PPARγ protein expression in FGR placenta derived EVs is impaired

To explore the differences in human placenta derived EVs between normal and FGR pregnancy, the EVs were firstly isolated from normal and FGR term placenta via gradient centrifugation. Electron microscopy showed these that EVs exhibit a typical double-membrane structure (Fig. S1B). Western blot confirmed high expression of EV-specific biomarkers, including CD9, CD81, CD63 and HSP90β as well as the placenta trophoblast biomarker placental alkaline phosphatase (PLAP), While the negative marker Tubulin was barely detectable (Fig. S1C). Moreover, we ascertained that the size of the extracted EVs from normal and FGR term placenta was primarily within the 80 to 130 nm range, as measured by laser scattering using the NanoSight tracking system (NTA, supplement Table 5, Fig. S1D). To identify the proteins enriched in human normal and FGR term placenta derived EVs, we conducted proteomic analysis of EVs derived from human normal and FGR term placenta using Gas Chromatography-Mass Spectrometry (GC-MS). Wayne’s analysis revealed that 634 proteins were commonly expressed in both normal and FGR placenta derived EVs (Fig. 1A). The Gene Ontology (GO) molecular function analysis of the commonly expressed proteins in normal and FGR term placenta derived EVs indicated a predominant focus on protein binding and fatty acid metabolism (Fig. 1B). Among the fatty acid metabolism-related proteins, PPARγ was noted for the high expression level and its crucial role in adipogenesis (Tables 1 and 2).

Table 1 PPARγ expression in human normal term placenta derived EVsTable 2 PPARγ expression in human FGR term placenta derived EVs

To confirm the expression of PPARγ in the human placenta, primary cytotrophoblasts (CTBs) were isolated from normal and FGR placenta tissues at late pregnancy. The purity of the extracted CTBs was validated by immunofluorescence assay, demonstrating high levels of epithelial cell markers CK7 and E-cadherin (98.09 ± 0.56% and 97.89 ± 0.65% of the cell population, respectively), with minimal expression of the mesenchymal cell marker vimentin (1.85 ± 0.61%) (Fig. 1C). Subsequently, EVs were isolated from primary CTBs (Fig. S1E-G, supplement Table 5). Western blot results showed that PPARγ protein levels in EVs from FGR-derived CTBs as well as FGR CTBs were significantly lower compared to the normal group (Fig. 1D and E), aligning with the lipogenesis disorders in FGR fetuses reported in previous studies [5, 6]. Furthermore, a notable decrease in PPARγ protein expression was observed in EVs derived from FGR placental tissues compared to the normal group (Fig. 1F).

Coat protein complex I (COPI) subunit proteins were enriched in both normal and FGR trophoblast derived EVs

As PPARγ was identified within EVs, we investigated the mechanism by which PPARγ protein enters EVs in trophoblasts. Through proteomic analysis of EVs from normal and FGR term placenta, we identified proteins associated with intracellular protein transport, including COPB2 (β’-COP), ARCN1 (δ-COP), COPG1 (γ-COP), COPA (α-COP) and COPE (ε-COP), all of which are components of the COPI complex (Tables 3 and 4). Western Blot analysis showed that all COPI complex subunits were expressed in both normal and FGR term placenta tissues as well as in placental-derived EVs (Fig. 1G-H). There were no significant differences in their expression levels between the two groups, suggesting that the transportation of PPARγ into EVs may be linked to the COPI complex.

Table 3 COPI complex subunits expression in human normal term placenta derived EVsTable 4 COPI complex subunits expression in human FGR term placenta derived EVsβ’-COP interacts directly with PPARγ in trophoblast starting from the early stage of EVs formation

To investigate the role of the COPI complex in sorting PPARγ into EVs, Co-IP was conducted to confirm the interactions between COPI complex subunits and PPARγ. The interaction of each COPI complex subunit with PPARγ was individually examined in primary CTBs from human normal term placenta. The results revealed a direct interaction between PPARγ and the β’-COP subunit in primary CTBs, with no involvement of other subunits (Fig. 2A-G). This association was further supported by reciprocal immunoprecipitation of COPI complex subunits with PPARγ antibody (Fig. 2H). Immunofluorescence staining demonstrated co-localization of β’-COP and PPARγ in human placenta tissues and primary CTBs from both normal and FGR groups (Fig. 2I), providing spatial evidence of their binding. These findings indicate a specific interaction between β’-COP and PPARγ in human trophoblast cells.

