Inositol phosphates and phosphoinositides activate -degrading , while phosphoinositides also mediate binding to endosomes

Eun Suk Songa, HyeIn Janga, Hou-Fu Guoa, Maria A. Julianob, Luiz Julianob, Andrew J. Morrisc, Emilia Galperina, David W. Rodgersa,d,1, and Louis B. Hersha,d,1

aDepartment of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536; bDepartment of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, Brazil; cDivision of Cardiovascular Medicine, University of Kentucky College of Medicine, Lexington, KY 40536; and dCenter for Structural Biology, University of Kentucky, Lexington, KY 40536

Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved February 24, 2017 (received for review August 12, 2016)

Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, PtdInsPs activate IDE by interacting with the same polyanion- including insulin, amylin, and the amyloid β peptides. Polyanions that binds ATP and triphosphate. activate IDE toward some substrates, yet an endogenous polyan- Found mainly in the cytosol, IDE also has been reported to be ion activator has not yet been identified. Here we report that associated with various subcellular compartments, including inositol phosphates (InsPs) and phosphatdidylinositol phosphates endosomes (23–25) and peroxisomes (26). It also is reported to be (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact secreted from cells. Localization ofIDEtodifferentintracellular with the polyanion-binding site located on an inner chamber wall compartments determines access to substrates, although the of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting mechanism whereby IDE traffics to and becomes associated with intracellular compartments remains largely unknown. Here we primarily Vmax. The extent of activation and binding affinity cor- relate with the number of phosphate groups on the inositol ring, provide evidence that IDE binding to PtdInsPs facilitates its lo- calization to endosomes. Thus, the interaction of IDE with InsPs with phosphate positional effects observed. IDE binds PtdInsPs and PtdInsPs can modulate the function of IDE by affecting both from solution, immobilized on membranes, or presented in lipo- its activity and its spatial distribution. somes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly en- Results counters physiological substrates. Thus, InsPs and PtdInsPs can InsPs Activate IDE. Because of their anionic nature and prominent serve as endogenous modulators of IDE activity, as well as regu- role in signaling, we tested a series of InsPs for their ability to lators of its intracellular spatial distribution. increase the activity of IDE toward the synthetic quenched fluo- rescent substrate Abz-Gly-Gly-Leu-Arg-Lys-His-Gly-Gln-EDDnp insulin-degrading enzyme | inositols | phosphatidylinositols | activation | (Table 1). All of the tested InsPs increased IDE activity, exhibiting subcellular localization a hyperbolic response, with the extent of activation generally greater as the number of phosphates on the inositol was in- nsulin-degrading enzyme (IDE), also known as insulysin (EC creased. For example inositol 3-monophosphate [Ins(3)P] pro- I3.4.24.56), hydrolyzes a broad range of bioactive peptides in vitro, duced a maximal sevenfold increase in activity, whereas inositol including angiotensin, glucagon, β-endorphin, amylin, and amyloid 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] produced a maximal β peptides (Aβ), with the two most established in vivo substrates being insulin and Aβ. Evidence supporting a role for IDE in Significance degrading insulin and Aβ in vivo includes associations of IDE gene ’ variants with type 2 diabetes (1, 2) and Alzheimer s disease (3, 4), A diverse collection of peptides mediates cell–cell communica- decreased clearance of the two peptides in IDE-deficient mice (5, tion. that cleave these peptides modulate their sig- 6), and antidiabetic activity produced by IDE inhibitors (7, 8), al- nals and thus play an important role in the physiology of though some reports have questioned its role in insulin degradation multicellular organisms. Insulin-degrading enzyme (IDE) is one (9, 10). In addition, IDE has been reported to have noncatalytic such enzyme that cleaves a number of bioactive peptides. IDE is functions, such as acting as a receptor for varicella-zoster virus (11) activated by polyanions, but physiological activators remain and serving as a heat shock protein in stressed cells (12). IDE also unidentified. Here we show that inositol-containing molecules, modulates the activity of the proteasome (13), reportedly in con- known to modulate various cellular functions, activate IDE, junction with the retinoblastoma tumor-suppressor protein (14). identifying them as potential physiological regulators. Inositol We previously established that polyanions, such as free ATP and phosphates are potent soluble activators of IDE. Phosphatidy- triphosphate, increase IDE activity by up to 100-fold toward a linositol phosphates, lipid components of cell membranes, also synthetic peptide substrate (15) and that ATP binds to a strongly activate but in addition facilitate the localization of IDE to in- electropositive inner surface of IDE that forms one-half of the tracellular compartments, where the enzyme gains access to substrate-binding chamber (16, 17). Significantly, mutations in IDE substrates, such as insulin, internalized by cells. that reduce its activation by polyanions also decrease its ability to

rescue production of a mature yeast mating factor, indicating that Author contributions: E.G., D.W.R., and L.B.H. designed research; E.S.S., H.J., H.-F.G., A.J.M., activation by polyanions plays an important physiological role in E.G., D.W.R., and L.B.H. performed research; M.A.J. and L.J. contributed new reagents/ cells (16). Physiological polyanionic activators of IDE have not yet analytic tools; E.G., D.W.R., and L.B.H. analyzed data; and D.W.R. and L.B.H. wrote been identified, however. the paper. Inositol phosphates (InsPs) are important intracellular second The authors declare no conflict of interest. messengers generated by activation of many cell surface receptors This article is a PNAS Direct Submission. (18, 19). Phosphatidylinositol phosphates (PtdInsPs; phosphoino- 1To whom correspondence may be addressed. Email: [email protected] or lhersh@ sitides) participate in signaling and help define the identity of uky.edu. subcellular compartments by enrichment of their membranes with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. one or more PtdInsPs (20–22). We report here that both InsPs and 1073/pnas.1613447114/-/DCSupplemental.

E2826–E2835 | PNAS | Published online March 21, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1613447114 Downloaded by guest on September 23, 2021 Table 1. Activation of IDE by myo-InsPs In general, the more phosphates present on an InsP, the tighter the PNAS PLUS K Maximal fold binding (i.e., lower observed A). In contrast, the binding of IDE to immobilized PtdIn monophosphates in general gave a stronger activation, K , μM, A signal than observed with immobilized PtdIn bisphosphates, Myo-inositol mean ± SD mean ± SD which in turn exhibited a stronger signal than PtdIn(3,4,5)P3. 3-monophosphate 6.2 ± 0.8 94.5 ± 21.5 To determine whether IDE could interact with PtdInsPs in bi- 1,2-bisphosphate 3.1 ± 0.4 56.8 ± 17.4 layer membranes, we tested the ability of IDE to bind to liposomes 1,3-bisphosphate 6.1 ± 0.5 27.2 ± 6.0 containing PtdIns(4,5)P2. IDE bound to PtdIns(4,5)P2-containing A 4,5-bisphosphate 13.8 ± 3.4 83.3 ± 24.2 liposomes, but not to liposomes containing only PC (Fig. 3 ), 1,3,5-trisphosphate 12.9 ± 0.7 7.8 ± 0.8 demonstrating an interaction with a PtdInsP present in a lipid 1,4,5-trisphosphate 30.6 ± 1.4 39.9 ± 4.3 bilayer. 1,3,4,5-tetrakisphosphate 58.6 ± 1.5 18.7 ± 1.5 PtdInsPs also have the ability to increase the rate of the IDE re- action with the fluorogenic Abz substrate. Activation by PtdIns(4,5)P2 1,3,4,5,6-pentakisphosphate 83.3 ± 4.0 8.2 ± 1.5 (di-C8) followed a hyperbolic curve, with a maximum 31-fold 1,2,3,4,5,6-hexakisphosphate 72.0 ± 4.0 0.7 ± 0.1 rate enhancement and a KA of 5.7 μM(Fig.3B). The longer- (phytic acid) acyl chain PtdIns(4,5)P2(di-C16) also activated IDE up to a ± ± 1-diphosphoinositol 79.7 3.1 0.9 0.1 concentration of ∼3 μM, after which the extent of activation pentakisphosphate decreased, leading to inhibition at the highest concentrations 5-diphosphoinositol 94.7 ± 4.1 1.7 ± 0.2 tested. The loss of activation began at the critical micelle con- pentakisphosphate centration (CMC) of this longer-acyl chain lipid (Fig. S1), in- dicating that the decrease in free lipids as micelles form accounted for this effect. Although a limited series was studied, K ∼60 fold increase in activity. The activation constant (KA)was the more phosphates present, the tighter the A of the PtdInsPs, decreased from ∼100 μM for Ins(3)P to ∼1 μM for inositols con- as with the InsPs but in contrast to the immobilized lipids. The taining six or more phosphate moieties. maximum level of activation also did not follow this trend. K μ ∼ To test for positional effects regarding the placement of the PtdIns(3)P(di-C8) had a A of 30 M with 15-fold activation, K μ ∼ phosphate group, we compared inositol bisphosphates with phos- and PtdIns(3,4,5)P3(di-C8) had a A of 0.5 M with 20-fold phate moieties at the 1 and 2, the 1 and 3, and the 4 and 5 positions. activation. These values and those noted above for PtdIns(4,5)P2(di- K ∼ μ ∼ Positional effects were observed, with the 4,5-bisphosphate pro- C8) can be compared with the A of 90 M and 6-fold acti- K ∼ μ ∼ ducing the highest level of activation, followed by the 1,2-bisphos- vation for Ins(3)P, A of 83 M and 14-fold activation for K μ ∼ phate and then the 1,3-bisphosphate. The K did not correlate with Ins(4,5)P2, and A of 7.8 Mand 30-fold activation for Ins(3,4,5)P3. A K the level of activation, being highest for the 4,5-bisphosphate, fol- The tighter A values for the lipids suggest that moieties other lowed by the 1,3-bisphosphate and then the 1,2-bisphosphate. A than the InsPs in their head groups contribute to binding and, at comparison of two triphosphate isomers gave similar results, with least in some cases, may make a modest contribution to activa- Ins(1,4,5)P3 producing greater activation than Ins(1,3,5)P3, but tion. Neither PS nor PC had an effect over the same concen- with the latter having a lower affinity based on its KA value. tration range (Fig. 3), showing that the observed effects were

