-like lasso peptide biosynthetic pathway.

Lasso peptides belong to a peculiar family of ribosomally synthesized and post-translationally modified peptides (RiPPs)-natural products with an unusual isopeptide-bonded slipknot structure. Except for assembling of this unusual lasso fold, several further post-translational modifications of lasso peptides, including C-terminal methylation, phosphorylation/poly-phosphorylation, citrullination, and acetylation, have been reported recently. However, most of their biosynthetic logic have not been elucidated except the phosphorylated paeninodin lasso peptide. Herein, we identified two novel lassomycin-like lasso peptide biosynthetic pathways and, for the first time, characterized a novel C-terminal peptide carboxyl methyltransferase involved in these pathways. Our investigations revealed that this new family of methyltransferase could specifically methylate the C terminus of precursor peptide substrates, eventually leading to lassomycin-like C-terminal methylated lasso peptides. Our studies offer another rare insight into the extraordinary strategies of chemical diversification adopted by lasso peptide biosynthetic machinery and predicated two valuable sources for methylated lasso peptide discovery.

GATA3 acetylation at K119 by CBP inhibits cell migration and invasion in lung adenocarcinoma.

GATA3 is a transcriptional factor involved in the development of multiple organs. Post translational modifications of GATA3 are critical to its function. Here, we report that GATA3 interacts with and is acetylated by the acetyltransferase CBP. Class I deacetylases HDAC1, HDAC2 and HDAC3 deacetylate GATA3. The major acetylated site of GATA3 in lung adenocarcinoma cells was determined at lysine 119 (AcK119). Functionally, GATA3-acetylation mimics K119Q mutant was found to inhibit lung adenocarcinoma cell migration and invasion with concomitant downregulation of EMT-controlling transcriptional factors Slug, Zeb1 and Zeb2. Taken together, we demonstrated that GATA3 acetylation at lysine 119 by CBP hinders the migration and invasion of lung adenocarcinoma cells.

Quantitative protein profiling and pathway analysis of spinal arteriovenous malformations.

Spinal arteriovenous malformations (sAVM) are rare and heterogeneous group of blood vessel disorders that affect spinal cord function directly or indirectly; however, the pathogenesis of sAVM is still unclear. In this study, we compared four sAVM specimens obtained during surgery and donated control samples in a Tandem Mass Tag (TMT)-labeled proteomic analysis. We identified 3101 proteins, 654 of which were differentially expressed in sAVM samples compared with the controls. Of these, 96 proteins were upregulated and 358 proteins were downregulated. Gene ontology (GO) analysis revealed that extracellular matrix organization in the biological process category and integrin-binding proteins in the molecular function category were the most enriched items. Two significant differentially expressed proteins (MYLK and MMP9) were verified by Western blot analysis. The pathway analysis indicated that the differentially expressed proteins in the pathways of angiogenesis, focal adhesion and cytoplasmic ribosome contributed to sAVM. The changes in protein profiles identified in this proteomic study provide an improved understanding of the pathogenesis of sAVM. The proteomics data are available via ProteomeXchange with identifier PXD007982.

Application of panoramic radiographs in the diagnosis of temporomandibular disorders.

To evaluate the feasibility of temporomandibular disorder (TMD) diagnosis with panoramic radiography, and provide standardized data for artificial intelligence-assisted diagnosis by measuring the differences in the condylar and mandibular ramus heights. A total of 500 panoramic radiographs (219 male and 281 female participants) of healthy individuals were examined. The panoramic machine compatible measurement software, SCANORA 5.2.6, was used to measure the bilateral condylar height and mandibular ramus height, and SPSS 27.0 was used to calculate the left- and right-side differences in condylar height and mandibular ramus height of healthy individuals. Magnetic resonance images of the temporomandibular joint region obtained from 46 outpatients in the Stomatology Department were selected along with their corresponding panoramic radiographs. The left- and right-sided differences were measured and compared with the magnetic resonance imaging results. The measurement data are expressed as mean ±â€…standard deviation (mm). t Tests were used to analyze data from healthy male and healthy female groups. The findings revealed that while there was no significant difference (P > .05) in the height of the condyle between men and women, there was a significant difference (P  < .05) in the height of the mandibular ramus. In healthy population, the difference in height between the left and right condyle was 1.09 ±â€…0.99 mm. The difference in height of mandibular ramus in men was 1.26 ±â€…0.85 mm and that in women was 1.19 ±â€…0.87 mm. For the diagnosis of TMD, the sensitivity of panoramic radiographs was 94.74% (36/38), specificity was 75.00% (6/8), and diagnostic accuracy was 91.30% (42/46). The height of the right and left lateral condyles was not identical in healthy individuals, resulting in a discernible height discrepancy. In addition, the height of the mandibular ramus varied. By considering the left-right lateral height differences identified in this study along with clinical examination, it is possible to employ this metric as a preliminary screening tool for patients with TMD. Further, the use of panoramic radiographs for initial TMD screening is both viable and significant.