Fig. 2

β’-COP interacts directly with PPARγ in trophoblast starting from the early stage of EVs formation. A-G Co-IP shows the interaction between PPARγ and β’-COP (A), α-COP (B), δ-COP (C), γ-COP (D), β-COP (E), ε-COP (F), ζ-COP (G). H Reciprocal immunoprecipitation of β’-COP, α-COP, δ-COP, γ-COP, β-COP, ε-COP, ζ-COP with PPARγ. I Immunofluorescence and corresponding scatter plot and statistical analysis charts of the localization of β’-COP and PPARγ in human placenta tissues and human placenta derived primary CTBs from normal and FGR group. J-K Immunofluorescence and corresponding scatter plots of the localization of CD63, EEA1 and PPARγ (J) as well as CD63, EEA1 and β’-COP (K) in HTR8/SVneo trophoblast cells. L Co-IP shows the interaction between PPARγ and β’-COP in HTR8/SVneo trophoblast cells. M-N Immunofluorescence and corresponding scatter plots of the localization of LAMP1, LC3 and β’-COP (M) as well as LAMP1, LC3 and PPARγ (N) in HTR8/SVneo trophoblast cells

Studies have shown that EV formation begins with the transition from early endosomes to late endosomes and multivesicular endosomes (LE/MVEs), culminating in EV production [35]. To investigate the stage at which β’-COP and PPARγ interact during EV formation, we performed an immunofluorescence assay to assess their co-localization with the early endosome marker EEA1 and the LE/MVE marker CD63 in HTR8/SVneo trophoblast cells. The assay confirmed the co-localization of PPARγ and β’-COP with EEA1 and CD63 (Fig. 2J-K). Furthermore, a Co-IP assay demonstrated a direct interaction between PPARγ and β’-COP in HTR8/SVneo trophoblast cells (Fig. 2L). These results suggest that the interaction between PPARγ and β’-COP occurs early in EV formation. However, no co-localization was observed with LAMP1, a lysosomal marker, or LC3 puncta, an indicator of autophagosome, in HTR8/SVneo trophoblast cells, indicating that PPARγ and β’-COP specifically localize to early endosomes and MVEs rather than lysosomes or autophagosomes (Fig. 2M-N). In conclusion, β’-COP directly binds to PPARγ in trophoblast cells, influencing PPARγ localization to early endosomes and MVEs during EV formation.

β’-COP mediates ab initio loading of PPARγ into trophoblast EVs

Subsequently, we investigated the role of β’-COP in loading PPARγ into EVs. Through lentiviral transfection, we successfully generated a β’-COP knockout (β’-COP-KO) HTR8/SVneo trophoblast cell line (Fig. 3A-C). The EVs derived from HTR8/SVneo were also characterized (Fig. S1H-J, supplement Table 5). However, immunofluorescence staining revealed a lack of co-localization of PPARγ with EEA1 and CD63 following β’-COP knockout (Fig. 3D-E). Western Blot analysis demonstrated a significant reduction in PPARγ protein expression in trophoblast EVs from the β’-COP-KO group compared to the control group (Fig. 3F). These findings suggest that β’-COP facilitates the transport of PPARγ into EVs during the early stages of EV formation.

Fig. 3

β’-COP mediated ab initio loading of PPARγ into EVs depending on intact COPI complex. A Confocal microscopy shows the GFP and bright phase of negative control (NC) and β’-COP-KO HTR8/SVneo cells. B Western blot of the expression level of β’-COP protein in control (Con), NC and β’-COP-KO HTR8/SVneo cells. C RT-qPCR analysis of the β’-COP mRNA expression level in Con, NC and β’-COP-KO HTR8/SVneo cells. D-E Immunofluorescence and corresponding scatter plot and statistical analysis charts of the localization of CD63 (D), EEA1 (E) and PPARγ in NC and β’-COP-KO HTR8/SVneo cells. F Western blots of the PPARγ, CD63 and PLAP protein expression in EVs of NC and β’-COP-KO HTR8/SVneo cells. G Confocal microscopy of the GFP and bright phase of NC and δ-COP-KO HTR8/SVneo cells. H Western blot of the expression level of δ-COP protein in Con, NC and δ-COP-KO HTR8/SVneo cells. I RT-qPCR analysis of the δ-COP mRNA expression level in Con, NC and δ-COP-KO HTR8/SVneo cells. J Confocal microscopy of the GFP and bright phase of NC and ε-COP-KO HTR8/SVneo cells. K Western blot of the expression level of ε-COP protein in Con, NC and ε-COP-KO HTR8/SVneo cells. L RT-qPCR analysis of the ε-COP mRNA expression level in Con, NC and ε-COP-KO HTR8/SVneo cells. M-N Co-IPs show the interaction between PPARγ and β’-COP in δ-COP-KO (M) and ε-COP-KO (N) HTR8/SVneo trophoblast. O-R Immunofluorescence and corresponding scatter plot and statistical analysis charts of the localization of CD63 (O), EEA1 (P) and PPARγ in δ-COP-KO HTR8/SVneo cells as well as CD63 (Q), EEA1 (R) and PPARγ in ε-COP-KO HTR8/SVneo cells. S Western blots of the PPARγ, CD63 and PLAP protein expression level in δ-COP-KO or ε-COP-KO HTR8/SVneo trophoblasts. These data are shown as mean ± SEM. Two-tailed Student’s t test, *p < 0.05

