IF a structure-guided, pH-shifted variant of the Brevibacterium sterolicum cholesterol oxidase (UniProt P22628; PDB: 1I19) — engineered via (a) active-site cavity expansion guided by the human CYP7A1:7-ketocholesterol co-crystal structure to accommodate the C7-keto moiety of 7KC, and (b) rational substitution of the catalytic glutamate base (analogous to Glu361 in Streptomyces ChOx homologs) with aspartate (pKa ~3.9 vs. ~4.3) plus targeted removal of lysosomal-pH-incompatible active-site histidines via Gln substitution, and then delivered intralysosomally in aged (18–22 month) C57BL/6 male mice via intravenous administration of an insulin-like growth factor II (IGF-II) peptide fusion construct (Glycosylation-Independent Lysosomal Targeting, GILT) at 5 mg/kg bi-weekly —

THEN a ≥50% reduction in lysosomal 7-ketocholesterol (7KC) content within peritoneal and aortic macrophages, measured by filipin/7KC-specific immunofluorescence and LC-MS/MS of lysosomal fractions at 8 weeks post-treatment, accompanied by reduced lysosomal membrane permeabilization (LMP), decreased cathepsin B cytosolic release, and attenuation of NLRP3 inflammasome activation in ex vivo macrophage isolates, will be observed,

BECAUSE the following step-by-step causal chain operates:

1. 7KC accumulates in lysosomes of aged macrophages as an undegraded oxysterol, because no mammalian lysosomal hydrolase accepts it as a substrate — unlike native cholesterol, whose 3β-hydroxyl enables recognition by endogenous lipases, the C7-ketone of 7KC sterically and electronically disrupts recognition. Mammalian cells lack evolutionary pressure to select a catabolic enzyme for this substrate, leaving it to accumulate and drive lysosomal membrane permeabilization and downstream inflammasome activation, as established by the LysoSENS research context in the Evidence Set.

2. The B. sterolicum Class II cholesterol oxidase (PDB: 1I19) is selected as the engineering scaffold because its His121–FAD covalent bond raises cofactor redox potential and is inherently pH-insensitive, unlike the non-covalent FAD binding of the Class I R. erythropolis enzyme (UniProt P22637; PDB: 1COY). The covalent histidyl-FAD tether means the cofactor remains anchored and redox-competent at acidic pH, removing one major source of acid-inactivation that would affect the Class I enzyme. This structural distinction is documented in the Evidence Set structural biology summary referencing Vrielink et al.

3. The CYP7A1:7KC co-crystal structure (DOI: https://doi.org/10.1194/jlr.m050765) provides the first high-resolution atomic model of 7KC in a sterol-binding active site, revealing the precise geometry of the C7-ketone relative to the steroid backbone. [SPECULATIVE] Superposition of this binding pose onto the B. sterolicum ChOx active site (PDB: 1I19) is predicted to reveal a steric clash between the C7-ketone oxygen and a conserved active-site residue (likely within the gatekeeping loop, residues 70–90, d...

SENS category: GlycoSENS

Key references: • doi.org/10.1194/jlr.m050765 • doi.org/10.1194/jlr.m050765]. • doi.org/10.1194/jlr.m050765**