We examined in more detail the effects of Ins(1,4,5)P3 and Ins specific to the PtdInsPs. BIOCHEMISTRY (1,2,3,4,5,6)P6 (phytic acid) on the kinetics of the IDE reaction Three small physiological IDE substrates—bradykinin, angio- with the fluorogenic Abz substrate. As shown in Fig. 1 and tensin, and dynorphin B9—as well as four large physiological — β β — summarized in Table 2, Ins(1,4,5)P3 increased Vmax for the re- substrates insulin, -endorphin, A 1–40 and glucagon were action by ∼40-fold, with little effect on the substrate K or the tested with PtdIns(4,5)P2(di-C8) as an activator. At 25 μM M K extent of (Hill coefficient). Phytic acid had the same PtdIns(4,5)P2, which is fivefold above its A, both bradykinin effect at low substrate concentrations, producing an increase in and angiotensin hydrolysis were increased by ∼20-fold, whereas ∼ Vmax in the substrate range of 1–25 μM, but decreased reaction the hydrolysis of dynorphin B9 was increased by 60-fold. With rates at substrate concentrations >25 μM. Competition between insulin as the substrate, there was a small (1.4-fold) increase in β β phytic acid and substrate at high substrate concentrations may the hydrolytic rate, whereas with -endorphin and A 1–40, the account for the observed biphasic effect on activity. We used phytic rate was increased by 1.3-fold, and there was essentially no acid to test activation with respect to physiological substrates, be- change in the rate of glucagon hydrolysis (1.1-fold increase). causeithasthelowestKA as well as high activation with the Abz fluorogenic substrate. With smaller peptides, namely angiotensin, IDE Interaction with InsPs and PtdInsPs Is Mediated by the Polyanion- bradykinin, and dynorphin B9, phytic acid stimulated hydrolysis by Binding Site. An IDE variant, IDEK898A,K899A,S901A, with muta- ∼6-fold to as much as 26-fold (Table 3). In contrast, there was no tions in the polyanion-binding site shows reduced activation by significant effect of phytic acid with the larger peptides insulin, Ins(1,4,5)P3 and phytic acid, supporting the concept that these β-endorphin, and glucagon, whereas an ∼twofold increase in ac- activators bind at the same site as ATP (Table 4). In addition to tivity was observed with β-amyloid peptide1–40 (Aβ1–40). the polyanion-binding site, IDE contains another allosteric site, the distal binding site, which interacts with the carboxyl terminus PtdInsPs also Activate IDE. The observation that InsPs activate IDE of extended peptide substrates (27) or, in the case of shorter prompted us to examine the effects of phospholipids with inositol substrates, binds a second peptide (27, 28). Binding of a peptide head groups. PtdIns and its phosphorylated derivatives share the at this distal site produces a threefold to fivefold increase in configuration of the InsP activators, and the substrate-binding cavity activity toward the Abz substrate (29). of IDE could accommodate these larger lipid molecules (or portions Mutations at either the polyanion-binding or distal binding of them when associated with membranes). We initially tested the sites reduce activation at the other site, indicating they are coupled binding of IDE to a panel of PtdInsPs immobilized on a membrane in some way (16). Thus, we tested the effect of a mutation in the using an anti-IDE antibody. A strong signal was obtained for IDE distal site, IDEY609F, on activation by Ins(1,4,5)P3 and phytic acid. binding to immobilized PtdIn3P, 4P, or 5P and to PtdIn(3,4)P2, Activation by these InsPs was decreased in IDEY609F,furtherestab- PtdIn(3,5)P2, and PtdIn(4,5)P2 (Fig. 2). Weak signals were observed lishing the link between the two sites. with PtdIns(3,4,5)P3, phosphatidic acid, phosphatidylserine (PS), The polyanion-binding site and distal site mutations also af- lysophosphatidic acid, and phosphatidylethanolamine, and no signal fected the ability of IDE to bind to PtdIns(4,5)P2 immobilized on was observed with PtdIns, phosphatidylcholine (PC), lysophospha- a membrane. By measuring the amount of IDE bound to in- tidylcholine, and sphingosine-1-phosphate. The strength of the ob- creasing amounts of immobilized PtdIns(4,5)P2 (Fig. 4 A and B), served binding signal was the reverse of the pattern seen with InsPs. we found that, relative to IDEwt, the polyanion-binding site

Song et al. PNAS | Published online March 21, 2017 | E2827 Downloaded by guest on September 23, 2021 site. Somewhat surprisingly, the distal site mutant IDEY609F showed reproducibly increased binding; for example, a ratio of 3.0 in in- tensity relative to IDEwt for the concentrations noted above was obtained. Perhaps this region interacts with the fatty acid portion of bound lipid, and mutating to a more hydrophobic residue en- hanced the interaction. (In fact, residue 609 is positioned to in- teract with one acyl chain of PtdIns in a binding model described below.) To obtain more direct evidence that IDE binds to PtdInsPs through the polyanion-binding site, we measured the ability of PtdIns(4,5)P2 to compete with the fluorescent ATP analog trinitrophenyl-ATP (TNP-ATP) for binding to IDE. It was pre- viously established that the fluorescence of TNP-ATP increases when bound to IDE at the polyanion-binding site (30). When PtdIns(4,5)P2 was added to IDE in the presence of TNP-ATP, there was a decrease in fluorescence consistent with decreased TNP-ATP binding (Fig. 4C). Phytic acid and PtdIns(3)P also competed with TNP-ATP for binding to IDE. Free 40 μM PtdIns(4,5)P2 has no effect on the fluorescence of free TNP-ATP.

IDE Localization to Endosomes Involves the Polyanion-Binding Site. Given that IDE has been reported to be present in endosomes (23–25), we hypothesized that the enzyme initially might be recruited to endosomes by binding to PtdIns(3)P known to be present in the outer leaflet of the endosomal membrane (21, 22, 31). Discontinuous sucrose density centrifugation was used to enrich endosomes from COS-1 cells as described by Jang et al. (32). In COS-1 cells transfected with wild-type (WT) IDE, the enzyme was detectable in the endosomal fraction (Fig. 5A and Fig. S2), which showed the expected enrichment in the endo- somal markers Rab-5 and early endosome antigen 1 (Fig. S3). To ensure that the endosomal fractions were free of any significant amount of plasma membrane, we subjected samples to Western + + blot analysis with antibodies against the Na /K ATPase (Fig. S3). We were unable to detect Na-K ATPase in the endosomal fraction, estimating a detection limit of 0.01% contamination based on the Na-K ATPase staining intensity in the plasma membrane fraction. In contrast, the estimated endosomal con- tent of IDE was 0.22% of the total IDE applied to the sucrose gradient. These data indicate that IDE found in the endosomal fraction is not an artifact due to contamination of endosomes with other cellular components. Fluorescence microscopy confirmed colocalization in COS-1 cells of immunostained IDEwt, with endosomes marked by cellular uptake of dye-labeled dextran. Images showed visible fluorescent dye spatial overlap and characteristic pixel color intensity corre- lation scatterplots (Fig. 5 D–G). For 18 masked cells from six 3D image stacks, the average Pearson correlation coefficient of dye Fig. 1. Activation of IDE by Ins(1,4,5)P3 and phytic acid [Ins(1,2,3,4,5,6)P6]. color values was 0.68 (+/− 0.13), indicating strong spatial corre- IDE activity was measured using the fluorogenic peptide Abz-GGFLRKHGQ- lation of the dye intensities consistent with colocalization. Eddnp at the indicated concentrations in 50 mM Tris buffer pH 7.4 with and To provide evidence that endosomal binding occurred through without the addition of 50 μM Ins(1,4,5)P3 or 5 μM phytic acid. an interaction with the polyanion-binding site, we compared the amount of endosomal IDE in COS-1 cells transfected with IDEwt K898A,K899A,S901A with that in COS-1 cells transfected with either of the IDE mutant IDE exhibited reproducibly decreased mutants, IDEK898A,K899A,S901A or IDEY609F. As noted above, binding at all levels of PtdIns(4,5)P2. For example, using IDEK898A,K899A,S901A contains mutations within the polyanion- 150 pmol of immobilized PtdIns(4,5)P2 and 0.5 μg of IDE, a binding site and shows decreased activation by polyanions, K898A,K899A,S901A wt ratio of IDE to IDE intensity of 0.5 was whereas IDEY609F has a mutation in the distal site that affects obtained. This loss of binding in IDEK898A,K899A,S901A suggests activation by polyanions, but not by directly affecting the that the phosphatidylinositol is interacting with the polyanion-binding polyanion-binding site. We assessed the distribution of these

