Proteolytic Potential of the MSC Exosome Proteome: Implications for an Exosome-Mediated Delivery of Therapeutic Proteasome

Abstract

Mesenchymal stem cells (MSCs) are used in many of the current stem cell-based clinical trials and their therapeutic efficacy has increasingly been attributed to secretion of paracrine factors. We have previously demonstrated that a therapeutic constituent of this secretion is exosome, a secreted bilipid membrane vesicle of ~50-100 nm with a complex cargo that is readily internalized by H9C2 cardiomyocytes. It reduces infarct size in a mouse model of myocardial ischemia/reperfusion (MI/R) injury. We postulate that this therapeutic efficacy is derived from the synergy of a select permutation of individual exosome components. To identify protein candidates in this permutation, the proteome was profiled and here we identified 20S proteasome as a protein candidate. Mass spectrometry analysis detected all seven α and seven β chains of the 20S proteasome, and also the three beta subunits of "immunoproteasome" with a very high confidence level. We demonstrated that a functional proteasome copurified with MSC exosomes with a density of 1.10-1.18 g/mL, and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Circulating proteasomes in human blood also copurified with exosomes. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents to ameliorate tissue damage.

Figure 1

Exosome proteins. (a) Venn diagram of number of proteins detected by LC-MS/MS in 3 independent batches of exosomes, CM15, CM17, and CM18. There are 379, 432, and 420 proteins detected in CM15, CM17 and CM18, respectively. The combined protein number is 766. (b) Venn diagram shows the overlap of proteins detected by LC-MS/MS and antibody array. There are 101 proteins detected by antibody array in CM15. 10 of the 101 proteins detected are overlapping with proteins detected by LC-MS/MS. (c) Intersection of the 739 proteins previously identified in MSC conditioned medium versus the 857 proteins identified in purified exosomes.

Figure 2

20S proteasome in MSC exosome. (a) Western blot analysis of MSC conditioned medium (CM) and exosome (Exo) using an antibody specific for PMSA 1–7 peptides. (b) Protein analysis of exosome fractionated on a sucrose gradient density. Exosome or exosome pretreated with lysis buffer was loaded on a sucrose density gradient prepared by layering 14 sucrose solutions of concentrations from 22.8 to 60% (w/v) in a SW60Ti centrifuge tube and then ultracentrifuged for 16.5 h at 200 000 g, 4°C, in a SW60Ti rotor. The gradients were removed from the top and the density of each fraction was calculated by weighing a fixed volume of each fraction. The fractions were analyzed by western blot analysis for CD9, PSMA1–7, CD81, CD59, and CD63 in exosome (upper panel) and pretreated exosomes (lower panel). (c) pI of 20S proteasome and CD9 in exosome. Exosome sample was separated by liquid-phase isoelectric focusing into 10 fractions from pH1–14. Each fraction was then concentrated and analyzed by western blot analysis for CD9, PSMA1–7. (d) Proteasome activity in MSC exosome was determined using a commercially available proteasome activity assay kit as described in the Materials and Methods section. Proteasome activity was measured by the rate of degradation of a fluorogenic peptide in the absence or presence of lactacystin, a proteasome inhibitor. One unit (U) enzyme activity is defined as the activity to generate 1 μmole product per minute at 37°C. Each bar represents mean ± SEM of 2 independent assays with each assay performed in triplicate. *P = 0.00023.

Figure 3

Exosome reduced oxidized protein in vivo. Myocardial ischemia/reperfusion injury was induced by ligation of the left coronary artery (LCA) for 30 min and subsequent reperfusion by releasing the ligation. Five minutes before reperfusion, mice (n = 4/group/time point) were intravenously infused with exosome or saline. At 15 min, 1 hour, 1 day, or 3 days after reperfusion, the animal was scarified and the area at risk (AAR) was excised and extracted for protein. One μg of each sample was applied onto a nitrocellulose membrane, stained with Ponceau S, destained and probed with rabbit antioligomer antibody and a HRP reporting system as described in material and method. The intensity of the antibody binding was quantified using Image Lab and normalized against the intensity of Ponceau S staining. (a) Representative dot plots. (b) Each bar represented mean ± SEM of 4 animal.

Figure 4

Human plasma contained exosome-associated 20S proteasome. (a) Human plasma was loaded on a sucrose density gradient prepared by layering 14 sucrose solutions of concentrations from 22.8 to 60% (w/v) in a SW60Ti centrifuge tube and then ultracentrifuged for 16.5 h at 200 000 g, 4°C, in a SW60Ti rotor. 330 μL fractions were removed sequentially from the top and the density of each fraction was calculated by weighing a fixed volume of each fraction. The fractions were analyzed by Western blot analysis for PSMA1–7. (b) Relative amount of CD81 and PSMA1–7 in plasma exosome. Exosomes which are known to have membrane enriched in GM1 gangliosides were isolated from the sucrose density gradient by the high affinity of GM1 gangliosides for cholera toxin B (CTB) chains conjugated to magnetic beads. The CTB extract was assayed by ELISA for the amount of PSMA1–7 and CD81. Each point represents mean ± SEM (n = 2).