Proteomic analysis of the amyloid precursor protein fragment C99: expression in yeast

C99 is a membrane-bound peptide generated from the amyloid precursor protein (APP)1 by β-secretase cleavage. The production of this C99 fragment from APP is believed to be the rate-limiting step in releasing smaller aggregation-prone peptide fragments, such as Aβ peptide, from the membrane [1]. The accumulation of these APP fragments to critical concentrations is thought to ultimately lead to the neurodegeneration that is evident in Alzheimer's disease (AD) [2]. The structure and aggregation propensity of many APP fragments have been well characterized in vitro [3–7] though the generation of all of these fragments in vivo under physiological conditions has not been elucidated. This project aimed to address this shortfall. Also, by engineering mammalian cell culture systems some of the intracellular processing pathways for APP have been identified [8–13]. However, given the use of pharmacological agents in most of these studies, the role of side effects from the agents used cannot be excluded. Nevertheless, these studies have provided useful information on the diverse spectrum of cellular proteases that contribute to the generation and/or degradation of APP and C99, including α-secretase, the γ-secretase protein complex, neprilysin (NEP; neutral endopeptidase), the neprilysin-like proteases (NEPLP), and insulysin [1,14–22]. The γ-secretase protein complex is an aspartyl membrane protease whose activity results in the production and release of the Aβ peptide [23]. In contrast, the α-secretase releases a lumenal fragment of APP, which can prevent the formation of the toxic Aβ peptide [16,24]. NEP, NEPLP, and insulysin (which degrades proteins in a manner similar to that of NEP) [19,25,26] are also membrane-bound enzymes but they preferentially cleave the N-terminal peptide bonds of bulky hydrophobic residues into small (<40 to 60-residue) peptides [27]. Since NEP and insulysin have been shown to catalyze Aβ degradation in vivo [14,15,19,27–30], identifying fragments that result from these enzymatic activities is important in modeling the mechanisms of AD [30]. However, the complete panel of in vivo-generated APP fragments and their propensities to generate toxic, aggregation-prone species, remains ill defined. In addition, while the contributions of some fragments to disease pathology are understood, the physiological relevance of many fragments remains to be elucidated. Another protease that may contribute to APP catabolism in mammalian cells is the proteasome [16,31]. The proteasome is a large (26S), multicatalytic cytoplasmic/nuclear protease known to process aggregation-prone secreted proteins that may otherwise lead to neurodegeneration [32]. Since proteasomal activity decreases with age and age is the biggest risk factor in the development of neurodegenerative diseases [22], it is not surprising that this abundant enzyme has been implicated in AD. Although there are clear advantages to using mammalian cell systems to explore the cellular processing pathways for APP [8–13], the use of the yeast system can and has allowed for rapid initial pilot studies for many proteins linked to human disease [33]. Hypotheses developed using the yeast system can then be tested in mammalian cells. Because the secretory pathways and protein quality control machineries are conserved between yeast and mammals, because specific and/or rapidly acting temperature-sensitive yeast mutants exist that allow for the inactivation of specific cellular component(s), and because yeast do not express APP or C99 but can be engineered to synthesize these proteins [34,35], they are an excellent system for these studies. Yeast have also been engineered to express the γ-secretase complex [23] and were found to process APP (and its fragments) using endogenous α-[36–39] and β-[40] secretase activities that are similar to those identified in human cells. Yeast have also been shown to package APP into secretory vesicles in the same manner as seen in human cells [41]. In this study, we generated a C99 yeast expression system to directly examine the role of the proteasome in the processing of C99. The vector system chosen was one that has been shown to successfully express and secrete full-length APP from Saccharomyces cerevisiae. The gene is under the control of a galactose-regulated promotor and the protein contains an N-terminal “pre pro” sequence for the yeast α mating factor, which directs the protein into the secretory pathway (Fig. 1). This pre pro region is cleaved and removed by Kex2 in the Golgi [34]. We have now reengineered this vector system for the expression of C99 and have combined this system, with proteomic technologies, to directly investigate the role of the proteasome in the processing of C99. Because our system does not require the long-term addition of pharmacological agents, nonspecific side effects are minimized. Specifically, we have investigated how yeast compensate for compromised proteasome activity with respect to C99 processing. The results of our study support a role for the proteasome in C99 catabolism, provide new insights into the fragments generated in vivo in the presence of all existing cellular proteases, and provide information on the important and to date overlooked role played by compensatory processing pathways that occur when proteasome activity is ablated. Fig. 1 C99 expression vector and encoded sequence