ZFYVE21 is a Novel Complement-Induced Rab5 Effector Protein Mediating Cardiac Allograft Vasculopathy

Abstract

Purpose Complement promotes vascular inflammation in transplant organ rejection, a process leading to cardiac allograft vasculopathy. In this study, we identify a new Rab5 effector protein, ZFYVE21, that mediates this process. We elucidate a ZFYVE21-SMURF2-pAkt signaling axis which activates non-canonical NF-kB, and we define the clinical relevance of this sequence. Methods We developed FACS-assisted protocols enabling proteomic analysis of MAC+Rab5+ endosomes and semi-quantitative analyses of the phosphoinositide content of these structures. A humanized mouse model incorporating human coronary artery segments and human lymphoid cells was employed to define a role for ZFYVE21-associated signaling in cardiac allograft vasculopathy. Software-assisted analyses of human transplant biopsies analyzing ZFYVE21 colocalization was performed using a computer algorithm we helped to developed in-house. Results In response to deposition of membrane attack complexes (MAC) on endothelial cells (EC), ZFYVE21 is post-translationally stabilized via recruitment to MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of the phosphoinositide content of MAC+Rab5+ endosomes with miltefosine reduces ZFYVE21 induction, EC activation, and MAC-induced allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC- but not ligand-induced non-canonical NF-κB and is a biomarker for complement-mediated endothelial signaling in transplant biopsies with antibody-mediated rejection. Conclusion Our data identifies ZFYVE21 as a novel Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway as a drug target and biomarker for allograft vasculopathy.