TRX2/Rab35 Interaction Impairs Exosome Secretion by Inducing Rab35 Degradation

Abstract

Given that exosomes mediate intercellular communication by delivering cellular components to recipient cells or tissue, they have the potential to be engineered to deliver therapeutic payloads. However, the regulatory mechanism of exosome secretion is poorly understood. In addition, mitochondrial components have been found in exosomes, suggesting communication between mitochondria and exosomes. However, the molecular mechanism of the mitochondria and vesicle interaction remains unclear. Here, we showed that mitochondrial thioredoxin 2 (TRX2) decreased exosome concentrations and inhibited HCT116 cell migration. Coimmunoprecipitation/mass spectrometry (Co-IP/MS) showed that TRX2 interacted with Rab35. TRX2 and Rab35 bound to each other at their N-terminal motifs and colocalized on mitochondria. Furthermore, TRX2 induced Rab35 degradation, resulting in impaired exosome secretion. Additionally, Rab35 mediated the suppressive effects of TRX2 on cell migration, and TRX2 suppressed cell migration through exosomes. Taken together, this study first found an interaction between TRX2 and Rab35. These results revealed a new role for TRX2 in the regulation of exosome secretion and cell migration and explained the upstream regulatory mechanism of Rab35. Furthermore, these findings also provide new molecular evidence for communication between mitochondria and vesicles.

Keywords: Rab35; TRX2; exosome; migration; mitochondria; protein–protein interaction.

Figure 1

TRX2 plays a role in exosome secretion and cell migration in HCT116 cells. (A) HCT116 cells were transfected with pcDNA3.1+ or FLAG−TRX2. After 72 h, exosome pellets were resuspended in the corresponding volume of PBS according to cell numbers to ensure that the same volume of PBS contained exosomes from the same cell number. The lysis (20 μg) and exosomes (20 μL) proteins were detected by WB. (B) HCT116 cells were transfected with siNC, siTRX2−247, or siTRX2, plus low levels of siRNA−resistant FLAG−TRX2. After 48 h, the transcription level of TRX2 was detected by real−time quantitative PCR. (C) HCT116 cells were transfected with siNC or siTRX2. After 72 h, exosome pellets were resuspended in the corresponding volume of PBS according to cell numbers to ensure that the same volume of PBS contained exosomes from the same cell number. The lysis (20 μg) and exosome (20 μL) proteins were detected by WB. (D) HCT116 cells were transfected with pcDNA3.1+, FLAG−TRX2, siNC, siTRX2, or siTRX2, and the FLAG−TRX2 EVs (extracellular vesicles) were isolated from the 10 mL cell culture supernatants of 50 million cells by serial ultracentrifugation and resuspended in 100 μL PBS. Exosome size and particle concentration were analyzed using NS300 nanoparticle characterization system. (E) HCT116 cells were transfected with pcDNA3.1+, FLAG−TRX2, siNC, siTRX2, or siTRX2, plus the FLAG−TRX2, and migration was measured by a scratch test. NC: pcDNA3.1+ as negative control; siNC: siRNA negative control. The scale bar = 50 μm. The experiments were performed for three biological replicates. The error bars represent means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 using the t test.

Figure 2

TRX2 interacted with Rab35 and they colocalized on mitochondria. (A) Silver staining. IP was performed with anti−FLAG magnetic beads. The * represents the specific protein bands of HCT116 cells transfected with FLAG−TRX2. The red boxes represent the gel piece excised for mass spectrometry. (B) HCT116 and HEK293T cells were transfected with pcDNA3.1+ or FLAG−TRX2. After 72 h, coimmunoprecipitation was performed, and Rab35 was detected. (C) HCT116 cells were transfected with pcDNA3.1+ or FLAG−TRX2. After 72 h, coimmunoprecipitation was performed, and Rab7A and Rab11B were detected. (D) HCT116 cells were transfected with EGFP−tagged TRX2 and mCherry−tagged Rab35. Rab35 (magenta), TRX2 (green), and nuclei (blue) are shown separately or merged. The scale bar = 10 μm. Magnification (lower panels) was ×5. (E) HCT116 cells were transfected with mCherry−tagged Rab35 or both mCherry−tagged Rab35 and CFP−tagged TRX2. After 48 h, the mitochondria were labelled with MitoTracker Green. Rab35 (magenta), mitochondria (green), and TRX2 (blue) are shown separately or merged. The experiments were performed for three biological replicates (indicated by arrows). The scale bar = 10 μm. Magnification (lower panels) was ×5.

Figure 3

TRX2 and Rab35 bound to each other through N−terminal motifs. (A) Schematic diagram of Rab35. Amino acids 33 and 38–46 are switch I domain, and amino acids 66 and 68–78 are switch II domain. Green indicates the G box and pink indicates RABF, the Rab family motif. Amino acids 1–92 are the truncated N−terminal (N−ter) region of Rab35, which contains the switch domain. Amino acids 93–201 are the truncated C−terminal (C−ter) domain of Rab35. (B) HCT116 cells were transfected with FLAG−tagged full−length (FL) TRX2 and HA−tagged full−length Rab35, Rab35 N−ter, or Rab35 C−ter. After 72 h, coimmunoprecipitation was performed with anti−FLAG beads. The pellet was analyzed by WB. (C) Schematic diagram of TRX2. In the pink region, amino acids 1–59 represent the signal peptide (to mitochondria). In the green region, amino acids 72–162 represent the thioredoxin family domains. The 90th and 93rd amino acids are the catalytic active sites of TRX2. The 1st to 100th amino acids are the truncated N−terminal (N−ter) region of TRX2, which contains the signal sequence and catalytic active sites. (D) HCT116 cells were transfected with HA−flagged Rab35 and FLAG−tagged full−length TRX2, TRX2 N−ter, or TRX2 C−ter. After 72 h, coimmunoprecipitation was performed with anti−FLAG beads. The pellets were analyzed by WB. The experiments were performed for three biological replicates.

