Proteomic identification of proteins specifically oxidized in Caenorhabditis elegans expressing human Aβ(1–42): Implications for Alzheimer's disease
Introduction
Protein oxidation [13] is extensive in Alzheimer's disease (AD) [14], [26], [30], [39]. Recent proteomics studies in our laboratory have identified specific targets of protein oxidation in AD brain, assessed by increased carbonyls, such as, creatine kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidase-related protein 2, α-enolase, and heat shock cognate 71, indicating that a number of cellular mechanisms are affected including energy metabolism, protein turnover, and neuronal communication [16], [17]. Alzheimer's disease brain is characterized pathologically by synapse loss and by the presence of senile plaques, neurofibrillary tangles, and neuropil threads. Extracellular senile plaques are composed primarily of fibrilar deposits of amyloid beta peptide (1–42) [Aβ(1–42)], a product of proteolytic cleavage of the transmembrane protein, amyloid precursor protein (APP). On the other hand, intracellular neurofibrillary tangles are composed of paired helical filaments formed from hyperphosphorylated tau, a microtubule associated protein [25], [33], [57].
Aβ(1–42) has been proposed to play a central role in the pathogenesis of AD as a mediator of oxidative stress [11], [12], [15]. However, the mechanism by which Aβ(1–42)-induced protein oxidation occurs is unknown. Several hypotheses have been proposed including the aggregation state of the peptide. Early studies demonstrated an association between fibrillar peptide aggregates and toxicity [36], [46]. More recently, however, it has been shown in C. elegans expressing Aβ(1–42) that protein oxidation precedes fibrillar deposition of the peptide suggesting that small, soluble oligomers of the peptide are the toxic species [21].
In this study, we utilized proteomic techniques to identify proteins that are specifically oxidized in a transgenic C. elegans expressing human Aβ(1–42) in body wall muscle (CL4176). Moreover, in order to evaluate the role of protein aggregation per se in oxidative stress, we analyzed a transgenic C. elegans expressing a green fluorescent protein (GFP) fusion protein, which forms rapid aggregates of GFP in the worms (CL2337). Finally, to control for non-specific protein oxidation resulting from muscle dysfunction itself, we examined the oxidative effects in transgenic C. elegans expressing the ypkA subunit of Yersinia pseudotuberculosis, a serine/threonine kinase known to affect the cytoskeleton (XA1440) [28]. We identified 16 proteins to be oxidatively modified in C. elegans expressing Aβ(1–42), five proteins to be oxidatively modified in C. elegans expressing aggregated GFP, and 1 protein was oxidatively modified in C. elegans expressing ypkA. The proteins identified in this study are involved in a variety of cellular functions including cytoskeletal integrity, scavenging of oxidants, signal transduction, lipid metabolism, proteasome function, and energy metabolism. Protein oxidation has been shown to alter protein conformation leading to loss of function [1], [16], [26], [31], [41], [49]. Thus, it is likely that oxidation of the proteins identified in this study will lead to loss of function. The implications of oxidation of these proteins are discussed with respect to Alzheimer's disease.
Section snippets
Chemicals
All chemicals were of the highest purity and were obtained from Sigma (St. Louis, MO, USA) unless otherwise noted. The oxyblot protein oxidation detection kit was purchased from Chemicon International (Temecula, CA, USA).
Transgenic strain construction and sample preparation.
Stable (chromosomally integrated) transgenic C. elegans strains with temperature-inducible transgene expression were constructed using the smg-1ts system as previously described [35]. Induction of strain CL4176 results in intramuscular accumulation of human Aβ(1–42). Strain
Results
Comparison of protein oxidation levels in C. elegans expressing Aβ(1–42) (CL4176) and control C. elegans (CL1234) was carried out by first identifying carbonylated proteins via anti-DNP immunochemical development of proteins transferred to a nitrocellulose membrane, or 2D-oyxblot analysis (Fig. 1B). Individual protein spots were matched between the 2D-PAGE maps and the 2D-oxyblots and the carbonyl immunoreactivity of each spot was normalized to the protein content in the 2D-PAGE (Fig. 1A).
Discussion
Aβ(1–42) has been proposed to play a central role in the oxidative stress evident in AD [14], [15]. However, little is know about the specific in vivo effects of Aβ(1–42) especially with respect to the specific protein targets of oxidation. In this study, we used proteomics techniques to identify proteins that are specifically oxidatively modified in C. elegans expressing Aβ(1–42) (CL4176). This model expresses intracellular Aβ(1–42), and thus we have specifically identified intracellular
Acknowledgements
This work was supported in part by NIH grants to D.A.B. [AG-05119; AG-10836] and C.D.L [AG12423, AG21037]. We are indebted to Creg Darby for providing the XA1440 strain.
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