Abstract
The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.
Highlights
Most newly synthesized acetylcholinesterase molecules are catalytically inactive and rapidly degraded intracellularly; how this is regulated is not known
We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE
The remaining 20 –30% of the enzyme molecules become catalytically active and consist of both stable and unstable forms that transit the secretory pathway to the cell surface where they are inserted into cholinergic synapses such as the neuromuscular junction or nicotinic and muscarinic synapses in the central and peripheral nervous systems
Summary
Most newly synthesized acetylcholinesterase molecules are catalytically inactive and rapidly degraded intracellularly; how this is regulated is not known. When muscle cells or cell lines expressing AChE catalytic subunits are incubated in the presence of the PRAD-KDEL peptides, the peptides are taken up by the cells and retrogradely transported back to the ER where they induce assembly of AChE tetramers This appears to occur through increased folding efficiency and induced oligomerization of the catalytic subunits, which in turn rescue the enzyme from intracellular degradation. Pulse-chase studies of radiolabeled AChE show that PRAD peptides dramatically increase the half-life of the enzyme indicating that the synthetic PRAD peptides rescue AChE catalytic subunits from intracellular degradation Together, these studies indicate that binding of the noncatalytic AChE targeting subunits to the enzymatic subunits provides an important post-translational regulatory step for the expression of the catalytically active enzyme and provides a mechanism for regulating levels of synaptic AChE without changes in the translational rate of the protein. They suggest a novel means of enhancing expression of AChE in vivo with potential novel therapeutic applications
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