Project description:This experiment aimed to identify GH5/10/30 arabinoxylan and glucuronoxylan-specific xylanases from secretomes produced by Bacillus subtilis using decorated activity based probes.
Project description:This experiment aimed to identify new GH13 alpha-amylaases from a complex compost sample using activity based probes. The substrate specificities of these novel enzymes could also be assigned by using -1 and -2 branched activity based probes.
Project description:Ubiquitin-specific protease 30 (USP30) is a deubiquitylating enzyme (DUB) localized in the mitochondrial membrane, which is related to PINK1/Parkin-mediated mitophagy, pexophagy, BAX/BAK-dependent apoptosis, and IKKβ-USP30-ACLY-regulated lipogenesis/tumorigenesis. Mission therapeutics pointed their in-house screened MTX652 as USP30 inhibitor for Phase I clinical trial in early 2022. Activity-based probes (ABPs) provide a powerful tool for screen USP30 inhibitors. Here, we report the first small molecule ABPs (ABP 2 and ABP 4) for profiling activity of USP30. Through in-gel fluorescence, target-enrichment and proteomics analysis, we demonstrate that ABP 2 and ABP 4 selectively engage USP30 at nanomolar concentration for only 10 min incubation time in live cells. This cellular USP30-engagement is selectively depending on the catalytic cysteine of USP30. Interestingly, DESI1 and DESI2, the small ubiquitin-related modifier (SUMO) proteases, are also engaged by ABP 2 and ABP 4, providing the novel strategy for these probes as DESIs ABPs. We use proteomics analysis to identify the probes targeted proteins by DIA analysis.
Project description:Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides towards breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling (ABPP) provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of b-xylosidases and endo-xylanases in the secretome of Aspergillus niger, by the use of chemical probes inspired by the b-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
Project description:Xanthohumol, the principle prenylflavonoid found in hops (Humulus lupulus), is a potential anti-inflammatory agent in the gut. To investigate the interactions between xanthohumol and catabolic gut bacteria, and its role in the anti-inflammatory pathway, a suite of xanthohumol-derived activity-based probes (ABPs) was synthesized and characterized in a model gut microbe. Through direct alkylation of both xanthohumol and structural isomer isoxanthohumol, an alkyne or diazirine-alkyne linker was attached to multiple alkylation sites to generate a suite of click chemistry enabled ABPs. LC-MS/MS data was analyzed with MaxQuant. This research was conducted by the "Discovery and Biological Signatures of Microbiome-Derived Xanthohumol Metabolites and their Role in Ameliorating Inflammatory Bowel Disease" project under the National Center for Complementary and Integrative Health (NCCIH) grant (R01AT010271). User facility data acquisition was performed under the EMSL project award 51663 (10.46936/reso.proj.2020.51663/60000245) at the Environmental Molecular Sciences Laboratory (https://ror.org/04rc0xn13), a DOE Office of Science User Facility sponsored by the Biological and Environmental Research program under Contract No. DE-AC05-76RL01830.
Project description:The nucleophilic thiol-alkyne addition reaction can form a covalent thiovinyl bond with cysteine reactivity specific,as the classical standards for cysteine-reactive warheads in activity-based probes (ABPs).Utilizing this classical reactivity property, we designed two potentially reactive unnatural amino acids containing alkyne moiety in its side chain, named as 4-propargyloxy-L-phenylalanine (pPR) and enrichable 4-propynyloxy-L-phenylalanine (epPR),for capturing deubiquitinases or ubiquitin-like protein specific proteases in the ubiquitin (Ub) and ubiquitin-like (Ubl) modification.The technology was extended to polyubiquitin chains through Sortase A mediated transpeptidation using reduced N6-((2-azidoacetyl)glycyl)-L-lysine (AzGGK) to triglycine tail (GGK),constructing ubiquitin conjuates,such as di diubiquitin (Diub). Rare codon recoding techonology (RCR) can be used to incorporation unnatural amino acid to mammalian cell, utilizing the rare codons to introduce multiple unnatural amino acids into proteins, creating multireactive and high-efficiency crosslinking probes. Here,we genetically encoded and sortase-mediated transpeptidation to develope crosslinkable and enrichable Ub/Ubl/Diub ABPs in E.coli and HEK293T cells.Meanwhile,the alkyne cysteine specificity of the covalent binding to the deubiquitinases allows for the preliminary screening of pan and irreversible inhibitors.Since most chemical Ub/Ubl/Diub ABPs are generally not cell-permeable, we performed functional complementation studies using genetically encoded biosynthetic Ub/Ubl/Diub ABPs. Meanwhile, the insertion of unnatural amino acids at multiple sites of UFM1 multi-reactive crosslinking probe was realized to be expressed in living cells by using the RCR technology, providing a novel methodology for comprehensive mapping of UFM1 interaction networks.
Project description:We have examined and compared the transcriptome of T. reesei growing on wheat straw and lactose as carbon sources under otherwise similar conditions. Gene expression on wheat straw exceeded that on lactose, and 1619 genes were found to be only induced on wheat straw but not on lactose. They comprised 30 % of the CAZome, but were also enriched in genes associated with phospholipid metabolism, DNA synthesis and repair and iron homeostatis. Two thirds of the CAZome was expressed both on wheat straw as well as on lactose, but 60 % of it at least >2-fold higher on the former. Major wheat straw specific genes comprised xylanases, chitinases and M-CM-^_-mannosidases. Interestingly, the latter two CAZyme families were significantly higher expressed in a strain in which xyr1 encoding the major regulator of cellulase and hemicellulase biosynthesis is non-functional, demonstrating that XYR1 is a repressor of these genes. We used two biological replicas of four T. reesei strains growing on glucose, lactose, and on wheat straw