Parkin Induces Ubiquitination and Large Extracellular Vesicle Release of HMGB1 to Activate Antitumor Immunity

Abstract

Parkin (PRKN) is a mitochondria-associated E3 ubiquitin ligase that mediates mitophagy and organelle quality control. More recently, PRKN has been implicated in stimulating antitumor immunity and reprogramming the tumor immune microenvironment. In this study, we showed that PRKN ubiquitinates the alarmin molecule, high-mobility group box-1 (HMGB1) on Lys146 (K146) using predominantly K48 linkages. By molecular modeling, the in-between-ring domain of PRKN (Gln326-Leu358) made extensive contacts with the amino-terminus A-box of HMGB1 (Met1-Ser42), forming a mitochondria-associated PRKN-HMGB1 complex that juxtaposes K146 to ubiquitin active site residues Gly76 and Arg74. Instead of proteasomal degradation, PRKN ubiquitination of K146 enabled the loading of HMGB1 but not HMGB1 K146A mutant, onto autophagy- and mitochondria-derived large extracellular vesicles (LEV). In turn, released PRKN-HMGB1-LEV stimulated a potent IFN and cytokine response in recipient cells, expanding CD8+ T-cell subsets with effector (CD69+/KLRG1+), self-renewal (TCF1+/PD-1+), and cytotoxic (KLRG1+/GrzB+) properties. Conditional expression of PRKN induced HMGB1 release, activated intratumoral CD8+ T cells, and suppressed syngeneic tumor growth in vivo in a response that was abolished by HMGB1 silencing. These data identify that PRKN-LEV-regulated release of HMGB1 reprograms antitumor immunity via stimulation of IFN signaling and expansion of specialized CD8+ T-cell subsets.

Significance: Parkin ubiquitinates the alarmin molecule HMGB1 to enable its regulated release in large extracellular vesicles that activate interferon signaling, expand specialized CD8+ T-cell subsets, and promote antitumor immunity.

Figure 1.. PRKN-HMGB1 ubiquitination.

(A) MS/MS spectra of ubiquitination peptides of HMGB1 in PC3 cells transfected with PRKN (light label) or vector (heavy label) identified by SILAC-based ubiquitin remnant motif enrichment (K-ε-GG). The Lys146 (K146) ubiquitination site is in red. b- and y-ions are indicated by horizontal bars above and below the sequence, respectively. (B) PC3 cells transfected with Flag-HMGB1 and reconstituted with Myc-PRKN were immunoprecipitated (IP) with IgG or an antibody to Flag and analyzed with antibodies to ubiquitin (Pan-Ub), Ub-K48- or Ub-K63 by Western blotting. (C and D) The conditions are as in (B) except that PC3 cells expressing Flag-HMGB1 were reconstituted with WT PRKN or PRKN C431S mutant (C) or WT HMGB1 or HMGB1 K112A or K146A mutant (D), followed by IP with IgG or an antibody to Flag and Western blotting. For panels B-D, the position of ubiquitinated HMGB1 species is indicated by a side bracket. (E) PC3 cells expressing vector or PRKN were analyzed for co-association with endogenous HMGB1 by Duolink proximity ligation assay (PLA) and fluorescence microscopy. The number of co-associated spots per condition is indicated (mean±SD, p<0.0001). Scale bar, 30 μm. Inset, magnification of indicated areas. (F and G) PC3 cells separately expressing Flag-HMGB1 or GFP-PRKN were analyzed by Western blotting (F) and IP with an antibody to Flag followed by by Western blotting (G). (H and I) PC3 cells expressing vector or PRKN were analyzed for co-localization with TOM20 and HMGB1 by confocal microscopy (H) and a Pearson Correlation Coefficient (PCC) of protein colocalization was quantified (I). Bottom, quantification of signal intensity. Representative images. Scale bar, 20 μm. A p value by two-tailed unpaired t test is indicated. (J and K) Subcellular fractions containing nuclei (Nucl), cytosol (Cyto) or mitochondria (Mito) were isolated from PC3 cells transiently (J) or conditionally (Doxy-induced) (K) expressing PRKN and analyzed by Western blotting. Bottom, densitometric quantification of HMGB1 protein bands.

Figure 2.. PRKN-HMGB1 complex.