β’-COP mediated transportation of PPARγ into EVs relies on intact COPI complex

To investigate the relationship between β’-COP mediated loading of PPARγ into EVs and the COPI complex, we constructed δ-COP knockout (δ-COP-KO) and ε-COP knockout (ε-COP-KO) HTR8/SVneo trophoblast cell lines by lentiviral transfection (Fig. 3G-L). Co-IP assays indicated that the knockout of δ-COP or ε-COP could significantly interfere with the interaction of PPARγ and β’-COP (Fig. 3M-N). Importantly, the deficiency of δ-COP or ε-COP can also result in an inhibition in the EVs transportation of PPARγ in trophoblast which manifested as declined co-localization of PPARγ with EEA1 and CD63, as well as reduced PPARγ protein expression in δ-COP-KO and ε-COP-KO HTR8/SVneo trophoblast (Fig. 3O-S). These findings demonstrated the essential role of intact COPI complex in the loading of PPARγ proteins into EVs.

The binding of PPARγ to β’-COP depends on solvation free energy and van der Waals forces

To investigate the binding modes between PPARγ and β’-COP, we conducted molecular dynamics simulations using Gromacs 2019.6 software. The root means square deviation (RMSD) analysis revealed that equilibrium in the β’-COP-PPARγ binding was reached after 20 ns of simulation (Fig. 4A), with the RMSD value stabilizing around 1.0 nm. Further assessment of the stability of the complex was performed through radius of gyration (Rg) and solvent accessible surface area (SASA) analysis (Fig. 4B-C). The Rg fluctuated within 4.7 nm post 20 ns of simulation, indicating sustained stability of the β’-COP-PPARγ binding complex (Fig. 4B). Additionally, the SASA of the complex remained constant throughout the simulation (after 20 ns) (Fig. 4C), suggesting persistent binding of PPARγ to the β’-COP surface (Fig. 4D-E).

Fig. 4

The binding of PPARγ to β’-COP depends on solvation free energy and van der Waals forces. A-C The RMSD (A), Rg (B) and SASA (C) analysis of β’-COP-PPARγ complex. D Binding surface image of the β’-COP-PPARγ complex. E 3D interaction interface diagram of the β’-COP-PPARγ complex. F The hydrogen bonding (H-bond) analysis of β’-COP-PPARγ complex. G The ribbon diagram of β’-COP-PPARγ complex. H 2D interaction interface diagram of β’-COP-PPARγ complex. I Energetic components analysis of β’-COP-PPARγ complex. J-K The bar diagram (J) and heat map (K) of the β’-COP-PPARγ complex binding free energy decomposition into residues

To investigate the binding force between β’-COP and PPARγ, we conducted simulations to analyze hydrogen bond distribution during the binding process (Fig. 4F). The results show that β’-COP-PPARγ typically forms 7–8 hydrogen bonds over the simulation period of 0-100ns, indicating stable binding of PPARγ to the β’-COP protein surface (Fig. 4F-H). Upon further analysis, the BFE between PPARγ and β’-COP was determined to be -38.55 kcal/mol, primarily driven by solvation free energy (ΔGSOLV, -32.87 kcal/mol) and Vander Waals forces (ΔVDWAALS, -31.60 kcal/mol) (Table 5). The Coulomb force was found to be the main repulsive force (ΔEEL, 25.92 kcal/mol) (Fig. 4I; Table 5). Decomposition of the BFE of the β’-COP-PPARγ complex into residues highlighted key amino acids crucial for their interaction (Fig. 4J-K). Nine of these amino acids were identified as hot spots with a binding energy less than − 1 kcal/mol, namely GLU175, HIS178, ASP203, ARG204, ASP207, ASN238, and ARG244 for β’-COP, and LYS273, GYN281, and PHE285 for PPARγ (Fig. 4J-K). This analysis and subsequent bioinformatics investigation identified the binding domains of PPARγ and β’-COP to be located between amino acid residues 258–286 on PPARγ.

