Proteasome regulates turnover of toxic human amylin in pancreatic cells

Abstract

Toxic human amylin (hA) oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Although recent studies demonstrated a causal connection between hA uptake and toxicity in pancreatic cells, the mechanism of amylin’s clearance following its internalization and its relationship to toxicity is yet to be determined, and hence was investigated here. Using pancreatic rat insulinoma β-cells and human islets as model systems, we show that hA, following its internalization, first accumulates in the cytosol followed by its translocation into nucleus, and to a lesser extent lysosomes, keeping the net cytosolic amylin content low. An increase in hA accumulation in the nucleus of pancreatic cells correlated with its cytotoxicity, suggesting that its excessive accumulation in the nucleus is detrimental. hA interacted with 20S core and 19S lid subunits of the β-cell proteasomal complex, as suggested by immunoprecipitation and confocal microscopy studies, which subsequently resulted in a decrease in the proteasome’s proteolytic activity in these cells. In vitro binding and activity assays confirmed an intrinsic and potent ability of amylin to interact with the 20S core complex thereby modulating its proteolytic activity. Interestingly, less toxic and aggregation incapable rat amylin (rA) showed a comparable inhibitory effect on proteasome activity and protein ubiquitination, decoupling amylin aggregation/toxicity and amylin-induced protein stress. In agreement with these studies, inhibition of proteasomal proteolytic activity significantly increased intracellular amylin content and toxicity. Taken together, our results suggest a pivotal role of proteasomes in amylin’s turnover and detoxification in pancreatic cells.

The Molecular Physiopathogenesis of Islet Amyloidosis

“…Similar to RIN-m5F cells, internalized hA predominantly localized in the nucleus of human islet cells (Figure 7C, bottom panel). Biochemical analysis confirmed that cytosolic and organelle enriched fractions together accumulated less than 10% of the total internalized hA in both cell types (Singh et al 2016). …”

“…By using this experimental set up we examined how cells defend themselves against high molecular weight toxic hA species. Following hA’s internalization into the cells, nuclear, cytosolic and organelle fractions were isolated using detergent-based approach (Singh et al 2016). Fraction purity and redistribution of organelles and other cellular components within fractions were determined by western blot analysis (Singh et al 2016).…”

“…Following hA’s internalization into the cells, nuclear, cytosolic and organelle fractions were isolated using detergent-based approach (Singh et al 2016). Fraction purity and redistribution of organelles and other cellular components within fractions were determined by western blot analysis (Singh et al 2016). Interestingly, hA specific ELISA revealed a large twenty-fold increase in absolute hA levels in the nucleus and to a much smaller extent (three-fold increase) in the cytosol of hA -treated (for 24h) RIN-m5F cells as compared to control rA-producing cells (Figure 8A).…”

“…Because in both these fractions the externally applied hA dwarfs the production of a native rA, this allowed us to study turnover of internalized hA in β-cells. hA accumulation in the large-organelle fraction of RIN-m5F cells was comparable to the cytosol, ~ 3% of the total internalized hA (Singh et al 2016). This biochemical finding was reconfirmed by indirect immunocytochemistry (Figure 7C).…”

“…Cells were treated with two distinct hA preparations (having small or large oligomeric/fibrilar content) for increasing periods of time (0 to 24h), cell fractions prepared as described previously (Singh et al 2016), and hA content in both nuclear and cytosolic fractions analyzed by ELISA. Biochemical analysis demonstrated that irrespective of the method of hA preparation, hA progressively translocates in the nucleus over a period of 24 hours, with a lag phase of ~4 hours (Figure 8C, D).…”