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Multi-granulin Domain Peptides Bind to Pro-cathepsin D and Stimulate its Enzymatic Activity in vitro Victoria Butler, Wilian Augusto Cortopassi, Sushmitha Gururaj, Austin L Wang, Olivia M Pierce, Matthew P Jacobson, and Aimee W Kao Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00275 • Publication Date (Web): 17 May 2019 Downloaded from http://pubs.acs.org on May 21, 2019
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Biochemistry
Multi-granulin Domain Peptides Bind to Pro-cathepsin D and Stimulate its Enzymatic Activity in vitro Victoria J. Butler1, Wilian A. Cortopassi2, Sushmitha Gururaj1, Austin L. Wang1, Olivia M. Pierce2#, Matthew P. Jacobson2* and Aimee W. Kao1* 1Department
of Neurology, University of California, San Francisco, California, 94143, U.S.A. of Pharmaceutical Chemistry, University of California, San Francisco, California, 94143, U.S.A. #Current address: Schrodinger, Inc., 120 West 45th Street, New York, New York, 10036, U.S.A. 2Department
*Co-corresponding authors:
[email protected] (Phone: 415-476-3568) and
[email protected] (Phone: 415-502-7123)
KEYWORDS. Progranulin, cathepsin D, granulin, neurodegeneration
ABSTRACT: Progranulin (PGRN) is an evolutionarily conserved glycoprotein associated with several disease states, including neurodegeneration, cancer and autoimmune disorders. This protein has recently been implicated in the regulation of lysosome function, whereby PGRN may bind to and promote the maturation and activity of the aspartyl protease cathepsin D (proCTSD – inactive precursor, matCTSD – mature, enzymatically active form). As the full-length PGRN protein can be cleaved into smaller peptides, called granulins, we assessed the function of these granulin peptides in binding to proCTSD and stimulating matCTSD enzyme activity in vitro. Here, we report that full-length PGRN and multi-granulin domain peptides bound to proCTSD with low- to sub-micromolar binding affinities. This binding promoted proCTSD destabilization, which was greater in magnitude for multi-granulin domain peptides than for full-length PGRN. Such destabilization correlated with enhanced matCTSD activity at acidic pH. The presence and function of multi-granulin domain peptides has typically been overlooked in previous studies. This work provides the first in vitro quantification of their binding and activity on proCTSD. Our study highlights the significance of multi-granulin domain peptides in the regulation of proCTSD maturation and enzymatic activity and suggests that attention to PGRN processing will be essential for future understanding of the molecular mechanisms leading to neurodegenerative disease states with loss-of-function mutations in PGRN.
Progranulin (PGRN) is an evolutionarily conserved protein implicated in diverse biological processes, including neurodegeneration, cancer, inflammation and wound healing.1, 2 Recent studies have highlighted a role for progranulin in regulating lysosome function.3, 4 Specifically, PGRN has been shown to physically
interact with the lysosomal aspartyl protease cathepsin D (CTSD), whereby it may stimulate enzymatic maturation and/or activity.5-7 This implicates a dysregulation of CTSD activity and aberrant lysosomal function in neurodegenerative diseases linked to PGRN loss-offunction mutations.3, 8 PGRN itself is a pro-protein that can be cleaved into smaller domains called granulins (A through G and p).9 Granulins share a highly disulfide-rich, evolutionarilyconserved beta-sheet fold.10, 11 Such characteristics are often found in highly stable proteins that can withstand heat and pH changes.11 Indeed, recent studies have highlighted that progranulin cleavage, and therefore granulin production, occurs in the acidic environment of the lysosome12, through the action of lysosomal cathepsins.13, 14 Individual granulins may oppose the function of the full-length protein in cell growth and inflammation.11 Alternatively, some evidence suggests that granulin domains also bind to and stimulate CTSD enzymatic activity.5 Although multi-granulin-sized peptides have been reported in highly degenerative brain regions from AD patients15, the actual molecular functions of individual granulins, multi-granulin fragments and the full-length protein are still incompletely understood. We previously demonstrated a role for full-length PGRN in stimulating the maturation and activity of proCTSD.6 We proposed that this results from a destabilizing effect of PGRN on proCTSD, facilitating CTSD propeptide cleavage and the production of mature, active CTSD. Here, we sought to clarify the role of PGRN and its cleavage products in proCTSD binding and CTSD enzymatic activity. We now report for the first time the binding affinities for full-length PGRN and multi-granulin domain peptides with proCTSD in vitro at neutral pH. These recombinant peptides, particularly the multi-granulin fragments BAC and CDE, induce a significant destabilizing effect on proCTSD, resulting in
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a negative shift for proCTSD in thermal stability assays. At acidic pH, we demonstrate that multi-granulin domain peptides promote CTSD activity above that of full-length PGRN. We propose a mechanism whereby multi-granulin domain peptides more effectively promote destabilization of the CTSD propeptide from the proCTSD catalytic core. In recent studies, our lab and others have demonstrated that full-length PGRN can bind to proCTSD.5-7 To quantitatively analyze this binding interaction, we utilized microscale thermophoresis (MST).16 MST measures the directed movement of a fluorescently-labeled protein and/or protein-ligand complex within a temperature gradient. Interaction with a ligand changes the diffusion of the protein–ligand complex with respect to the unbound protein, and this can be used to derive equilibrium binding constants. We performed MST assays at neutral pH to prevent the autoactivation of proCTSD, which occurs at acidic pH.17 After confirming the binding of fluorescently-labeled proCTSD with full-length PGRN, we measured the binding affinity between proCTSD and the recombinant multi-granulin (pG, BAC, CDE) or granulin (C) peptides that are commercially available (Figures 1, S1, S2 and Table 1). PGRN and multi-granulin domain peptides bound to proCTSD at low- or sub-micromolar affinity. Interestingly, peptides pG and CDE bound to proCTSD with a higher affinity than that of full-length PGRN. Furthermore, ligand addition induced a change in the initial fluorescence of these protein complexes during MST capillary scans (Figure S3), which was eliminated after protein denaturation (Figure S4), suggesting that a physical interaction between proCTSD and ligand results in a conformational change in proCTSD. Neutral pH (7.4) Peptide
MST - Kd (µM)
Acidic pH (3.5)
DSF ∆Tm (°C)
Normaliz ed Vmax
Lag time (sec)
proCTSD
-
-
1.00 ± 0.00
1712 ± 31.42
+PGRN
0.95 ± 0.34
-0.41 ± 0.05
1.29 ± 0.06
1585 ± 34.65
+pG
0.33 ± 0.03
-0.23 ± 0.02
1.27 ± 0.11
1611 ± 30.01
+BAC
2.80 ± 0.89
-3.38 ± 0.08
1.89 ± 0.09
1343 ± 51.57
***
** 1376 ± 48.23
+CDE
0.13 ± 0.01
-2.78 ± 0.36
1.84 ± 0.09 ***
*
+C
-
0.05 ± 0.07
1.13 ± 0.17
1488 ± 117.20
Table 1. Summary table showing binding and activity of multi-granulin domain peptides on proCTSD.a
a Values shown are mean ± SEM. Normalized V max and lag time were analyzed for significant difference to proCTSD alone using one-way ANOVA with Tukey’s multiple comparisons test, ***=P
