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Elod Kortvely, Lili Feng, Stefanie M Hauck, Behler Jennifer, Matteo Gorza, Karsten Boldt, Marcel Blindert, Marius Ueffing; Autophagy-related secretion of ARMS2. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1327.
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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.
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.
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.
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.
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