Autophagy-related secretion of ARMS2

Abstract

Purpose: Age-related maculopathy susceptibility 2 (ARMS2) is a small (11 kDa), primate-specific protein implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness in elderly. We have previously reported that ARMS2 is actively secreted, although it lacks a typical signal peptide. Here we report that autophagy-related processes are instrumental in conveying ARMS2. Autophagosomes are dynamically formed and serve as a bulk degradation pathway. However, emerging evidence suggests that they are also involved in the biogenesis of transport organelles destined to export a specific group of proteins including ARMS2.

Methods: Several human cell lines were transfected with plasmids coding for ARMS2. Plasmids coding for pro-interleukin (IL)-1β and ARMS2 carrying the desired substitutions were constructed by molecular cloning. The intracellular trafficking of the synthesized protein was monitored by co-staining of well-established markers for different cellular compartments. Digital images of immunostained cells were acquired on a Zeiss Axioscope. The classical secretory pathway was inhibited by using brefeldin A (BFA) in some experiments. ARMS2-positive carriers were isolated for proteomic analysis by native electrophoresis.

Results: Golgi reassembly stacking protein proteins (GRASPs) are the only known markers for unconventional protein secretion. ARMS2-positive vesicle-like structures proved to be positive for this marker. Furthermore, the major proinflammatory cytokine interleukin-1β (IL-1β) is a prototypical example of secretory autophagy. Co-expression of ARMS2 and IL-1β leads to the redistribution of these two proteins into the same vesicle-like structures suggesting a common secretory pathway. On the other hand, co-expression of ARMS2 and HTRA1 (classically secreted protein) or eGFP (non-secreted protein) does not give rise to the colocalization of the two proteins (data not shown), indicating an active cargo selection mechanism. Critical residues within ARMS2 for becoming a client of this transport machinery have also been identified.

Conclusions: Our data suggest that ARMS2 belongs to the group of proteins being secreted by autophagy related mechanisms. Strikingly, all other known proteins hauled by this pathway act pro-inflammatory. Accordingly, ARMS2 might exert its physiological function by regulating immune cells within the eye.

Keywords: 412 age-related macular degeneration • 660 proteins encoded by disease genes • 663 proteomics