Table 2. Kinetic parameters for activation of IDE by two InsPs

Vmax, nmol/min/μg, Km, μM, Addition mean ± SD mean ± SD Hill coefficient, mean ± SD

None 29.1 ± 2.8 11.1 ± 1.8 1.6 ± 0.4 Ins(1,4,5)P3 1,276 ± 99 8.1 ± 1.3 1.9 ± 0.5 Ins(1,2,3,4,5,6)P6 (phytic acid)* 1,153 ± 119 8.5 ± 1.1 1.6 ± 0.2

*Based on a substrate concentration range of 0–25 μM.

E2828 | www.pnas.org/cgi/doi/10.1073/pnas.1613447114 Song et al. Downloaded by guest on September 23, 2021 Table 3. Effect of phytic acid on the hydrolysis of physiological serves as an activator of IDE, by demonstrating an increase in PNAS PLUS peptides by IDE activity when MES was added to Tris buffer. Thus, IDE can Rate, nmol/min/mg IDE exhibit significant activity at low pH, as is seen in endosomes, particularly when activators are present. Plus phytic acid Substrate No addition (fold increase) Discussion IDE is known to be allosterically activated in vitro by both peptides Angiotensin 6.1 38.3 (6.3) and polyanions (15, 29). The results of this study now clearly es- Bradykinin 9.6 254 (26.4) tablish InsPs as polyanion activators of IDE. As previously ob- Dynorphin B9 35 340 (9.7) served with nucleotide phosphates (15), the greater the number of Insulin 22.4 22.4 (1.0) phosphates present on inositol, the greater the extent of activation β-endorphin 1,400 1,200 (0.86) up to six phosphate moieties. There are also positional effects of Amyloid β peptide (1–40) 1,500 2,720 (1.8) phosphate placement, although these effects are smaller than the Glucagon 3,200 2,672 (0.8) total charge effect. The dependence on the number of negative charges, the competitive binding with a fluorescent ATP analog, The concentration of phytic acid, when present, was 5 μM. and the finding that a polyanion-binding site mutant binds less well to InsPs all indicate that InsPs bind to the previously identified mutants in enriched endosomal fractions (Fig. 5 B and C). The polyanion-binding region of IDE. This site/region lies on one-half IDEK898A,K899A,S901A mutant showed a decreased amount of of the inner surface of the substrate-binding chamber formed by IDE in the endosomal fraction relative to the WT enzyme, the C-terminal two domains of the enzyme (16). As noted pre- whereas the amount of IDEY609F present in the endosomal viously, this appears to be not a single binding site in the classical fraction was comparable to that of IDEwt. We quantified this by sense, but rather a positively charged region of the enzyme that can comparing the ratio of Western blot intensity of endosomal IDE accommodate polyanion binding in at least several different bind- relative to total input IDE. The distal site mutant yielded a ratio ing modes (16, 17). corresponding to 0.18%, close to that of WT IDE (0.22%). In Given that a series of peptides exhibits quite different rates of contrast, a lower value (0.09%) was observed for the polyanion- hydrolysis by IDE, the rate-determining step of the reaction must binding site mutant. We also compared the ratio of Western blot be dependent on the substrate or products. This could be cleavage intensity for endosomal IDE relative to Rab5, a known endosomal of the peptide itself, conversion of the IDE-substrate complex from protein. Again, the WT and distal site mutants gave similar values (0.13% and 0.10%, respectively), whereas the polyanion site mu- tant exhibited a lower value (0.04%). Wortmannin is a potent inhibitor of phosphoinositide 3-kinases (PI3Ks), lowering cellular PtdIns3P levels. Thus, if PtdIns3P were involved in IDE recruitment to endosomes, presumably through binding to the polyanion site, then wortmannin would be expected to decrease the amount of IDE bound to endosomes. That this is the case is demonstrated by our measurements of the amount of BIOCHEMISTRY IDE in the endosomal fraction derived in COS-1 cells treated with 200 nM wortmannin (Table 5). There was a time-dependent de- crease in endosomal IDE in wortmannin-treated cells. Because the sucrose gradient system used in this study also generates a Golgi/ER fraction, we compared the IDE content in this fraction between WT IDE and the polyanion and distal site mutants. In this case, there was no discernible difference between WT IDE and the two mutants (Fig. S4). This suggests that locali- zation to the Golgi/ER does not involve the polyanion-binding site and likely is not mediated by PtdInsPs. Because IDE is reported to be in peroxisomes, we determined the levels of two peroxisomal enzymes, catalase and thiolase, in the endosomal and Golgi/ER fractions. We found no detectable thiolase or catalase in the endosomal fraction, but detected catalase and thiolase in the Golgi/ ER fraction, showing that it contained peroxisomes, which likely accounts for much of the IDE in this fraction. The lack of de- pendence of peroxisomal IDE on the polyanion-binding site is consistent with the report that IDE has a C-terminal peroxisome- targeting signal (33).

IDE Retains Activity at Endosomal pH in Some Buffer Systems. The level of IDE activity in endosomes as they acidify is an important consideration in its role in degrading endosomal peptides, and IDE has been reported to lose substantial activity at low pH (34). Thus, we examined the effect of pH on IDE activity using the quenched fluorogenic peptide substrate, and found that IDE activity was dependent on the buffer as well as on the pH (Table Fig. 2. IDE binds to immobilized phosphatidylinositols. IDE was incubated with S1). For example, we observed activity in MES buffer at a pH of commercial lipid strips (100 pmol/spot) as described in Materials and Methods. close to 5, but no activity in acetate or citrate buffer at this pH. Binding was determined using anti-IDE antibody in a Western blot-like pro- Similarly, at a pH of ∼5.5, we observed activity in citrate buffer, cedure. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphati- and much higher activity at this pH in MES buffer. We also dylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1-phosphate; PA, observed high activity at pH 6 in MES buffer, ∼10-fold greater lysophosphatidic acid; PS, phosphatidylserine; dye, blue marker dye. Numbers in than the activity at pH 7.5 in Tris buffer. We found that MES parentheses indicate intensities of the spots relative to the intensity for PC.