Figure 4

TRX2 induced Rab35 ubiquitination and degradation. (A) HCT116 cells were transfected with pcDNA3.1+ or FLAG−TRX2. Rab35 was detected by WB. (B) HCT116 cells were transfected with siNC, siTRX2, or siTRX2, plus low levels of FLAG−TRX2. Rab35 was detected by WB. (C) HCT116 cells were transfected with pcDNA3.1+ or FLAG−TRX2 and treated with or without MG132. Rab35 was detected by WB. (D) HCT116 cells were transfected with pcDNA3.1+ or FLAG−TRX2− and treated with cycloheximide (CHX) for the indicated times. Rab35 was detected by WB. (E) HCT116 cells were transfected with siNC or siTRX2 and treated with CHX for the indicated times. Rab35 was detected by WB. (F) HCT116 cells were transfected with V5−TRX2, FLAG−Rab35, or HA−tagged ubiquitin. After 48 h, the cells were treated with MG132. The ubiquitination of Rab35 was detected by IP. (G) HCT116 cells were transfected with FLAG−Rab35, HA−tagged ubiquitin, siTRX2, or siTRX2, plus low levels of V5−FLAG. After 48 h, the cells were treated with MG132. The ubiquitination of Rab35 was detected by IP. The experiments were performed for three biological replicates. The error bars represent means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 using the t test or one−way ANOVA with Šídák’s multiple comparisons test.

Figure 5

TRX2 impaired exosome secretion through Rab35 degradation. (A) HCT116 cells were transfected with pcDNA3.1+, FLAG−TRX2, or HA−Rab35. The indicated proteins were detected by WB. (B) HCT116 cells were transfected with pcDNA3.1+, FLAG−TRX2, or HA−Rab35. The EVs were isolated from the 10 mL cell culture supernatants of 25 million cells by serial ultracentrifugation and resuspended in 100 μL PBS. The total exosomal protein and exosome concentrations were measured. (C) HCT116 cells were transfected with siNC, siTRX2, or siRab35. The indicated proteins were detected by WB. (D) HCT116 cells were transfected with siNC, siTRX2, or siRab35. The EVs were isolated from the 10 mL cell culture supernatants of 25million cells by serial ultracentrifugation and resuspended in 100 μL PBS. The total exosomal protein and exosome concentrations were measured. (E) HCT116 cells were transfected with pcDNA3.1+, FLAG−TRX2, or HA−Rab35, and the exosomes were purified. The biomarkers of the exosomes were detected by WB in whole cell lysates (20 μg) and exosomes (20 μL). (F) HCT116 cells were transfected with siNC, siTRX2, or siRab35, and the exosomes were purified. The biomarkers of the exosomes were detected by WB in whole cell lysates (20 μg) and exosomes (20 μL). The experiments were performed for three biological replicates. The error bars represent means ± SD. ns, no significant difference; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one−way ANOVA with Šídák’s multiple comparisons test.

Figure 6

Rab35 mediated the TRX2-induced suppression of cell migration. (A) HCT116 cells were transfected with pcDNA3.1+, FLAG-TRX2, or HA-Rab35. Cell migration was measured by scratch tests. (B) HCT116 cells were transfected with siNC, siTRX2, or siRab35. Cell migration was measured by scratch tests. The experiments were performed for three biological replicates. The error bars represent means ± SD. ns, no significant difference; * p < 0.05 and ** p < 0.01 using one-way ANOVA with Šídák’s multiple comparisons test.

Figure 7

Effect of exosomes on HCT116 cell migration. (A) HCT116 cells were transfected with pcDNA3.1+ or FLAG-TRX2, and exosomes were purified. Then, 2 × 10⁵ particles of exosomes were added to HCT116 cells cultured in exosome-free media. Cell migration was measured by scratch tests. (B) HCT116 cells were transfected with siNC or siTRX2, and exosomes were purified. Then, 2 × 10⁵ particles of exosomes were added to HCT116 cells cultured in exosome-free media. Cell migration was measured by scratch tests. The experiments were performed for three biological replicates. The scale bar = 50 μm. The error bars represent means ± SD. EXO, exosome; * p < 0.05, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA with Šídák’s multiple comparisons test.

Figure 8

Schematic representation of the role of the TRX2/Rab35 interaction in the regulation of exosome secretion. Rab35 (at the cytosolic face of endosome-MVBs) mediates MVB docking to the cell membrane to promote exosome secretion. Mitochondrial TRX2 (on the mitochondrial membrane) interacts with Rab35 and induces Rab35 ubiquitination and degradation, resulting in impaired exosome secretion.