Elucidation of the molecular logic by which misfolded α1-antitrypsin is preferentially selected for degradation

Abstract

The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized ␣1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation. Transiently expressed mutant and WT AAT variants underwent rapid destabilization in response to an artificially elevated ERManI concentration in the murine hepatoma cell line, Hepa1a. Based on the mannosidase-and lactacystin-sensitive properties of intracellular turnover, a stochastic model is proposed in which the delayed onset of the glycan modification, relative to the duration of nonnative protein structure, coordinates the preferential degradation of the misfolded monomer and spares the native molecule from destruction. Newly synthesized endogenous transferrin underwent degradation in response to an elevated concentration of ERManI, whereas the nonglycosylated secretory glycoprotein albumin was not affected. Taken together, these findings indicate that efficient conformational maturation might function as the initial quality control standard for a broad population of glycoproteins.

Inhibition of proteasome by bortezomib causes intracellular aggregation of hepatic serpins and increases the latent circulating form of antithrombin

“…34 In addition to this, our observation that antithrombin and a1-antitrypsin intracellularly accumulate and aggregate after bortezomib treatment further supports the requirement of a proteasome-dependent, ER-associated protein degradation machinery (ERAD) for the correct processing and quality control of serpins. [4][5][6][7][8][9] All these results, together with similar findings for another conformationally sensitive protein such as fibrinogen, 24 but minor consequences on non-structurally sensitive proteins such as prothrombin, support the notion that impairment of proteasome mainly affects conformationally sensitive proteins, leading to the intracellular accumulation of misfolded molecules. Moreover, the minor effect that bortezomib has on circulating proteins secreted from the liver, and the absence of intracellular accumulation of prothrombin, suggest that although the ER may be partially blocked with misfolded proteins, the secretion of properly folded proteins seems to be not significantly impaired, at least after 2 days of treatment.…”

“…2 The ubiquitin-proteasome system has been characterised as the most important cell machinery to degrade misfolded secretory proteins. 3 As expected for structurally sensitive molecules, this machinery has been shown to be extremely important for intracellular degradation of abnormal conformers, proper folding and release of serpins, such as antithrombin, [4][5][6] a1-antitrypsin, 7,8 and neuroserpin. 9 Impairment of this system has been one of the main contributing factors to a number of conformational diseases, mainly neurological disorders.…”

Intracellular Processing of 1-Antitrypsin

“…The modified glycans promote extraction of glycoproteins from the calnexin cycle (18), and, in combination with nonnative protein structure, are suspected of completing the formation of a proposed bipartite glycoprotein ERAD (GERAD) signal. The temporal manner in which the bipartite signal is formed is suspected of distinguishing chronically misfolded glycoproteins from nonnative wild-type folding intermediates, such that only the former population is degraded (29) (Figure 1, step 4). Subsequent dislocation of the tagged proteins into the cytosol for proteasomal destruction involves the cooperation of additional events and proteins, some of which are currently under investigation.…”

“…That the glycosidase's intracellular concentration plays a central role in the establishment of an equitable glycoprotein quality control standard was recently substantiated by the capacity of experimentally overexpressed ERManI to selectively target nascent molecules of wild-type AAT and transferrin into GERAD (29).…”

“…Notably, a delay in the degradation of the PI Z variant has been observed in transduced fibroblasts from patients who eventually underwent liver transplantation (29), implying that the degradation process might very well contribute to disease pathogenesis. Because synthesis of the PI Z variant does not activate the unfolded response, the hepatocytes of homozygous ZZ patients must rely on the low basal concentration of ERManI to target monomers into GERAD for the purpose of preventing the formation of polymers.…”