Song et al. PNAS | Published online March 21, 2017 | E2829 Downloaded by guest on September 23, 2021 example, glucagon, given that the number and conformation of or- dered residues for the two peptides are similar when bound to IDE. Ins(1,4,5)P3 has been established as a second messenger that mobilizes calcium pools, and recently more highly phosphorylated InsPs have been shown to be important effectors in a number of signaling pathways (18, 40). For example, activation of the insulin receptor stimulates, among other effects, generation of highly phosphorylated inositols, such as 5-diphosphoinositolpentaki- sphosphate (InsP7) (40–42). Our present finding that InsPs stim- ulate IDE catalysis suggests that they may serve as physiological activators of the cytosolic pool of the enzyme. Concentrations of various InsPs in mammalian cells range from submicromolar to >100 μM, and their levels can be modulated substantially by various events, such as receptor activation and progression through the cell cycle (43, 44). These InsP concentrations, including the potent activator phytic acid, are in ranges shown here to produce IDE activation, consistent with a possible physiological role. The most well-documented role for cytosolic IDE is in degrada- tion of the amyloid precursor protein intracellular domain (AICD) (6, 45), which serves as a transcriptional regulatory factor. In- terestingly, AICD recently has been shown to activate the kinase PIKfyve (46), which increases the production of PtdIns(3,5)P2, a signaling lipid and a source of soluble Ins(5)P (47). This connection to PtdIns and InsP production suggests a possible regulatory system for control of AICD levels involving IDE activation. Other roles for cytosolic IDE are not well established, but given the broad range of substrates in vitro, it seems likely that additional peptides are sub- strates within cells. It also may be that interactions with InsPs affect noncatalytic IDE functions, such as regulating proteasome activity (13, 48, 49) and serving as a heat shock-like protein in stressed cells (12). The functions of cytosolic IDE and the role of allosteric modulation of its activity likely will be promising areas for future investigation. Fig. 3. IDE binds to phosphatidylinositol-containing liposomes and PtdIns(4,5)P2 Ever since IDE was identified as a cytosolic enzyme (50), the activates the enzyme. (A) IDE binding to liposomes was tested using 260 μgof question of how it gains access to substrates that are degraded in total lipid and 0.9 μg of IDE. Liposomes isolated after incubation were sepa- other subcellular compartments has been of considerable interest rated on a polyacrylamide gel and stained by Western blotting with anti-IDE (51). For example, insulin is metabolized primarily in early to late antibody. Lane 1 (MW Std), molecular weight markers; lane 2 (empty well), endosomes after internalization of the peptide bound to its receptor no IDE or liposomes; lane 3 (PC), IDE plus liposomes containing only DOPC; lane (23, 52, 53) and thus is inaccessible to cytosolic IDE. However, small 4 (PtdIns 4,5 diP), IDE plus DOPC liposomes with PtdIns(4,5)P2 at a ratio of 10:1; pools of IDE have been found in endosomes (23–25) as confirmed lane 5 (IDE alone), enzyme only; lane 6 (input), 0.1 μg of purified IDE with- here, as well as in peroxisomes (26, 54) and mitochondria (55). In out incubation. (B) Comparison of IDE activation by PtdIns(4,5)P2 (di-C16 or addition, low levels of IDE are reportedly secreted from some cell di-C8 acyl chains), Ins(4,5)P2, phosphatidylserine, and phosphatidylcholine. IDE types through an unconventional pathway (56) that may involve activity was measured by the hydrolysis of Abz-GGFLRKHGQ-Eddnp substrate routing through multivesicular bodies and release in association with at varying concentrations of the indicated lipid or Ins(4,5)P2. The curves for exosomes (25). As noted above, a targeting sequence at the C ter- activation by PtdIns(4,5)P2 (di-C8 acyl chains) and Ins(4,5)P2 are the fits to a minus that localizes some IDE to peroxisomes has been reported hyperbolic function. Curves are drawn through the data points for activation by other compounds to show trends. previously (26, 33, 54), and a potential alternative splice form with an extended N terminus was shown to direct IDE to mitochondria (55). Otherwise, determinants of IDE intracellular localization have not an open conformation to a closed conformation, or conversion of been defined conclusively. We found here that a portion of cellular the IDE-product complex from a closed conformation to an open IDE localizes to the endosomal system (Fig. 5 and Fig. S3), and our conformation, permitting product release (17). One of these steps observation that IDE binds to PtdInsPs suggests a mechanism for intracellular localization of the enzyme. One role of PtdInsPs is to must be the one increased by InsPs (or PtdInsPs). InsPs increase the hydrolytic rate of the small peptides tested (9– 12 aa), but have no effect or only a mild effect with larger peptides. β Table 4. Effect of polyanion-binding site and distal site An unexplained exception is A 1–40, the rate of which was increased mutations on activation by InsPs by approximately twofold by phytic acid. In crystal structures of IDE with bound peptides (28, 35–39), larger substrates are seen to make Fold activation contacts at both the and the distal site (which mediates Activator IDEwt IDEK898A,K899A,S901A IDEY609F activation by peptides). It is possible that activity is not stimulated for large peptides because they themselves activate in a manner similar Ins(4,5)P2 7.8 1.6 10 † to polyanions, particularly because their hydrolysis rates are often PtdIns(4,5)P2 3.9* 1.7 1.6 higher than those for small peptides. The basis for such activation is Ins(1,4,5)P3 32.6 6.7 9.4 not clear from existing structures, however. Generally, only substrate Phytic acid 68.6 6.4 29.4 residues at the active and distal site are ordered, and the larger μ μ μ peptides do not make contacts with the anion-binding surface. Intact Assays were conducted with 50 M Ins(4,5)P2, 5 M PtdIns(4,5)P2, 50 M PtdIns(1,4,5)P3, and 5 μM phytic acid. insulin, with its disulfide cross-linking, is mostly well ordered in the *Based on a concentration range of 0–3 μM above which inhibition was binding chamber of the enzyme, but it also does not interact with the observed (Fig. 3B). † anion-binding surface. There is no obvious reason why the Aβ1–40 Data could not be fit to a hyperbola, because an increase in rate was observed peptide should behave differently in terms of activation than, for only at 1.5 and 3.0 μM. Reported increase is based on a single value at 3 μM.

E2830 | www.pnas.org/cgi/doi/10.1073/pnas.1613447114 Song et al. Downloaded by guest on September 23, 2021 PNAS PLUS

Fig. 4. Phosphoinositides interact with the polyanion-binding site. (A) Mutation of the polyanion-binding site, but not the distal site, reduces BIOCHEMISTRY binding to PtdIns(4,5)P2. PtdIns(4,5)P2 was spotted at the indicated amounts on a hydrophobic membrane along with a phosphatidylcholine control. Membrane strips were incubated with IDEwt, the polyanion site mutant IDEK898A,K899A,S901A, or the distal site mutant IDEY609F at the amounts in- dicated. Bound enzyme was visualized by incubation with anti-IDE antibody. (B) Coomassie blue-stained gel indicating the same amount of the three IDE constructs used for membrane binding. Amounts of IDE for the trials were all within 15.5% of the mean. (C) Displacement of TNP-ATP from the IDE polyanion-binding site by PtdIns(4,5)P2. IDE (66 μg) was mixed with 10 μM TNP-ATP in 50 mM Tris buffer, pH 7.4, and the fluorescent spectra were recorded on an LS 55 Luminescence Spectrometer (PerkinElmer) over the indicated wavelengths. Also shown are the fluorescent spectra of IDE alone, TNP-ATP alone, and IDE with TNP-ATP in the presence of the indicated concentration of PtdIns(4,5)P2. Fig. 5. Endosomal IDE content of COS-1 cells. (A) Western blot of the PNS and endosomal fractions isolated from COS-1 cells transfected with IDEwt. establish the identities of subcellular compartments by enrichment of (B) Western blot of fractions isolated from COS-1 cells transfected with their membranes with differently phosphorylated forms of the lipid polyanion-binding site mutant IDEK898A,K899A,S901A.(C) Western blot of (20, 22, 57). These markers direct a diverse set of protein domains to fractions isolated from COS-1 cells transfected with the distal site mutant target membranes based on preferential binding to the enriched IDEY609F. The total protein loaded in each lane is indicated. (D)Green PtdInsP (21, 57). We postulate that in the same manner, PtdInsP channel indicating labeled endosomes for a representative micrograph binding can recruit IDE to subcellular locations through binding at (single plane from a 3D image stack), supporting the localization of IDE to the polyanion site. In particular, the binding to immobilized PtdIns(3) endosomes. Endosomes in COS-1 cells were labeled by uptake of Alexa P is consistent with initial recruitment to early endosomes (22, 31, Fluor 488-dextran. A number of cells are visible in the image. (Scale bar: 5 μm.) (E) Red channel indicating IDE distribution for the same micrograph. 57), and this possibility is supported by our finding that wortmannin wt treatment, which reduces PtdIns(3)P levels, decreases IDE localiza- Expressed IDE was labeled by immunostaining with Alexa Fluor 549- tion to endosomes (Table 5). Binding to PtdIns(3,4)P2 also would conjugated antibody. (F) Merged red and green channels from the same micrograph. In D–F,theInsets show magnified views of the areas indi- cause early endosome localization, because this phospholipid is pre- cated by the smaller rectangles to emphasize the degree of dye overlap. sent in vesicles just after endocytosis. (G) Scatterplot showing distribution of red and green channel intensities in Recruitment to endosomes and downstream compartments is voxels from a single COS-1 cell masked in a 3D image stack. The distribu- particularly relevant to substrate access by IDE. As noted above, tion shows a strong spatial correlation between channel intensities con- insulin degradation occurs largely in the endosomal system (23, sistent with localization of IDE with endosomes. Vertical and horizontal 52, 53). Similarly, Aβ peptides are generated primarily in endo- lines indicate threshold cutoffs for statistical analysis. Symbol colors rep- somes, and one mechanism for clearance of extracellular Aβ is resent the number of voxels in color intensity bins, progressing from blue uptake into endocytic compartments with degradation in endo- (lowest number) to red (highest number).

