M24B aminopeptidase inhibitors selectively activate the CARD8 inflammasome

Nat Chem Biol 2022 May;18(5):565-574.PMID:35165443DOI:10.1038/s41589-021-00964-7.

Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.

Optimized M24B Aminopeptidase Inhibitors for CARD8 Inflammasome Activation

J Med Chem 2023 Feb 23;66(4):2589-2607.PMID:PMC10149104DOI:10.1021/acs.jmedchem.2c01535.

Inflammasomes are innate immune signaling platforms that trigger pyroptotic cell death. NLRP1 and CARD8 are related human inflammasomes that detect similar danger signals, but NLRP1 has a higher activation threshold and triggers a more inflammatory form of pyroptosis. Both sense the accumulation of intracellular peptides with Xaa-Pro N-termini, but Xaa-Pro peptides on their own without a second danger signal only activate the CARD8 inflammasome. We recently reported that a dual inhibitor of the Xaa-Pro-cleaving M24B aminopeptidases PEPD and XPNPEP1 called CQ31 selectively activates the CARD8 inflammasome by inducing the build-up of Xaa-Pro peptides. Here, we performed structure-activity relationship studies on CQ31 to develop the optimized dual PEPD/XPNPEP1 inhibitor CQ80 that more effectively induces CARD8 inflammasome activation. We anticipate that CQ80 will become a valuable tool to study the basic biology and therapeutic potential of selective CARD8 inflammasome activation.

High-level expression and enzymatic properties of a novel thermostable xylanase with high arabinoxylan degradation ability from Chaetomium sp. suitable for beer mashing

Int J Biol Macromol 2021 Jan 31;168:223-232.PMID:33309660DOI:10.1016/j.ijbiomac.2020.12.040.

A novel thermostable xylanase gene from Chaetomium sp. CQ31 was cloned and codon-optimized (CsXynBop). The deduced protein sequence of the gene shared the highest similarity of 75% with the glycoside hydrolase (GH) family 10 xylanase from Achaetomium sp. Xz-8. CsXynBop was over-expressed in Pichia pastoris GS115 by high-cell density fermentation, with the highest xylanase yield of 10,017 U/mL. The recombinant xylanase (CsXynBop) was purified to homogeneity and biochemically characterized. CsXynBop was optimally active at pH 6.5 and 85 °C, respectively, and stable over a broad pH range of 5.0-9.5 and up to 60 °C. The enzyme exhibited strict substrate specificity towards oat-spelt xylan (2, 489 U/mg), beechwood xylan (1522 U/mg), birchwood xylan (1067 U/mg), and showed relatively high activity towards arabinoxylan (1208 U/mg), but exhibited no activity on other tested polysaccharides. CsXynBop hydrolyzed different xylans to yield mainly xylooligosaccharides (XOSs) with degree of polymerization (DP) 2-5. The application of CsXynBop (200 U/g malt) in malt mashing substantially decreased the filtration time and viscosity of malt by 42.3% and 8.6%, respectively. These excellent characteristics of CsXynBop may make it a good candidate in beer industry.

Structural and biochemical insights into the substrate-binding mechanism of a glycoside hydrolase family 12 β-1,3-1,4-glucanase from Chaetomium sp

J Struct Biol 2021 Sep;213(3):107774.PMID:34329700DOI:10.1016/j.jsb.2021.107774.

β-1,3-1,4-Glucanases are a type of hydrolytic enzymes capable of catalyzing the strict cleavage of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in β-D-glucans and have exhibited great potential in food and feed industrials. In this study, a novel glycoside hydrolase (GH) family 12 β-1,3-1,4-glucanase (CtGlu12A) from the thermophilic fungus Chaetomium sp. CQ31 was identified and biochemically characterized. CtGlu12A was most active at pH 7.5 and 65 °C, respectively, and exhibited a high specific activity of 999.9 U mg-1 towards lichenin. It maintained more than 80% of its initial activity in a wide pH range of 5.0-11.0, and up to 60 °C after incubation at 55 °C for 60 min. Moreover, the crystal structures of CtGlu12A with gentiobiose and tetrasccharide were resolved. CtGlu12A had a β-jellyroll fold, and performed retaining mechanism with two glutamic acids severing as the catalytic residues. In the complex structure, cellobiose molecule showed two binding modes, occupying subsites -2 to -1 and subsites + 1 to + 2, respectively. The concave cleft made mixed β-1,3-1,4-glucan substrates maintain a bent conformation to fit into the active site. Overall, this study is not only helpful for the understanding of the substrate-binding model and catalytic mechanism of GH 12 β-1,3-1,4-glucanases, but also provides a basis for further enzymatic engineering of β-1,3-1,4-glucanases.

High level expression of a novel β-mannanase from Chaetomium sp. exhibiting efficient mannan hydrolysis

Carbohydr Polym 2012 Jan 4;87(1):480-490.PMID:34662993DOI:10.1016/j.carbpol.2011.08.008.

A novel β-mannanase gene (CsMan5A) was cloned from Chaetomium sp. CQ31 and expressed in Pichia pastoris. It had an open reading frame of 1251bp encoding 416 amino acids and contained two introns. The deduced amino acid sequence shared the highest similarity (73%) with the β-mannanase from Emericella nidulans and belongs to glycosyl hydrolase family 5. The recombinant β-mannanase (CsMan5A) was secreted at extremely high levels of 50,030UmL-1 and 6.1mgmL-1 in high cell density fermentor. The purified enzyme was optimally active at pH 5.0 and 65°C and displayed broad pH stability (pH 5.0-11.0) and exhibited specificity towards locust bean gum (Km=3.1mgmL-1), guar gum (Km=9.3mgmL-1) and konjac powder (Km=10.5mgmL-1). It efficiently degraded mannan polysaccharides into mannose and mannooligosacccharides, and also hydrolyzed mannotriose and mannotetraose. These properties make CsMan5A highly useful in food, feed and paper/pulp industries.