Table 5 Analysis of the free energy of binding between PPARγ and β’-COPASP258, LYS273, GLN281, GLY282, PHE285 and ARG286 of PPARγ protein were the key binding sites of β’-COP-PPARγ complex

In order to identify the essential amino acid binding sites for the PPARγ-β’-COP complex within the 258–286 binding domains, an analysis was conducted to assess the binding energies contributed by amino acid sites within this domain. Through mutation of all amino acids in the binding domain to alanine, we observed a significant impact on the BFE at positions ASP258, ILE279, PHE280, GLY282, PHE285, and ARG286 (Table 6). Additionally, we investigated the effect of amino acid residue mutation on protein stability and BFE using ΔΔG, revealing that mutations at ASP258, LYS273, GLN281, GLY282, GLN284, PHE285, and ARG286 significantly influence the binding stability of the β’-COP-PPARγ complex (Table 7). Overall, our findings highlight the critical role of ASP258, LYS273, GLN281, GLY282, PHE285, and ARG286 in facilitating the transport of PPARγ into EVs mediated by β’-COP.

Table 6 Analysis of the BFE between PPARγ and β’-COP after amino acid sites mutationTable 7 ΔΔG analysis of the BFE between PPARγ and β’-COP after amino acid sites mutationThe mutation of ASP258, LYS273, GLN281, GLY282, PHE285 and ARG286 of PPARγ weaken the binding between PPARγ and β’-COP

To elucidate the functions of the six amino acids in the β’-COP-PPARγ interaction, we employed bioinformatics to simulate the mutations of these six amino acids in the PPARγ protein to alanine. The stability of the system was assessed using RMSD, revealing initial stability from 0 to 30 ns, followed by significant fluctuations after 35 ns (Fig. 5A). These fluctuations were attributed to the impact of mutations on system stability, leading to partial dissociation and substantial RMSD fluctuations. Similar trends were observed for Rg and SASA, with Rg increasing significantly after 35 ns and SASA showing an increasing trend after 30 ns, indicating system instability post-mutation (Fig. 5B-C). Analysis of hydrogen bond distribution revealed a gradual decrease in the number of hydrogen bonds from 8 to 10 at the start of the simulation to approximately 1–2 by the end (Fig. 5D-H), aligning with the dissociation process observed in RMSD, Rg, and SASA.

Fig. 5

The mutation of ASP258, LYS273, GLN281, GLY282, PHE285 and ARG286 of PPARγ weaken the binding between PPARγ and β’-COP. A-C The RMSD (A), Rg (B) and SASA (C) analysis of β’-COP-PPARγ complex after PPARγ mutation. D Binding surface image of the β’-COP-PPARγ complex after PPARγ mutation. E 3D interaction interface diagram of the β’-COP-PPARγ complex after PPARγ mutation. F The hydrogen bonding (H-bond) analysis of β’-COP-PPARγ complex after PPARγ mutation. G The ribbon diagram of β’-COP-PPARγ complex after PPARγ mutation. H 2D interaction interface diagram of β’-COP-PPARγ complex after PPARγ mutation. I Energetic components analysis of β’-COP-PPARγ complex after PPARγ mutation. J The binding free energy change of β’-COP-PPARγ complex after PPARγ mutation. K-L The bar diagram (K) and heat map (L) of the β’-COP-PPARγ complex binding free energy decomposition into residues after PPARγ mutation

We investigated the impact of a PPARγ mutation on BFE of PPARγ and β’-COP. The BFE was − 31.11 kcal/mol, primarily influenced by solvation free energy (ΔGSOLV, -42.63 kcal/mol) and van der Waals force (ΔVDWAALS, -26.42 kcal/mol), with Coulomb force (ΔEEL, 37.94 kcal/mol) as the main repulsive force (Table 8; Fig. 5I). The BFE initially increased within the first 20ns, followed by a subsequent decrease to a minimum of approximately − 8 kcal/mol at 42 ns. It then rose again, stabilizing at around − 32 kcal/mol (Fig. 5J), weaker than the BFE of the unmutated β’-COP-PPARγ complex (-38.55 kJ/mol). Key amino acids responsible for the bonding between the β’-COP-PPARγ complex were also altered and reduced (Fig. 5K-L). The mutation led to a significant decrease in the BFE between PPARγ and β’-COP, indicating a weakening of their interaction compared to the pre-mutation state.