(A) Predicted PRKN-HMGB1 contact interaction matrix across 30 docking poses. Color coding, residue sequence numbers. Only interacting residues are shown. (B) Predicted PRKN (green)-HMGB1 (grey) binding interface comprising 31 residues from the A-Box of HMGB1 and 35 residues from the PRKN IBR domain. (C) RMSD plot obtained during 200 ns molecular dynamics (MD) simulations. (D) Predicted HDOCK alignment of K146 of HMGB1 and Ub active site residues, R74 and G76. (E and F) Predicted HDOCK model of WT HMGB1 (E) or K146A mutant HMGB1 (F) binding to PRKN. (G and H) PC3 cells expressing PRKN and reconstituted with WT HMGB1 or K146A HMGB1 mutant were analyzed by confocal fluorescence microscopy (G) and a PCC of PRKN co-localization with WT or mutant HMGB1was quantified (H). Scale bar, 10 μm. Representative images. A p value by two-tailed unpaired t test is indicated. (I and J) PC3 cells expressing Flag-HMGB1 or Flag-HMGB1 K146A mutant were reconstituted with Myc-PRKN, IP with IgG or an antibody to Flag (I) or Myc (J) and immune complexes were analyzed by Western blotting. Bar graphs, densitometric quantification of WT HMGB1 (WT) or HMGB1 K146A mutant (Mut) protein band. (K) Mitochondrial fractions (Mito) of PC3 cells expressing Flag-HMGB1 or Flag-HMGB1 K146A mutant and Myc-PRKN were analyzed by Western blotting. Bar graph, densitometric quantification of WT HMGB1 (WT) or HMGB1 K146A mutant (Mut) protein band.

Figure 3.. PRKN-mediated extracellular release of HMGB1.

(A) PC3 cells expressing PRKN were reconstituted with Flag-HMGB1 (WT) or Flag-HMGB1 K146A mutant (Mut) and analyzed by Western blotting. TCE, total cell extracts. *, nonspecific band. (B) PC3 cells expressing PRKN were transfected with HMGB1-directed siRNA (siHMGB1) and reconstituted with Flag-HMGB1 or Flag-HMGB1 K146A mutant (Mut) and analyzed by Western blotting. CM, conditioned medium. Both endogenous HMGB1 and Flag-HMGB1 bands are indicated. (C-E) PC3 cells expressing vector or PRKN were transfected with ATG5- (C), FIP200- (D) or Rab27a (E)-directed siRNA (siATG5, siFIP200, siRab27a) and aliquots of TCE or CM were analyzed by Western blotting. Bottom, densitometric quantification of HMGB1 protein bands in the CM under the various conditions. (F) PC3 cells expressing vector or PRKN were analyzed for co-localization with endogenous LC3 and HMGB1 by confocal fluorescence microscopy. Scale bar, 10 μm. Representative fields are shown. A PCC is indicated (mean±SD, p<0.0001). Bottom, quantification of signal intensity. (G and H) PC3 cells expressing vector (V) or PRKN (P) were analyzed for total LC3 pucta (G) or LC3-TOM20 colocalization (H) by fluorescence microscopy. For (H), a PCC was quantified. p values by two-tailed unpaired t test are indicated. (I) PC3 cells expressing PRKN or PRKN C431S mutant were analyzed with an antibody to HMGB1 by immuno-gold transmission electron microscopy (TEM). Arrows, localization of gold particles. Scale bars, 500 nm. Representative images. (J) The experimental conditions are as in (J) and the total number of membranous structures/mitochondria (n=533) positive or negative for HMGB1 labeling by TEM was quantified. Total number of cells, n=16; total number of images, n=56; average number of particles/labeled mitochondria, n=3.4.

Figure 4.. Characterization of membranous constituents in PRKN-HMGB1 extracellular release.

(A) Schematic diagram of sequential sucrose- and OptiPrep-gradient density isolation of total cellular vesicles from PRKN-expressing PC3 cells. Created in BioRender. Altieri, D. (2025) https://BioRender.com/9w6x2fw (B) The conditions are as in (A) and the indicated gradient density fractions (F1-F7) of total cellular vesicles were analyzed by Western blotting. (C) Densitometric quantification of the indicated protein bands in OptiPrep gradient density fractions isolated from total cellular vesicles (top) or large extracellular vesicles (LEV) (bottom). Representative experiment out of three independent determinations. (D) ZetaView distribution of LEV produced by PC3 cells expressing vector or PRKN. The median LEV size per condition is indicated. (E) LEV isolated from PC3 cells expressing vector or PRKN as in (D) were fractionated by gradient density ultracentrifugation followed by Western blotting. (F) LEV isolated from PRKN-expressing PC3 cells were analyzed for co-localization of PRKN and HMGB1 by confocal fluorescence microscopy with quantification of individual LEV size. Scale bar, 1 μm. A representative microscopy field is shown. Right, quantification of signal intensity. (G) LEV isolated from PC3 cells expressing WT or K146A HMGB1 mutant were analyzed by Western blotting. (H) LEV or small extracellular vesicles (SEV) isolated from PC3 cells expressing vector or PRKN were analyzed by Western blotting.

Figure 5.. PRKN extracellular release of HMGB1 stimulates IFN gene expression.