A cell-permeable Ub-Dha probe for profiling E1-E2-E3 enzymes in live cells.

Activity-based ubiquitin probes (Ub-ABPs) have recently been developed as effective tools for studying the capabilities of E1-E2-E3 enzymes, but most of them can only be used in cell lysates. Here, we report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which enable successful delivery into cells confirmed by a fluorophore at the N-terminus of Ub and live-cell fluorescence microscopy. A total of 18 E1-E2-E3 enzymes in live cells were labelled and enriched in combination with label-free quantification (LFQ) mass spectrometry. This work provided a new cell-penetrating Ub tool for studying the activity and function of Ub-related enzymes.

Fully Bio-based Poly(ketal-ester)s by Ring-opening Polymerization of a Bicylcic Lactone from Glycerol and Levulinic Acid.

A fully renewable bio-based bicyclic lactone containing a five-membered cyclic ketal moiety, 7-methyl-3,8,10-trioxabicyclo[5.2.1]decan-4-one (TOD), was synthesized through a two-step acid-catalyzed process from glycerol and levulinic acid. The ring-opening polymerization (ROP) of TOD at 30°C with benzyl alcohol (BnOH) as the initiator and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) as the catalyst can afford high molar mass PTOD with a cis-2.4-disubstitued 2-methyl 1,3-dioxolane moiety in its repeating unit. PTOD is an amorphous polymer with a glass transition temperature (Tg ) of 13°C. It can be hydrolyzed into structurally defined small molecules under acidic or basic conditions by the selective cleavage of either the cyclic ketal or the ester linkage respectively. The TBD-catalyzed copolymerization of L-lactide (L-LA) and TOD at -20°C was investigated. It was confirmed that L-LA polymerized quickly with racemization to form PLA, followed by a slow incorporation of TOD into the formed PLA chains via transesterification. By varying the feed ratios of L-LA to TOD, a series of random copolymers (PLA-co-PTOD) with different TOD incorporation ratios and tunable Tg s were obtained. Under acidic conditions, PLA-co-PTOD degrades much faster than PLA via the selective cleavage of the cyclic ketal linkages. This work provides insights for the development of more sustainable and acid-accelerated degradable alternatives to aliphatic polyesters.

Cancer-cell-derived fumarate suppresses the anti-tumor capacity of CD8+ T cells in the tumor microenvironment.

Metabolic alterations in the microenvironment significantly modulate tumor immunosensitivity, but the underlying mechanisms remain obscure. Here, we report that tumors depleted of fumarate hydratase (FH) exhibit inhibition of functional CD8+ T cell activation, expansion, and efficacy, with enhanced malignant proliferative capacity. Mechanistically, FH depletion in tumor cells accumulates fumarate in the tumor interstitial fluid, and increased fumarate can directly succinate ZAP70 at C96 and C102 and abrogate its activity in infiltrating CD8+ T cells, resulting in suppressed CD8+ T cell activation and anti-tumor immune responses in vitro and in vivo. Additionally, fumarate depletion by increasing FH expression strongly enhances the anti-tumor efficacy of anti-CD19 CAR T cells. Thus, these findings demonstrate a role for fumarate in controlling TCR signaling and suggest that fumarate accumulation in the tumor microenvironment (TME) is a metabolic barrier to CD8+ T cell anti-tumor function. And potentially, fumarate depletion could be an important strategy for tumor immunotherapy.

Gas chromatography-mass spectrometry analysis on compounds in volatile oils extracted from yuan zhi (radix polygalae) and shi chang pu (acorus tatarinowii) by supercritical CO2.

OBJECTIVE: To analyze the constituents of volatile oils extracted from Yuan Zhi (Radix Polygalae), Shi Chang Pu (Acorus Tatarinowii), and a mixture of the two herbs. METHODS: The volatile oils were extracted using supercritical fluid extraction (SFE) with CO2, and the constituents of the volatile oil extracts were analyzed by gas chromatography-mass spectrometry (GC-MS). The relative content of each component was calculated using peak area normalization. RESULTS: The optimized SFE conditions were 45 MPa at 35 degrees C for 2 h. Twenty-four compounds were identified in the extract from the Yuan Zhi (Radix Polygalae) and Shi Chang Pu (Acorus Tatarinowii) mixture, and six of these had relative contents >1. These compounds were 1,2-dimethoxy-4-(2-propenyl)-benzene; 1,2,3-trimethoxy-5-(2-propenyl)-benzene; beta-asarone; (Z,Z) 9,12-octadecadienoic acid; (Z) 6-octadecenoic acid; and ethyl oleate. Combination of the herbs increased the number of pharmacologically active substances in the extract and decreased the number of compounds with one benzene ring compared with the extracts from the individual herbs. CONCLUSION: These results indicate there is a synergistic relationship among the compounds in these herbs.