The heavy metal cadmium induces valosin-containing protein (VCP)-mediated aggresome formation

Abstract

Cadmium (Cd²⁺) is a heavy metal ion known to have a long biological half-life in humans. Accumulating evidence shows that exposure to Cd²⁺ is associated with neurodegenerative diseases characterized by the retention of ubiquitinated and misfolded proteins in the lesions. Here, we report that Cd²⁺ directly induces the formation of protein inclusion bodies in cells. The protein inclusion body is an aggresome, a major organelle for collecting ubiquitinated or misfolded proteins. Our results show that aggresomes are enriched in the detergent-insoluble fraction of Cd²⁺-treated cell lysates. Proteomic analysis identified 145 proteins in the aggresome-enriched fractions. One of the proteins is the highly conserved valosin-containing protein (VCP), which has been shown to colocalize with aggresomes and bind ubiquitinated proteins through its N domain (#1–200). Our subsequent examination of VCP's role in the formation of aggresomes induced by Cd²⁺ indicates that the C-terminal tail (#780–806) of VCP interacts with histone deacetylase HDAC6, a mediator for aggresome formation, suggesting that VCP participates in transporting ubiquitinated proteins to aggresomes. This function of VCP is impaired by inhibition of the deacetylase activity of HDAC6 or by over-expression of VCP mutants that do not bind ubiquitinated proteins or HDAC6. Our results indicate that Cd²⁺ induces the formation of protein inclusion bodies by promoting the accumulation of ubiquitinated proteins in aggresomes through VCP and HDAC6. Our delineation of the role of VCP in regulating cell responses to ubiquitinated proteins has important implications for understanding Cd²⁺ toxicity and associated diseases.

Introduction

Cadmium (Cd²⁺) poisoning is a serious health threat due to an increased level of industrial pollution and lack of an effective therapy (Shih et al., 2004). Cd²⁺ has an extremely long half-life in the human body (Elinder et al., 1976), and causes disorders of the renal, skeletal, vascular, and respiratory systems (Nordberg, 2004). Chronic exposure to Cd²⁺ is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (Bar-Sela et al., 1992, Bar-Sela et al., 2001) and Alzheimer's disease (Lui et al., 1990), which are characterized by the presence of protein inclusion bodies in the lesions (Li et al., 2004). It is not known, however, whether Cd²⁺ directly induces the formation of protein inclusion bodies, thus constituting an etiologic factor for neurodegenerative disorders. A common etiology of protein inclusion body formation in related neurodegenerative disorders is the generation of aggresomes, a type of cellular organelle in which ubiquitinated and misfolded proteins accumulate (Kopito, 2000, Kawaguchi et al., 2003). An aggresome is located near the centrosome and is formed through an active transport process involving multiple factors. When cells are treated with proteasome inhibitors, polyubiquitinated protein aggregates are transported to aggresomes via microtubules by the deacetylase HDAC6 and the dynein motor complex (Wojcik et al., 1996, Garcia-Mata et al., 1999, Johnston et al., 2002). Thus, depolarization of microtubules, inhibition of the deacetylase activity of HDAC6, and inactivation of the dynein motor all suppress aggresome formation. During the process of aggresome formation, HDAC6 interacts with the dynein motor complex through a dynein-binding domain and with ubiquitinated proteins through its ZnF-UBP zinc finger, which is a motif of the isopeptidase T class of deubiquitinating enzymes (Kawaguchi et al., 2003). Because the ZnF-UBP motif interacts not only with polyubiquitin chains attached to proteins (Kawaguchi et al., 2003) but also with free ubiquitin molecules (Seigneurin-Berny et al., 2001), the presence of free ubiquitin in the cell may decrease the capacity of HDAC6 to transport ubiquitinated proteins. Therefore, it has been postulated that other cellular proteins may facilitate the interaction between HDAC6 and ubiquitinated protein aggregates (Kopito, 2003).

Valosin-containing protein (VCP), an AAA family ATPase (Peters et al., 1990; and reviewed in Wang et al., 2004), binds to both ubiquitinated proteins and HDAC6. VCP also colocalizes with aggresomes induced by proteasome inhibitors (Kitami et al., 2006), suggesting that VCP may be involved in Cd²⁺-induced aggresome formation. Structurally, VCP consists of an N domain, two conserved ATPase domains D1 and D2, and a rather flexible C-terminal tail. The N domain is responsible for the binding of VCP to ubiquitin chains and to VCP cofactors, such as p47 and the Ufd1–Npl4 complex. Since these cofactors also bind to polyubiquitinated proteins, VCP is able to associate with large complexes containing ubiquitinated proteins and transport the complexes to aggresomes (Meyer et al., 2000, Dai and Li, 2001, Song et al., 2003; and reviewed in Wang et al., 2004).

In this study, we report the capacity of Cd²⁺ to directly induce aggresome formation in human cells. Proteomic analysis of Cd²⁺-induced aggresomes shows that VCP is present in detergent-insoluble fractions of aggresome-containing cell lysates. In addition, we found that VCP plays a key role in aggresome formation by acting as an adaptor to the deacetylase HDAC6 for transporting ubiquitinated protein aggregates. While the N domain of VCP (#2–207) binds ubiquitinated proteins, its C-terminal tail (#779–806) interacts with HDAC6. Consequently, VCP loads ubiquitinated cargo proteins onto the HDAC6/dynein complexes and transports the complexes to aggresomes via the microtubule network. Thus, VCP meditates Cd²⁺-induced aggresome formation and serves as a molecular target for the development of therapeutics for Cd²⁺ poisoning.