Song et al. PNAS | Published online March 21, 2017 | E2831 Downloaded by guest on September 23, 2021 Table 5. Effect of wortmannin on the endosomal level of IDE in ularly relevant to PtdInsP binding (Fig. 6A). One site occurs at COS-1 cells an edge of the anion-binding surface near the interface between Duration of Intensity of IDE band per the two halves of IDE. This interface would open first when IDE undergoes a hinge-like motion that must accompany substrate treatment, h milligram of endosomal protein binding and product release (Fig. 6A), and docking of InsPs to 0290this site persists in models of open forms of IDE generated by a 0.5 214 clamshell-like motion of the two halves of the enzyme. Rotating 4119the two halves of IDE by 6 degrees about the likely hinge point is the minimum amount to allow placement of a model PtdIns(3)P without steric clashes when the lipid head group is aligned with a high-scoring docked Ins(1,3)P2 (Fig. 6B). This model of PtdInsP somes, multivesicular bodies, and lysosomes (58, 59). Amylin, binding provides a plausible mechanism for IDE interaction with another IDE physiological substrate (49), also is taken up by membrane-bound lipids, closely resembling structural models for receptor-mediated endocytosis (60). interactions between known PtdInsP-binding domains and Once localized to endosomes, IDE would still have to gain membrane PtdInsPs (75). The hydrophobic acyl chains of the access to the interior of the compartment to encounter sub- lipid would remain outside the IDE inner chamber, allowing strates. A possible internalization mechanism is the budding of them to remain embedded in the lipid bilayer. Of potential sig- vesicles into the endosome that occurs during normal endosome nificance, we note that the molecular surface surrounding this maturation to multivesicular bodies and late endosomes (31, 61, proposed membrane lipid-binding site is the most extensively 62). This formation of intraluminal vesicles is a central aspect of conserved portion of the outer IDE surface (Fig. 6C), possibly the sorting process that determines whether endocytosed mem- reflecting its role in interaction with the membrane. brane proteins are targeted for lysosomal degradation or for Whereas the anion-binding surface site near the half-molecule recycling to other organelles, including the plasma membrane interface is a good candidate for mediating membrane lipid and Golgi (62, 63). Endosomal vesicle uptake (microautophagy) binding, and thus localization to endosomes, interaction with of cytosolic proteins bearing a targeting sequence or linked to PtdInsPs in this manner likely would not account for their ability ubiquitin occurs (64, 65). IDE could be internalized with these to activate IDE. The bound lipid would partially occlude the active targeted proteins by virtue of its attachment to PtdIns(3)P, which site and, by holding the enzyme in an open conformation, displace is known to be present on intraluminal vesicles (61). Recently, residues in domain IV that contribute to catalysis. It also is un- cytosolic proteins taken up by this microautophagy process in likely that a free PtdInsP would bind in a manner that places its late endosomes were observed in the endosome lumen (64), acyl tails in bulk solvent. However, a second high-scoring docking presumably through disruption of some luminal vesicles. The site on the anion-binding surface (Fig. 6A) allows placement of a K ∼ relatively weak interaction of IDE with PtdInsPs [ d of 3mM PtdIns(3)P in the inner chamber of IDE, again aligning the Ins(3) for PtdIns(4,5)P2-containing liposomes] found here would per- P group of the lipid with a docked Ins(1,3)P molecule (Fig. 6 D mit the enzyme to escape into the endosome lumen during this and E). In this position, the acyl chains of the bound lipid extend process. toward the distal site in domain 2, interacting with a relatively As noted above, considerable evidence from genetic associa- nonpolar portion of the inner chamber surface (Fig. 6F). This – tions (1, 2), knockout mouse and inhibitor studies (6 8, 66, 67), model has the virtue of not overlapping with known substrate- as well as characterization of hydrolysis products (23, 68), sup- binding surfaces in the active and distal sites and thus is compat- ports the role of IDE in metabolism of insulin. However, Dur- ible with activation of IDE. Mutations in the IDEK898A,K899A,S901A ham et al. (10) found that an IDE inhibitor did not increase variant occur adjacent to both proposed lipid head group inter- plasma insulin levels or improve insulin sensitization, and con- action sites, consistent with their effect on both types of PtdInsP- cluded that IDE plays only a limited role in insulin clearance. binding interaction observed in this work. Moreover, Steneberg et al. (9) reported that IDE knockout mice The finding that a number of different InsPs share some compu- did not exhibit elevated fasting insulin levels, in contrast to other tational docking sites (Fig. S5) has relevance to the observed en- findings. Our results supporting the presence of IDE in endo- hancement of interaction affinities and activation levels with an somes and providing a mechanism for localization strengthen the increasing number of phosphate groups found for the various InsPs case for its role in insulin degradation. The sensitivity of endo- tested (Table 1). If interactions occurprimarilyatthesamesiteor somal localization to mutations in the IDE anion-binding site sites, then enhancement of affinity by additional phosphates simply should provide a basis for a strong test of this function. reflects an increased number of interactions with positively charged IDE recruitment to endosomes also may be relevant to the or hydrogen-bond donor groups on the anion-binding surface. With mechanism underlying the reported secretion of IDE from cer- respect to activation, studies with adenine nucleoside phosphate li- tain cell types (25, 56, 69). Secreted IDE has been proposed to gands (15) have shown that increasing the number of phosphates be involved in extracellular Aβ degradation (6, 70, 71) and po- increases the extent of activation, and the results here with InsPs tentially some insulin hydrolysis as well (72, 73). IDE does not indicate that this effect likely reflects a general dependence on the contain any standard secretion signaling sequences, reportedly total charge or the charge density of the interacting ligand. On the being secreted by unconventional pathways (25, 56, 69, 71). other hand, there are subtle differences in computational docking Enzyme localized to the endosomal system could contribute to preferences for different InsPs, suggesting that different binding this secreted pool, given that some multivesicular bodies fuse modes may play roles in ligand affinity and the level of activation. with the cell plasma membrane, releasing their contents, in- Indeed, the finding of modest differences in affinity and activation cluding the intraluminal vesicles (exosomes), into the extracel- level between isomers bearing the same number of phosphate groups, lular medium (25, 74). Endosomal IDE also could be trafficked such as Ins(1,3,5)P3 and Ins(1,4,5)P3, supports this possibility. to the Golgi (or taken up directly into that organelle) and sub- Overall, the results presented here suggest InsPs and PtdInsPs sequently secreted, but this pathway does not appear to be sig- as potential physiological modulators of IDE, with InsPs serving as nificant in the cell types in which secretion of the enzyme was activators with respect to small peptide substrates and PtdInsPs studied (25, 56, 69). playing a role in the intracellular spatial regulation of IDE. Because the anion-biding site is not accessible in the closed form of IDE, the question naturally arises as to how IDE uses Materials and Methods this surface to bind PtdInsPs present in a lipid bilayer. Compu- Materials. InsPs were purchased from Cayman Chemical Company, and PtdInsPs tational docking of Ins(1,3)P2, representing the head group of were purchased from Echelon Bioscience. The PtdInsPs used in this study PtdIns(3)P, as well as several other InsPs to IDE shows a small contained palmitic acid as the fatty acid in both the 1 and 2 positions (di-C16) or number of preferred binding sites, two of which appear partic- octanoic acid in both position (di-C8). Avanti Polar Lipids was the source for

E2832 | www.pnas.org/cgi/doi/10.1073/pnas.1613447114 Song et al. Downloaded by guest on September 23, 2021 College London, London, UK. The anti-IDE antibody (rIDE4020) used in this study PNAS PLUS has been described previously (5). Western blot analysis established that this an- tibody exhibits the same avidity for IDE and the IDE mutants used in the present study (Fig. S2). Anti-His antibody was obtained from GE Healthcare Life Science. IDE and its mutant forms were expressed in Sf9 cells and purified to homogeneity as described previously (16, 29, 78). Dextran (molecular weight 10,000) labeled with Alexa Fluor 488 was obtained from Thermo Fisher Scientific.

Activity Measurements. IDE was routinely assayed using the fluorogenic pep- tide Abz-GGFLRKHGQ-Eddnp as described previously (79). Kinetic data were fit to the Hill or Michaelis–Menten equation using GraphPad Prism software.

Peptide Hydrolysis. The hydrolysis of unlabeled peptide substrates was fol- lowed by measuring the disappearance of the parent peptide by HPLC. Re- actions of 80 μL containing 10 μM peptide, IDE, and InsP as indicated in 50 mM Tris·HCl pH 7.4 were incubated at 37 °C for 5–40 min. The reaction was terminated by the addition of 8 μL of 5% TFA, and 75 μL was injected onto a Vydac C18 column. Peptides were eluted with a linear gradient of 5–50% acetonitrile (15), and quantified by measuring peak areas.

