Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Global protease activity profiling provides differential diagnosis of pancreatic cysts

doi: 10.1158/1078-0432.CCR-16-2987

Figure Lengend Snippet: (A) Substrate specificity of cathepsin D, cathepsin E, and gastricsin as determined by MSP-MS. Residues shown in iceLogo are statistically significant with p < 0.05. (B) Heatmap comparing the amino acid enrichment Z-scores for gastricsin relative to cathepsin D and cathepsin E. (C) Venn diagram depicting the unique and overlapping cleavages detected by MSP-MS with cathepsin D, cathepsin E, and gastricsin. (D) Cleavage of the fluorescent substrates by cathepsin D, cathepsin E, and gastricsin. Activity was normalized to 1.00 based on the maximal activity against each substrate. Red arrow indicates the site of cleavage. Error bars denote standard error of the mean (SEM) from triplicate analysis.

Article Snippet: For recombinant gastricsin (R&D Systems, 6186-AS), cathepsin D (R&D Systems, 1014-AS), and cathepsin E (R&D Systems, 1294-AS), the MSP-MS assay was performed as described above with slight modifications: 10 nM of recombinant protease in pH 3.5 acetate buffer was used and aliquots were removed after 15, 60, and 240 minutes.

Techniques: Activity Assay

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: Pulldown of proCTSD with PGRN expressed in HEK293FT cells. Immunoprecipitation was performed against the FLAG epitope on FLAG-tagged PGRN. Immunoblotting was performed with anti-FLAG and anti-CTSD antibodies. Immunoblots are representative of three independent experiments.

Article Snippet: Thermal denaturation assays (70μl) for 1.5μM proCTSD (R&D Systems Inc., #1014-AS) were measured in the presence and absence of PGRN (R&D Systems, #2420-PG-050) in 50mM Tris-HCl (Teknova #T5074), 150mM NaCl (Fisher #S271-3) pH 7.4.

Techniques: Immunoprecipitation, FLAG-tag, Western Blot

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A) Differential scanning fluorimetry (DSF) was used to obtain fluorescent intensity curves versus temperature, and the curve derivatives are plotted for recombinant proteins: 1.5μM HIS-tagged proCTSD alone (blue), 4.5μM HIS-tagged PGRN alone (black), and 1.5μM proCTSD with 4.5μM PGRN (red). DSF was performed at neutral pH 7.4. Assays were run in triplicate and values plotted are mean ± SEM. (B) Melting temperature, Tm, for PGRN:proCTSD complex at increasing molar ratios of PGRN.

Article Snippet: Thermal denaturation assays (70μl) for 1.5μM proCTSD (R&D Systems Inc., #1014-AS) were measured in the presence and absence of PGRN (R&D Systems, #2420-PG-050) in 50mM Tris-HCl (Teknova #T5074), 150mM NaCl (Fisher #S271-3) pH 7.4.

Techniques: Recombinant

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A-D) In vitro maturation time-course assays for recombinant HIS-tagged proCTSD at pH 3.4 in the (A) absence (n = 4) and presence of (B) 75nM (n = 3), (C) 100nM (n = 3) and (D) 150nM (n = 3) HIS-tagged PGRN. Membranes were immunoblotted with anti-CTSD antibody. Boxed regions in panel B indicate the signal measured for quantification of % 30kDa CTSD / total. (E) Michaelis-Menten kinetic curves for the conversion of proCTSD to matCTSD. (F) Correlation between initial velocity of reaction (V0) and PGRN concentration.

Article Snippet: Thermal denaturation assays (70μl) for 1.5μM proCTSD (R&D Systems Inc., #1014-AS) were measured in the presence and absence of PGRN (R&D Systems, #2420-PG-050) in 50mM Tris-HCl (Teknova #T5074), 150mM NaCl (Fisher #S271-3) pH 7.4.

Techniques: In Vitro, Recombinant, Concentration Assay

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A) ProCTSD undergoes an autocatalytic activation mechanism for the formation of initial matCTSD molecules at a low rate. (B) Once formed, matCTSD can convert proCTSD to matCTSD through an intermolecular cleavage of the propeptide. (C) PGRN binds around the propeptide region of proCTSD to destabilize its interaction with the enzyme catalytic core, (D) facilitating propeptide cleavage by matCTSD. Catalytic aspartyl residues are represented as orange dots, the propeptide in blue and the propeptide cleavage site in red.

Article Snippet: Thermal denaturation assays (70μl) for 1.5μM proCTSD (R&D Systems Inc., #1014-AS) were measured in the presence and absence of PGRN (R&D Systems, #2420-PG-050) in 50mM Tris-HCl (Teknova #T5074), 150mM NaCl (Fisher #S271-3) pH 7.4.

Techniques: Activation Assay