(A and B) Aliquots of CM from PC3 cells reconstituted with WT PRKN (A) or PRKN C431S mutant (B) were co-incubated with PRKN-negative recipient PC3 cells followed by analysis of IFN gene expression by RT-qPCR (mean±SD, n=3). (C) The conditions are as in (A) and aliquots of CM from PC3 cells expressing vector or PRKN were analyzed by multiplex Luminex assay for the indicated cytokines/chemokines. Right, densitometric quantification of band intensity (mean±SD, n=3). (D and E) PC3 cells expressing PRKN were silenced for endogenous HMGB1 and reconstituted with WT HBGB1 or HMGB1 K146A mutant followed by Western blotting (D) or analysis of IFN gene expression by RT-qPCR (E) (mean±SD, n=3). (F) Aliquots of CM from PC3 cells reconstituted with WT or K146A mutant HMGB1 as in (D and E) were incubated with parental PC3 cells followed by analysis of IFN gene expression by RT-qPCR. Mean±SD (n=3). (G) LEV isolated from PRKN-expressing cells were labeled with DiD, incubated with recipient PC3 cells and analyzed by fluorescence microscopy. Scale bar, 10 μm. Representative merged images are shown. The concentrations of LEV added per condition are indicated. (H) LEV (left) or SEV (right) isolated from PC3 cells expressing vector or PRKN were co-incubated with recipient PC3 cells followed by analysis of IFN gene expression by RT-qPCR. Mean±SD (n=6). (I) LEV isolated from PC3 cells expressing PRKN or PRKN C431S mutant (Mut) were incubated with recipient PC3 cells followed by analysis of IFN gene expression. For panels A, B and F, numbers are p values by unpaired two-tailed t test. For panels H and I, ***, p<0.0001 by two-tailed unpaired t test.

Figure 6.. Parkin-HMGB1 stimulation of T cell activation.

(A) CM from TRAMP-C2 cells conditionally expressing PRKN (Doxy) were reconstituted with HMGB1 or HMGB1 K146A mutant (Mut), incubated with syngeneic CD8 splenocytes from C57BL/6J mice and analyzed for the indicated markers by multiparametric flow cytometry. The percentage of cells is indicated. EM, effector memory. (B) The conditions are as in (A) and syngeneic CD8 splenocytes incubated with TRAMP-C2 CM were analyzed for expression of TCF1 and double positive TCF1/PD-1 and KLRG1/GrzB subsets, by flow cytometry. (C) The conditions are as in (B) and CD4 T cells harvested from splenocytes of C57BL/6J mice were incubated with CM from PRKN-expressing cells and analyzed for the indicated markers by multiparametric flow cytometry. (D and E) Aliquots of CM from reconstituted TRAMP-C2 cells as in (A) were incubated with CD8 (D) or CD4 (E) T cells from syngeneic IFNAR1^−/− mice and modulation of the indicated markers was quantified by multiparametric flow cytometry. For panels B-E, each point corresponds to an individual mouse determination (n=6). Numbers are p values by two-way ANOVA. ns, not significant.

Figure 7.. Parkin-HMGB1 antitumor immunity.

(A) Prostate cancer MPTEN1 cells transduced with Doxy-regulated conditional PRKN expression were engrafted onto the flanks of C57BL/6 mice and tumor growth was quantified with a caliper at the indicated time intervals. Mice were administered vehicle or Doxy in the drinking water (500 ng/ml) when tumors reached a volume of ~120–150 mm³ (arrow). Each line is an individual tumor. (B) MPTEN1 tumor samples harvested from vehicle (Veh) or Doxy-treated mice as in (A) were analyzed for expression of PRKN (top), HMGB1 (middle) or HMGB1 in the presence of control shRNA or HMGB1-directed shRNA (bottom) by immunohistochemistry. Representative images. Scale bar, 50 μm. (C and D) Intratumoral CD8 (C) or CD4 (D) T cell subsets were harvested from mice as in (A) and analyzed for the indicated markers by multiparametric flow cytometry. Each symbol is an individual mouse determination. Numbers correspond to p values by two-way ANOVA. ns, not significant. (E) MPTEN1 cells expressing Doxy-regulated PRKN were stably transduced with control shRNA (shCtrl) or HMGB1-directed shRNA (shHMGB1) and cells harvested after three passages (Psg) were analyzed with or without Doxy by Western blotting. (F) MPTEN1 cells as in (E) were engrafted onto the flanks of immunocompetent C57BL/6 mice and tumor growth was assessed with a caliper at the indicated time intervals. All animals in both groups were administered Doxy in the drinking water when tumors reached ~120–150 mm³. Each point is a single animal determination. (G and H) The conditions are as in (F) and intratumoral CD8 (G) or CD4 (H) T cells were analyzed for the indicated markers by multiparametric flow cytometry. Each symbol is an individual mouse determination. Numbers correspond to p values by two-way ANOVA. ns, not significant. For panels G and H, each point is an individual determination. Numbers are p value by two-way ANOVA. ns, not significant.