Lipid and Western Blot Analyses. Membranes containing 15 different bi- ologically active lipids at 100 pmol/spot (Echelon Bioscience) were first blocked with 5% fatty acid-free BSA prepared in PBS at room temperature for 2 h, after which 4 mL of fresh blocking solution containing 0.49 μg/μL IDE was added. The membrane was incubated at room temperature for 1 h and then at 4 °C overnight. The membrane was washed three times for 30 min each with PBS containing 0.1% Tween 20 (PBST). After washing, the membrane was treated with either anti-IDE antibody or anti-His antibody in PBS con- taining 5% fatty acid-free BSA for 1 h. After three washes with PBST for 30 min each, the membrane was incubated with horseradish peroxidase- conjugated anti-mouse antibody or peroxidase-conjugated anti-rabbit an- tibody (Invitrogen) in PBS containing 5% fatty acid-free BSA for 1 h, and then rinsed three times for 30 min with PBST. The signal was detected using ECL Plus Western Blotting Reagent (Thermo Fisher Scientific). Membranes containing varying amounts of PtdIns(4,5)P2 were prepared by spotting 2 μL of a lipid solution containing the indicated amount of lipid onto Hybond-C Extra membranes (Amersham). After drying, the membranes were incubated with IDE or its mutant forms and treated as above. Lipid solutions were prepared in 250 μL of a 1:2:1 chloroform:MeOH:water solu- BIOCHEMISTRY tion to which 2 μL of Ponceau S was added. For Western blots, when feasible protein concentrations were measured using the Coomassie Plus Protein Assay Kit (Thermo Fisher Scientific). Samples were subjected to electrophoresis on 10% polyacrylamide gels. Bands were transferred to PVDF membranes (GE Healthcare), and membranes were probed Fig. 6. Computational docking and models for lipid binding by IDE. (A) Binding with the appropriate primary and secondary antibodies and developed as site clusters for Ins(1,3)P2 computationally docked to IDE, with the ligands drawn described above for the lipid blots. In the case of subcellular fractionation as stick figures. The site that possibly mediates interaction with membrane-bound studies, the blots for expression of different IDE variants were done on separate PtdIns head groups is indicated by the red circle. The site that may mediate the membranes but compared as ratios with either the total amount of IDE protein head group interaction with activating lipid bound within the substrate-binding loaded or the amount of Rab5 as an endosomal marker. chamber is circled in black. Side chains of active site residues are shown in a stick Quantitation of lipid and Western blots was carried out on a Gel Doc XR+ gel representation. (B) A model for the interaction with membrane-bound PtdIns(3)P documentation system (Bio-Rad) with Image Lab software (Bio-Rad). Either box with the lipid in a space-filling representation and the protein in a ribbon rep- (Western) or circle (lipid) integration areas with minor adjustments across lanes resentation. The lipid is shown with 17:0 and 20:4 acyl chains and was placed were used with the automatic background subtraction function. Linearity was manually so that its head group matches the binding of Ins(1,3)P2, indicated by determined by comparing results for at least three different exposure times. the red circle in A. Side chains of active site residues are shown. (C) Space-filling view of the IDE surface that would interact with the membrane in the model Preparation of Liposomes. Liposomes were prepared as described by Buser shown in B. Residue atoms were colored based on conservation using the ConSurf and McLaughlin (80). In brief, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; server, with blue the least conserved and dark red the most conserved. The 6 μmol) with or without PtdIns(4,5)P2 (1 μmol) in chloroform were mixed and conservation of this surface is the highest for the any external surface of IDE. dried under vacuum in a rotary evaporator immersed in a 30 °C water bath. (D) A model for the internal binding of PtdIns(3)P with the lipid head group The crude liposomes were resuspended in a 6-mL sucrose solution (176 mM manually placed to match the binding of Ins(1,3)P2 in the docking cluster in- sucrose and 1 mM Mops, pH 7.0) and taken through five cycles of freezing in

dicated by the black circle in A. The lipid is shown with 17:0 and 20:4 acyl chains. liquid N2 and thawing in a 30 °C water bath. Large unilamellar vesicles were (E) Cutaway view of the IDE substrate-binding chamber showing another view of prepared by subjecting the mixture to 10 cycles of extrusion through a stack of the bound PtdIns(3)P model to emphasize the ability of the inner chamber to two polycarbonate filters (100-nm pore size) in a Lipex Biomembranes Ex- accommodate the lipid. (F) Cutaway view of the substrate-binding chamber truder. The sucrose solution on the outside of the vesicles was removed by showing the surface that would interact with PtdIns acyl chains in the model mixing the large unilamellar vesicles with a salt solution (100 mM KCI and shown in D and E. The surface is color-coded by electrostatic potential using the 1 mM Mops, pH 7.0) and recovering the vesicles by ultracentrifugation at adaptive Poisson–Boltzmann solver server with cutoffs of ±10 kT. The surface 100,000 × g for 1 h at 25 °C. The concentration of PtdIns(4,5)P2 in the lipo- proposed to interact with the acyl chains has a relatively low electrostatic po- somes was determined by measuring the phosphate content (81) and cor- tential, consistent with this interaction. rected for the number of phosphates in the inositol.

Liposome-Binding Assay. IDE (0.9 μg) was incubated with DOPC liposomes with 1,2-dioleoyl-sn-glycero-3-phosphocholine; 1-diphosphoinositol pentakisphosphate or without phosphatidylinositol 4,5-bisphosphate (total lipid concentration, and 5-diphosphoinositol pentakisphosphate were synthesized as described pre- 260 μM) in a volume of 280 μL for 1 h at room temperature with gentle viously (76, 77) by A. Saiardi, MRC Laboratory for Molecular Cell Biology, University rocking. After centrifugation at 112,000 × g for 1 h at 20 °C, the liposomes

Song et al. PNAS | Published online March 21, 2017 | E2833 Downloaded by guest on September 23, 2021 were pelleted and then resuspended in SDS/PAGE sample buffer. The pelleted dual filter wheels, and a 175W xenon light source, all controlled by SlideBook liposomes were subjected to SDS/PAGE and Western blot analysis. software (Intelligent Imaging Innovations). The detection of Alexa Fluor 488 fluorescence was performed using an FITC filter channel, and the de- Sucrose Gradient Subcellular Fractionation. Sucrose gradient subcellular frac- tection of Alexa 549 Fluor fluorescence was performed using a TRITC channel. tions were prepared as described by de Araujo et al. (82). In brief, COS-1 cells Images were acquired in 2 × 2 binning mode. Image analysis was performed were grown on 15-cm dishes, washed, and scraped with a rubber policeman in using SlideBook 6 software (Intelligent Imaging Innovations). Colocalization cold PBS. The cells were then pelleted, resuspended in homogenization buffer analysis was done with the interactive segmentation and colocalization (250 mM sucrose and 3 mM imidazole, pH 7.4, containing and phos- modules in SlideBook 6. Statistics for colocalization are averages for 18 cells phatase inhibitors), and homogenized until ∼90% of the cells were broken from six independent 3D images. Threshold levels were determined using without major breakage of the nucleus, as monitored by microscopy. The the method developed by Costes et al. (83). samples were centrifuged at 2,000 × g for 10 min at 4 °C, and the resulting supernatant was designated the postnuclear supernatant (PNS). The PNS sam- Computational Docking and Modeling of Phosphoinositide Binding. Compu- ples were adjusted to 40.6% sucrose and then overlaid with 1.5 volumes of 35% tational docking of InsPs to the WT unliganded IDE structure (Protein Data sucrose. The remaining volume of the centrifuge tube was then filled with 8.6% Bank ID code 3P7L) (27) was carried out with Autodock Vina 1.1.2 (84) as sucrose. Sucrose gradients were centrifuged at 100,000 × g for 6 h at 4 °C, and implemented in Yasara (85). The 3D structures for InsP ligands were gen- the endosomal and Golgi/ER membranes were collected. erated from SMILES representations, and energy was minimized in Yasara using the NOVA force field and the supplied em_runclean macro. Either Wortmannin Treatment. COS-1 cells were treated with 200 nM wortmannin for 25 or 100 docking runs were carried out with Ins(1,3)P2, Ins(1,4,5)P3, Ins 0, 0.5, and 4 h, after which the endosomal fraction was isolated by sucrose (1,3,5)P3, or phytic acid using the Yasara dock_run macro with the choice of density gradient centrifugation as described above. A constant amount Vina docking. A model and molecular descriptors for PtdIns(3)P were gen- of endosomal protein was subjected to Western blot analysis using anti- erated from a SMILES descriptor using the eLBOW (86) module of PHENIX IDE antibody. (87) and acyl chains were adjusted manually in Coot (88). The resulting model was then energy-minimized in Yasara as described for the InsPs. The Immunofluorescence Staining and Analysis. Cos-1 cells were grown on PtdIns(3)P ligand was manually modeled into the binding site IDE, aligning polylysine-coated coverslips in serum-free DMEM media and transformed with its head group with a high-scoring docked Ins(1,3)P2. Open models of IDE pCDNA-3.1 plasmid expressing IDEwt. For dextran uptake experiments, cells were generated manually in Coot. were pulse-labeled in serum-free DMEM with 2 mg/mL dextran-Alexa Fluor 488 (molecular weight 10,000, lysine fixable; Invitrogen) for 2 h at 37 °C in 5% CO2, ACKNOWLEDGMENTS. We thank Dr. A. Saiardi for providing the isomer of followed by permeabilization with 0.05% saponin before cell fixation. Cells InsP7 used in this study and acknowledge the use of facilities at the Univer- were then fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for sity of Kentucky Center for Structural Biology and the Center for Molecular 10 min at room temperature and then blocked with 10% normal serum, 1% Medicine Protein Core (supported by National Institutes of Health Grant P20 BSA, and 0.3 M glycine. For immunostaining, cells were incubated with the GM103486). This work was supported by National Institutes of Health grants GM 11787 (to L.B.H.), NS38041 (to D.W.R.), GM113087 (to E.G.); American primary antibody for 90 min in PBS containing 1% BSA, rinsed, and incubated Cancer Society Grant RSG-14-172-01-CSM (to E.G.); American Heart Associa- for 60 min with secondary Alexa Fluor 549-conjugated antibody (Jackson tion Grant 15PRE25090207 (to H.J.); and National Science Foundation Grant ImmunoResearch) in PBS with 1% BSA. Cells were rinsed and mounted for IIA-1355438 (to D.W.R.). Part of this work was supported by the Fundação de microscopy in Mowiol mounting medium. Amparo à Pesquisado Estado de São Paulo (Project 12/50191-4R) and the All images were acquired using a Mariannas Imaging system consisting of a Conselho Nacional de Desenvolvimento Científico e Tecnológico (Projects Zeiss inverted microscope equipped with a cooled CCD CoolSnap HQ (Roper), 443978-2014-0 and 467478-2014-7).