Table 8 Analysis of the free energy of binding between PPARγ and β’-COP after mutationThe mutation of ASP258, LYS273, GLN281, GLY282, PHE285 and ARG286 of PPARγ resulted in decreased expression of PPARγ in EVs

To verify the participation of the six amino acids in β’-COP-mediated EVs transportation, we utilized lentivirus overexpression to introduce mutated PPARγ with these residues substituted with alanine into HTR8/SVneo trophoblast cell line. Concurrently, the fluorescent protein mCherry was incorporated upstream of the PPARγ protein, and a Histag label protein was attached downstream, resulting in a labeled PPARγ ASP258, LYS273, GLN281, GLY282, PHE285 and ARG286 mutated HTR8/SVneo trophoblast cell line (PPARγM-OE HTR8/SVneo) (Fig. 6A). The mCherry-PPARγ-histag overexpressed HTR8/SVneo (PPARγ-OE HTR8/SVneo) were used as the control group (Fig. 6A).

Fig. 6

The mutations of ASP258, LYS273, GLN281, GLY282, PHE285 and ARG286 of PPARγ result in decreased expression of PPARγ in EVs. A Illustration of the PPARγM-OE HTR8/SVneo and PPARγ-OE HTR8/SVneo trophoblast cell line. B Confocal microscopy of the mCherry and bright phase of PPARγM-OE HTR8/SVneo and PPARγ-OE HTR8/SVneo cells. C Western blots of the expression level of mCherry, PPARγ and histag protein in control (N/A), PPARγM-OE HTR8/SVneo and PPARγ-OE HTR8/SVneo cells. D RT-qPCR analysis of the mCherry, PPARγ and histag mRNA expression level in control (N/A), PPARγM-OE HTR8/SVneo and PPARγ-OE HTR8/SVneo cells. E Co-IP of the interaction between Histag (represent the mCherry-PPARγM-Histag fused protein) and β’-COP in PPARγM-OE HTR8/SVneo trophoblast. F Immunofluorescence and corresponding scatter plots and statistical analysis charts of the localization of mCherry and β’-COP in PPARγM-OE and PPARγ-OE HTR8/SVneo cells. G-H Immunofluorescence and corresponding scatter plots and statistical analysis chart of the localization of CD63 (G), EEA1 (H) and mCherry in PPARγM-OE HTR8/SVneo and PPARγ-OE HTR8/SVneo cells. I Western blots of the PPARγ, CD63 and PLAP protein expression level in the EVs of control (N/A), PPARγM-OE HTR8/SVneo and PPARγ-OE HTR8/SVneo cells. These data are shown as mean ± SEM. Two-tailed Student’s t test, *p < 0.05

Confocal microscopy revealed the high transfection efficiency of the overexpressed letivirus in HTR8/SVneo trophoblast cells (Fig. 6B). Additionally, Western Blot and RT-qPCR analysis revealed a significantly overexpression of mCherry, PPARγ and histag both in PPARγM-OE and PPARγ-OE HTR8/SVneo cells (Fig. 6C-D). Co-IP experiment show a lack of interaction between Histag (represent the mCherry-PPARγM-Histag fused protein) and β’-COP after PPARγ mutation (Fig. 6E). In addition, the colocalization of PPARγ and β’-COP in HTR8/SVneo cells also vanished in PPARγM-OE group compared to PPARγ-OE group (Fig. 6F). After mutation, the immunofluorescence assay revealed a significantly reduction in the co-localization of mCherry fluorescent protein with the EEA1 and CD63 in the PPARγM-OE HTR8/SVneo group (Fig. 6G-H). In contrast, the PPARγ-OE HTR8/SVneo group exhibited colocalization of mCherry protein with EEA1 and CD63 (Fig. 6G-H). Western Blot analysis also revealed a significantly reduction of PPARγ expression level in the EVs of PPARγM-OE HTR8/SVneo group compared to the PPARγ-OE HTR8/SVneo group (Fig. 6I). Taken together, these findings indicate that ASP258, LYS273, GLN281, GLY282, PHE285 and ARG286 of PPARγ are crucial amino acid sites for the β’-COP-mediated transport of PPARγ into EVs.