Chapter one - Analysis of Serpin Secretion, Misfolding, and Surveillance in the Endoplasmic Reticulum

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Abstract

Biological checkpoints are known to function in the cellular nucleus to monitor the integrity of inherited genetic information. It is now understood that posttranslational checkpoint systems operate in numerous biosynthetic compartments where they orchestrate the surveillance of encoded protein structures. This is particularly true for the serpins where opposing, but complementary, systems operate in the early secretory pathway to initially facilitate protein folding and then selectively target the misfolded proteins for proteolytic elimination. A current challenge is to elucidate how this posttranslational checkpoint can modify the severity of numerous loss-of-function and gain-of-toxic-function diseases, some of which are caused by mutant serpins. This chapter provides a description of the experimental methodology by which the fate of a newly synthesized serpin is monitored, and how the processing of asparagine-linked oligosaccharides helps to facilitate both the protein folding and disposal events.

Introduction

The conformational maturation and secretion of an active serpin involves a multistep process. Initially, cellular machinery orchestrate a series of requisite events that eventually direct the cotranslational insertion of the nascent polypeptide across the membrane of the rough endoplasmic reticulum (ER) and into its lumen (Cabral et al., 2001). Transient physical interactions with a group of molecular chaperones promote correct protein folding by diminishing the frequency of unproductive folding events that can lead to inappropriate aggregation (Choudhury et al., 1977, Fewell et al., 2001).

Efficient conformational maturation functions as a quality control standard, permitting the progression of active proteins beyond the ER (Wu et al., 2003). Noncompliance results in the ER retention of misfolded polypeptides and unassembled protein subunits, both of which exhibit prolonged physical interaction with molecular chaperones and are eventually targeted for intracellular degradation (Liu et al., 1997). In recent years, a picture has emerged in which the addition of asparagine-linked oligosaccharides, and their covalent modification, functions to assist the productive folding of newly synthesized serpins by facilitating their physical interactions with a small family of lectins that contribute to the glycoprotein folding machinery (Cabral et al., 2001, Ellgaard et al., 1999).

Importantly, additional carbohydrate modifications are responsible for generating a tag that selectively targets the nonnative proteins for intracellular degradation (Cabral et al., 2001, Molinari, 2007). A model has been proposed in which the timing of the latter glycan modification, relative to the duration of nonnative glycoprotein structure, functions as an underlying stochastic mechanism by which serpins that exhibit permanent structural defects are selectively targeted for intracellular disposal (Wu et al., 2003). Later stages of the disposal process include retrotranslocation across the ER membrane and into the cytoplasm, polyubiquitination, and elimination by 26S proteasomes (Bonifacino and Weissman, 1998).

Almost all naturally occurring secretion-incompetent serpin variants misfold following biosynthesis (Sifers et al., 1992), and therefore fail to pass the quality control checkpoint. The serpins have provided a model system to elucidate how glycoprotein processing contributes to these surveillance mechanisms. The following information describes the experimental strategies and methodologies designed to elucidate the involvement of asparagine-linked oligosaccharide processing in regulating the fate of a newly synthesized serpin.

Section snippets

Transfection of mammalian cell lines

Because the biosynthesis of serpins is naturally expressed in only a few cell types, it will be necessary to transfect the serpin cDNA into a selected cultured mammalian cell line to generate a system for studying the protein's fate following biosynthesis. The wild-type cDNA can be utilized in the expression studies, or one that has undergone site-directed mutagenesis either to mimic a naturally occurring variant or to test a specific structural hypothesis. The cDNA is subcloned into any number

Concluding Remarks

Very little is known, or even appreciated, as to how biological systems that function exclusively at the level of encoded proteins can contribute to, or modify, gene expression. Conceivably, human biological variation at this level can influence the phenotypic expression of an individual's genotype (Cabral et al., 2002). The devised methodologies outlined above describe various experimental strategies that have successfully elucidated the roles played by asparagine-linked oligosaccharide

Acknowledgments

We acknowledge funding (to R. N. S.) by multiple grants from the National Institutes of Health (NIHLB and NIDDK), American Lung Association, American Heart Association, Alpha1-Foundation, and the Moran Foundation. S. P. was funded by a postdoctoral fellowship from the Alpha1-Foundation and a grant from the Baylor College of Medicine Digestive Disease Center (DK56338). M. J. I. is the recipient of a training grant from the Huffington Center on Aging, at Baylor College of Medicine.

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