1. Sladek R, et al. (2007) A genome-wide association study identifies novel risk loci for 17. Song ES, et al. (2015) An extended polyanion activation surface in insulin-degrading type 2 diabetes. Nature 445(7130):881–885. enzyme. PLoS One 10(7):e0133114. 2. Zeggini E, et al.; Wellcome Trust Case Control Consortium (2007) Replication of 18. Tsui MM, York JD (2010) Roles of inositol phosphates and inositol pyrophosphates genome-wide association signals in UK samples reveals risk loci for type 2 diabetes. in development, cell signaling, and nuclear processes. Adv Enzyme Regul 50(1): Science 316(5829):1336–1341. 324–337. 3. Kim M, et al. (2007) Decreased catalytic activity of the insulin-degrading enzyme in 19. Wilson MS, Livermore TM, Saiardi A (2013) Inositol pyrophosphates: Between sig- chromosome 10-linked Alzheimer disease families. J Biol Chem 282(11):7825–7832. nalling and metabolism. Biochem J 452(3):369–379. 4. Wang S, He F, Wang Y (2015) Association between polymorphisms of the insulin- 20. Behnia R, Munro S (2005) Organelle identity and the signposts for membrane traffic. degrading enzyme gene and late-onset Alzheimer disease. J Geriatr Psychiatry Nature 438(7068):597–604. Neurol 28(2):94–98. 21. Kutateladze TG (2010) Translation of the phosphoinositide code by PI effectors. Nat 5. Miller BC, et al. (2003) Amyloid-beta peptide levels in brain are inversely correlated Chem Biol 6(7):507–513. with insulysin activity levels in vivo. Proc Natl Acad Sci USA 100(10):6221–6226. 22. Balla T (2013) Phosphoinositides: Tiny lipids with giant impact on cell regulation. 6. Farris W, et al. (2003) Insulin-degrading enzyme regulates the levels of insulin, amy- Physiol Rev 93(3):1019–1137. loid beta-protein, and the beta-amyloid precursor protein intracellular domain in 23. Hamel FG, Mahoney MJ, Duckworth WC (1991) Degradation of intraendosomal vivo. Proc Natl Acad Sci USA 100(7):4162–4167. insulin by insulin-degrading enzyme without acidification. Diabetes 40(4): 7. Maianti JP, et al. (2014) Anti-diabetic activity of insulin-degrading enzyme inhibitors 436–443. mediated by multiple hormones. Nature 511(7507):94–98. 24. Fernández-Gamba A, Leal MC, Morelli L, Castaño EM (2009) Insulin-degrading en- 8. Deprez-Poulain R, et al. (2015) Catalytic site inhibition of insulin-degrading enzyme by zyme: Structure-function relationship and its possible roles in health and disease. Curr a small molecule induces glucose intolerance in mice. Nat Commun 6:8250. Pharm Des 15(31):3644–3655. 9. Steneberg P, et al. (2013) The type 2 diabetes-associated gene ide is required for 25. Bulloj A, Leal MC, Xu H, Castaño EM, Morelli L (2010) Insulin-degrading enzyme insulin secretion and suppression of α-synuclein levels in β-cells. Diabetes 62(6): sorting in exosomes: A secretory pathway for a key brain amyloid-beta degrading 2004–2014. protease. J Alzheimers Dis 19(1):79–95. 10. Durham TB, et al. (2015) Dual exosite-binding inhibitors of insulin-degrading enzyme 26. Morita M, et al. (2000) Insulin-degrading enzyme exists inside of rat liver peroxisomes challenge its role as the primary mediator of insulin clearance in vivo. J Biol Chem and degrades oxidized proteins. Cell Struct Funct 25(5):309–315. 290(33):20044–20059. 27. Noinaj N, et al. (2011) Identification of the allosteric regulatory site of insulysin. PLoS 11. Li Q, Ali MA, Cohen JI (2006) Insulin-degrading enzyme is a cellular receptor medi- One 6(6):e20864. ating varicella-zoster virus infection and cell-to-cell spread. Cell 127(2):305–316. 28. Shen Y, Joachimiak A, Rosner MR, Tang WJ (2006) Structures of human insulin- 12. Tundo GR, et al. (2013) Insulin-degrading enzyme (IDE): A novel heat shock-like degrading enzyme reveal a new substrate recognition mechanism. Nature 443(7113): protein. J Biol Chem 288(4):2281–2289. 870–874. 13. Sbardella D, et al. (2015) Proteasome activity is affected by fluctuations in insulin- 29. Song ES, Juliano MA, Juliano L, Hersh LB (2003) Substrate activation of insulin- degrading enzyme distribution. PLoS One 10(7):e0132455. degrading enzyme (insulysin): A potential target for drug development. J Biol 14. Radulescu RT, Duckworth WC, Levy JL, Fawcett J (2010) Retinoblastoma protein co- Chem 278(50):49789–49794. purifies with proteasomal insulin-degrading enzyme: Implications for cell pro- 30. Yao H, Hersh LB (2006) Characterization of the binding of the fluorescent ATP analog liferation control. Biochem Biophys Res Commun 395(2):196–199. TNP-ATP to insulysin. Arch Biochem Biophys 451(2):175–181. 15. Song ES, et al. (2004) ATP effects on insulin-degrading enzyme are mediated primarily 31. Bissig C, Gruenberg J (2013) Lipid sorting and multivesicular endosome biogenesis. through its triphosphate moiety. J Biol Chem 279(52):54216–54220. Cold Spring Harb Perspect Biol 5(10):a016816. 16. Noinaj N, et al. (2012) Anion activation site of insulin-degrading enzyme. J Biol Chem 32. Jang ER, et al. (2015) Spatial control of Shoc2 scaffold-mediated ERK1/2 signaling 287(1):48–57. requires remodeling activity of the ATPase PSMC5. J Cell Sci 128(23):4428–4441.

