Analysis of AlphaMissense data in different protein groups and structural context.

Single amino acid substitutions can profoundly affect protein folding, dynamics, and function. The ability to discern between benign and pathogenic substitutions is pivotal for therapeutic interventions and research directions. Given the limitations in experimental examination of these variants, AlphaMissense has emerged as a promising predictor of the pathogenicity of missense variants. Since heterogenous performance on different types of proteins can be expected, we assessed the efficacy of AlphaMissense across several protein groups (e.g. soluble, transmembrane, and mitochondrial proteins) and regions (e.g. intramembrane, membrane interacting, and high confidence AlphaFold segments) using ClinVar data for validation. Our comprehensive evaluation showed that AlphaMissense delivers outstanding performance, with MCC scores predominantly between 0.6 and 0.74. We observed low performance on disordered datasets and ClinVar data related to the CFTR ABC protein. However, a superior performance was shown when benchmarked against the high quality CFTR2 database. Our results with CFTR emphasizes AlphaMissense's potential in pinpointing functional hot spots, with its performance likely surpassing benchmarks calculated from ClinVar and ProteinGym datasets.

Synthesis and Biological Evaluation of Pyrazole-Pyrimidones as a New Class of Correctors of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

Cystic fibrosis (CF) is caused by the functional expression defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Despite the recent success in CFTR modulator development, the available correctors only partially restore the F508del-CFTR channel function, and several rare CF mutations show resistance to available drugs. We previously identified compound 4172 that synergistically rescued the F508del-CFTR folding defect in combination with the existing corrector drugs VX-809 and VX-661. Here, novel CFTR correctors were designed by applying a classical medicinal chemistry approach on the 4172 scaffold. Molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were conducted to propose a plausible binding site and design more potent and effective analogs. We identified three optimized compounds, which, in combination with VX-809 and the investigational corrector 3151, increased the plasma membrane density and function of F508del-CFTR and other rare CFTR mutants resistant to the currently approved therapies.

PtdIns4P is required for the autophagosomal recruitment of STX17 (syntaxin 17) to promote lysosomal fusion.

The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion.Abbreviations: Cardiolipin: 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO: dimethyl sulfoxide; GST: glutathione S-transferase; GUV: giant unilamellar vesicles; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); PI: L-α-phosphatidylinositol; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS: 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P2: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-3',5'-bisphosphate); PtdIns3P: 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate); PtdIns4P: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-4'-phosphate); SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17: syntaxin 17.