tmprss2 expression constructs  (Addgene inc)

Journal: Protein Science : A Publication of the Protein Society

Article Title: Positional scanning and computational modeling reveal determinants of legumain transpeptidase activity

doi: 10.1002/pro.70563

Figure Lengend Snippet: Asp160 in the S2' pocket of human legumain is a key determinant of substrate specificity. (a) Crystal structure of hLEG in complex with the covalent inhibitor YVAD‐cmk (Tyr‐Val‐Ala‐Asp‐chloromethylketone) (PDB: 4AW9 ). (b–d) AlphaFold 3 models of hLEG bound to the indicated peptides: (b) FPN 14 –GL, (c) FPN 14 –GK, and (d) FGN 14 –GK. (e) MALDI‐ToF mass spectrum of the unoptimized G 1 …N 14 –GLA SFTI precursor peptide after incubation with the V155G–D160Y mutant of hLEG. The corresponding spectrum for the reaction catalyzed by wild‐type hLEG is shown in the inset. (f) MALDI‐ToF mass spectrum of the SFTI opt precursor peptide after incubation with the V155G–D160Y variant of hLEG. A blue star indicates a peak corresponding to a demethylated (Δ14 Da) SFTI–GLA species, and a red star marks a demethylated (Δ14 Da) c‐SFTI species. (g) Reaction scheme of the SFTI‐cyclisation by hLEG. Protonation of Cys189 is regulated by the P2’ residue. Positively charged P2'/P2" residues may facilitate deprotonation of Cys189, either directly or indirectly by compensating the negative electrostatic influence of the nearby Glu190. In step 1 of the transpeptidation reaction, the Cys189 Sγ is attacking the scissile peptide bond leading to the formation of a thioester intermediate. In Step 2 of the reaction the intermediate is released by the N‐terminal P1" residue.

Article Snippet: Briefly, LEXSY P10 cells containing the wild‐type human legumain (hLEG), or the V155G‐D160Y legumain mutant expression construct or the AtLEGβ expression construct were grown in BHI medium (Jena Bioscience) supplemented with 5 mg/mL porcine Hemin (Jena Bioscience), 50 units/mL penicillin and 50 mg/mL streptomycin (Pen‐Strep, Jena Bioscience).

Techniques: Incubation, Mutagenesis, Variant Assay, Residue

Journal: medRxiv

Article Title: Gene-gene interactions protect against Familial Hypercholesterolemia: effect of lost- of-function PCSK9 variants

doi: 10.64898/2026.03.26.26348145

Figure Lengend Snippet: Pedigree of the family showing segregation of LDLR and PCSK9 variants. Squares represent males, circles are females and diamond symbols are used to represent family members without specifying their sex. Individuals fulfilling the clinical diagnosis criteria for familial hypercholesterolemia (FH) are indicated by half-filled symbols. The first row below each symbol correspond to the individual ID following by the genotype at LDLR, PCSK9 and APOB ..

Article Snippet: Human LDLR cDNA constructs were generated using a GFP-tagged plasmid vector (C-GFPSpark®, NM_000527; Sino Biological, Beijing, China).

Techniques: Biomarker Discovery

Journal: medRxiv

Article Title: Gene-gene interactions protect against Familial Hypercholesterolemia: effect of lost- of-function PCSK9 variants

doi: 10.64898/2026.03.26.26348145

Figure Lengend Snippet: Functional characterization of LDLR c.2479G>A ; (A) Flow cytometry histogram overlay showing DiI-LDL binding; (B) Quantification of LDL uptake relative to WT; (C) LDLR expression. The upper line represents the benignity cutoff (90%) established by the ACMG/ClinGen guidelines for functional studies. * <0.05; **** <0.0001.

Article Snippet: Human LDLR cDNA constructs were generated using a GFP-tagged plasmid vector (C-GFPSpark®, NM_000527; Sino Biological, Beijing, China).

Techniques: Functional Assay, Flow Cytometry, Binding Assay, Expressing