E2834 | www.pnas.org/cgi/doi/10.1073/pnas.1613447114 Song et al. Downloaded by guest on September 23, 2021 33. Kuo WL, Gehm BD, Rosner MR, Li W, Keller G (1994) Inducible expression and cellular 61. Falguières T, Luyet PP, Gruenberg J (2009) Molecular assemblies and membrane do- PNAS PLUS localization of insulin-degrading enzyme in a stably transfected cell line. J Biol Chem mains in multivesicular endosome dynamics. Exp Cell Res 315(9):1567–1573. 269(36):22599–22606. 62. Hu YB, Dammer EB, Ren RJ, Wang G (2015) The endosomal-lysosomal system: From 34. Grasso G, Satriano C, Milardi D (2015) A neglected modulator of insulin-degrading acidification and cargo sorting to neurodegeneration. Transl Neurodegener 4:18. enzyme activity and conformation: The pH. Biophys Chem 203-204:33–40. 63. Burd C, Cullen PJ (2014) Retromer: a master conductor of endosome sorting. Cold 35. Guo Q, Manolopoulou M, Bian Y, Schilling AB, Tang WJ (2010) Molecular basis for the Spring Harb Perspect Biol 6(2):a016774. recognition and cleavages of IGF-II, TGF-alpha, and amylin by human insulin- 64. Sahu R, et al. (2011) Microautophagy of cytosolic proteins by late endosomes. Dev Cell degrading enzyme. J Mol Biol 395(2):430–443. 20(1):131–139. 36. Manolopoulou M, Guo Q, Malito E, Schilling AB, Tang WJ (2009) Molecular basis of 65. Liu XM, et al. (2015) ESCRTs cooperate with a selective autophagy receptor to me- catalytic chamber-assisted unfolding and cleavage of human insulin by human diate vacuolar targeting of soluble cargos. Mol Cell 59(6):1035–1042. insulin-degrading enzyme. J Biol Chem 284(21):14177–14188. 66. Leissring MA, et al. (2010) Designed inhibitors of insulin-degrading enzyme regulate 37. Ralat LA, et al. (2011) Insulin-degrading enzyme modulates the natriuretic peptide- the catabolism and activity of insulin. PLoS One 5(5):e10504. mediated signaling response. J Biol Chem 286(6):4670–4679. 67. Abdul-Hay SO, et al. (2011) Deletion of insulin-degrading enzyme elicits antipodal, 38. Ralat LA, et al. (2011) Ubiquitin is a novel substrate for human insulin-degrading age-dependent effects on glucose and insulin tolerance. PLoS One 6(6):e20818. enzyme. J Mol Biol 406(3):454–466. 68. Hamel FG, Posner BI, Bergeron JJ, Frank BH, Duckworth WC (1988) Isolation of insulin 39. Liang WG, Ren M, Zhao F, Tang WJ (2015) Structures of human CCL18, CCL3, and degradation products from endosomes derived from intact rat liver. J Biol Chem CCL4 reveal molecular determinants for quaternary structures and sensitivity to 263(14):6703–6708. – insulin-degrading enzyme. J Mol Biol 427(6 Pt B):1345 1358. 69. Glebov K, Schütze S, Walter J (2011) Functional relevance of a novel SlyX motif in non- 40. Manning BD (2010) Insulin signaling: Inositol phosphates get into the Akt. Cell 143(6): conventional secretion of insulin-degrading enzyme. JBiolChem286(26):22711–22715. – 861 863. 70. Qiu WQ, Folstein MF (2006) Insulin, insulin-degrading enzyme and amyloid-beta 41. Chakraborty A, et al. (2010) Inositol pyrophosphates inhibit Akt signaling, thereby peptide in Alzheimer’s disease: Review and hypothesis. Neurobiol Aging 27(2): – regulating insulin sensitivity and weight gain. Cell 143(6):897 910. 190–198. 42. Illies C, et al. (2007) Requirement of inositol pyrophosphates for full exocytotic ca- 71. Tamboli IY, et al. (2010) Statins promote the degradation of extracellular amyloid – pacity in pancreatic beta cells. Science 318(5854):1299 1302. beta-peptide by microglia via stimulation of exosome-associated insulin-degrading 43. Sasakawa N, Sharif M, Hanley MR (1995) Metabolism and biological activities of enzyme (IDE) secretion. J Biol Chem 285(48):37405–37414. inositol pentakisphosphate and inositol hexakisphosphate. Biochem Pharmacol 50(2): 72. Yokono K, Roth RA, Baba S (1982) Identification of insulin-degrading enzyme on the 137–146. surface of cultured human lymphocytes, rat hepatoma cells, and primary cultures of 44. Barker CJ, Wright J, Hughes PJ, Kirk CJ, Michell RH (2004) Complex changes in cellular rat hepatocytes. Endocrinology 111(4):1102–1108. inositol phosphate complement accompany transit through the cell cycle. Biochem J 73. Goldfine ID, et al. (1984) Degradation of insulin by isolated mouse pancreatic acini: 380(Pt 2):465–473. Evidence for cell surface protease activity. Diabetes 33(1):64–72. 45. Edbauer D, Willem M, Lammich S, Steiner H, Haass C (2002) Insulin-degrading enzyme 74. Kowal J, Tkach M, Théry C (2014) Biogenesis and secretion of exosomes. Curr Opin Cell rapidly removes the beta-amyloid precursor protein intracellular domain (AICD). J Biol Biol 29:116–125. Chem 277(16):13389–13393. 75. Kutateladze TG (2012) Molecular analysis of protein-phosphoinositide interactions. 46. Currinn H, Wassmer T (2016) The amyloid precursor protein (APP) binds the PIKfyve Curr Top Microbiol Immunol 362:111–126. complex and modulates its function. Biochem Soc Trans 44(1):185–190. 76. Azevedo C, Burton A, Bennett M, Onnebo SM, Saiardi A (2010) Synthesis of InsP7 by 47. Zolov SN, et al. (2012) In vivo, Pikfyve generates PI(3,5)P2, which serves as both a the inositol hexakisphosphate kinase 1 (IP6K1). Methods Mol Biol 645:73–85. signaling lipid and the major precursor for PI5P. Proc Natl Acad Sci USA 109(43): 77. Saiardi A, Bhandari R, Resnick AC, Snowman AM, Snyder SH (2004) Phosphorylation of 17472–17477. proteins by inositol pyrophosphates. Science 306(5704):2101–2105. 48. Duckworth WC, Bennett RG, Hamel FG (1994) A direct inhibitory effect of insulin on a 78. Song ES, et al. (2001) Analysis of the subsite specificity of rat insulysin using fluoro- cytosolic proteolytic complex containing insulin-degrading enzyme and multicatalytic genic peptide substrates. J Biol Chem 276(2):1152–1155. proteinase. J Biol Chem 269(40):24575–24580. 79. Csuhai E, et al. (1999) New fluorogenic substrates for N-arginine dibasic convertase. 49. Bennett RG, Fawcett J, Kruer MC, Duckworth WC, Hamel FG (2003) Insulin inhibition Anal Biochem 269(1):149–154. of the proteasome is dependent on degradation of insulin by insulin-degrading en- 80. Buser CA, McLaughlin S (1998) Ultracentrifugation technique for measuring the zyme. J Endocrinol 177(3):399–405. 50. Kirschner RJ, Goldberg AL (1983) A high molecular weight metalloendoprotease from binding of peptides and proteins to sucrose-loaded phospholipid vesicles. Methods BIOCHEMISTRY – the cytosol of mammalian cells. J Biol Chem 258(2):967–976. Mol Biol 84:267 281. 51. Hersh LB (2006) The insulysin (insulin-degrading enzyme) enigma. Cell Mol Life Sci 81. Barenholz Y, Amselem S (1993) Quality control assays in the development and clinical 63(21):2432–2434. use of liposome-based formulation. Liposome Technology, ed Gregoriadis G (CRC 52. Bergeron JJ, Cruz J, Khan MN, Posner BI (1985) Uptake of insulin and other ligands Press, Boca Raton, FL), 2nd Ed, Vol I, p 527. into receptor-rich endocytic components of target cells: The endosomal apparatus. 82. de Araùjo ME, Huber LA, Stasyk T (2008) Isolation of endocitic organelles by density – Annu Rev Physiol 47:383–403. gradient centrifugation. Methods Mol Biol 424:317 331. 53. Duckworth WC, Bennett RG, Hamel FG (1998) Insulin degradation: Progress and po- 83. Costes SV, et al. (2004) Automatic and quantitative measurement of protein-protein – tential. Endocr Rev 19(5):608–624. colocalization in live cells. Biophys J 86(6):3993 4003. 54. Authier F, et al. (1995) Degradation of the cleaved leader peptide of thiolase by a 84. Trott O, Olson AJ (2010) AutoDock Vina: Improving the speed and accuracy of peroxisomal proteinase. Proc Natl Acad Sci USA 92(9):3859–3863. docking with a new scoring function, efficient optimization, and multithreading. 55. Leissring MA, et al. (2004) Alternative translation initiation generates a novel isoform J Comput Chem 31(2):455–461. of insulin-degrading enzyme targeted to mitochondria. Biochem J 383(Pt. 3):439–446. 85. Krieger E, Vriend G (2002) Models@Home: Distributed computing in bioinformatics 56. Zhao J, Li L, Leissring MA (2009) Insulin-degrading enzyme is exported via an un- using a screensaver-based approach. Bioinformatics 18(2):315–318. conventional protein secretion pathway. Mol Neurodegener 4:4. 86. Moriarty NW, Grosse-Kunstleve RW, Adams PD (2009) Electronic ligand builder and 57. Shewan A, Eastburn DJ, Mostov K (2011) Phosphoinositides in cell architecture. Cold optimization workbench (eLBOW): A tool for ligand coordinate and restraint gen- Spring Harb Perspect Biol 3(8):a004796. eration. Acta Crystallogr D Biol Crystallogr 65(Pt 10):1074–1080. 58. Tarasoff-Conway JM, et al. (2015) Clearance systems in the brain: Implications for 87. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- Alzheimer disease. Nat Rev Neurol 11(8):457–470. molecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221. 59. Nixon RA (2005) Endosome function and dysfunction in Alzheimer’s disease and other 88. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta neurodegenerative diseases. Neurobiol Aging 26(3):373–382. Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132. 60. Trikha S, Jeremic AM (2013) Distinct internalization pathways of human amylin 89. Goddard ED, Turro NJ, Kuo PL, Ananthapadmanabhan KP (1985) Fluorescence probes monomers and its cytotoxic oligomers in pancreatic cells. PLoS One 8(9):e73080. for critical micelle concentration determination. Langmuir 1(3):352–355.

Song et al. PNAS | Published online March 21, 2017 | E2835 Downloaded by guest on September 23, 2021