The DNA repair-ubiquitin-associated HR23 proteins are constituents of neuronal inclusions in specific neurodegenerative disorders without hampering DNA repair
Introduction
In mammals, two Homologues of the yeast gene Rad23 exist, designated HR23A and HR23B. Both have a function in DNA repair and in the ubiquitin–proteasome system (UPS) (Schauber et al., 1998). In DNA repair, part of the HR23 proteins are in complex with XPC and function in the DNA damage recognition step of the global genome sub-pathway of nucleotide excision repair (GG-NER), which recognizes and removes helix-distorting lesions from the entire genome (Ng et al., 2003, Sugasawa et al., 1997, Sugasawa et al., 1998). Both HR23A and HR23B stabilize the XPC protein in vivo and increase the affinity for damaged DNA of XPC in vitro (Li et al., 1997, Lommel et al., 2002, Ng et al., 2003, Sugasawa et al., 1996). It has been demonstrated that Rad23 interacts with the 26S proteasome and with ubiquitylated proteins as well (Elsasser et al., 2004, Saeki et al., 2002). In addition to a repair function, HR23B is also essential for development as indicated by the dramatic phenotype of the HR23B knock-out mouse, including impaired embryonic development and high rates of intrauterine death. In contrast, mouse models defective in XPC develop relatively normal and only exhibit a profound NER deficiency (Cheo et al., 1997, Ng et al., 2002, Sands et al., 1995). HR23B knock-out mice are smaller, display facial dysmorphology and male infertility but are GG-NER proficient due to the functional redundancy with HR23A (Ng et al., 2002). In contrast, HR23A knock-out mutants are indistinguishable from wild type because of complete functional compensation by HR23B (Ng et al., 2003).
Ubiquitylation of proteins is a general protein modification involved in many processes such as endocytosis, transcription, antigen presentation and protein degradation (Glickman and Ciechanover, 2002, Ravid and Hochstrasser, 2004). Ubiquitylation of target proteins is performed by the sequential action of the ubiquitin-activating enzyme (E1), one of a series of ubiquitin-conjugating enzymes (E2) in combination with a specific ubiquitin ligase (E3). Ubiquitin is usually covalently attached to internal lysine residues of the target protein. Since ubiquitin contains several internal lysines itself, a polyubiquitin tree can be formed (Glickman and Ciechanover, 2002). It is generally believed that proteins containing a lysine 48-branched ubiquitin tree are destined for degradation by the 26S proteasome. Proteins that contain a ubiquitin-interacting domain like the ubiquitin-associated domain (UBA) recognize ubiquitylated proteins creating new interactions and subsequent actions (Verma et al., 2004). Interestingly, both HR23A and HR23B contain besides an amino terminal ubiquitin-like domain (UBL), known to interact with the proteasome (Elsasser et al., 2002, Saeki et al., 2002), also two UBA domains. Recently, it has been shown that the carboxy terminal UBA domain of yeast Rad23 has a relatively high affinity for lysine 48 polyubiquitin trees (Raasi et al., 2004, Ryu et al., 2003, Varadan et al., 2005), whereas Rad23 itself is protected from degradation by an intrinsic stabilization signal (Heessen et al., 2005). This, together with the capacity to interact with the proteasome, makes HR23A and HR23B ideal as shuttles/chaperones for proteins to be degraded by the proteasome. In fact, it has been shown that Rad23, and others, are shuttles of the 26S proteasome (Varadan et al., 2005, Verma et al., 2004). Misregulation of protein degradation via the UPS is directly or indirectly involved in many human diseases including malignancies, auto-immune diseases and neurodegenerative disorders (Glickman and Ciechanover, 2002).
The formation of inclusions in specific parts of the brain is a pathological hallmark of many neurodegenerative diseases (Ciechanover and Brundin, 2003). Actually, the specific localization and the composition of these inclusions have been utilized as a diagnostic criterion of several neurodegenerative disorders, including frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), Parkinson disease (PD), Huntington disease (HD), Spinocerebellar ataxia (SCAs) and fragile X associated tremor/ataxia syndrome (FXTAS). Major constituent of the inclusions is often a disease-specific mutant protein (e.g. tau, α-synuclein, huntingtin) or mutant mRNA (e.g. FMR1 mRNAs in FXTAS). For instance, an important group of neurodegenerative disorders is the polyglutamine diseases characterized by neuronal intranuclear inclusions that consist of accumulations of insoluble aggregated polyglutamine-containing proteins. Next, to mutant protein, UPS components and molecular chaperones have been detected within the inclusions, including ubiquitin and the 20S catalytic core complex of the proteasome.
Many neurodegenerative diseases share these ubiquitin-positive structures; however, their origin and function in relation to the pathology remain unclear. Although ubiquitin-positive inclusions are the pathological hallmark of many neurodegenerative disorders, it might very well be that they are secondary responses to different causes (Ciechanover and Brundin, 2003). Inclusions have been found to positively influence cell viability supporting a protective function for the inclusions (Arrasate et al., 2004, Bowman et al., 2005). Therefore, it might be that they arise as a consequence of cellular stress, thereby trying to solve a toxicity problem in the cell. Indeed, it has been shown that accumulation of mutant proteins containing long polyglutamine tracts can cause impairment of the UPS leading to cellular stress in living cells (Bence et al., 2001). In neurodegenerative diseases such as HD and SCA 1–7, expanded polyglutamine tracts in target proteins are known to be the primary source of the inclusions (Ciechanover and Brundin, 2003). Purified 26S proteasomes fail to digest these long repeats and release them instantly (Venkatraman et al., 2004). Consequently, polyglutamine-containing (mutant) proteins accumulate in time, especially in non-dividing neurons, ultimately leading to neuronal cell death. In mouse models mimicking human SCA7, no proof for UPS impairment prior to or during onset of the disease was found, suggesting that the impairment is a secondary consequence (Bowman et al., 2005).
The issue whether the formation of inclusions is a cause or an effect of toxicity is of great importance for understanding the pathogenesis of these diseases. Knowledge of the formation and of the constituents of inclusions, regardless if they have a positive or a negative influence on cell viability, can ultimately lead to the development of novel therapeutic interventions. Since HR23A and HR23B have a prominent role in shuttling proteins to the proteasome, we wondered if HR23A and/or HR23B participate in the formation of inclusions.
Section snippets
Western blotting
Wild type and HR23B−/− mouse embryonic fibroblasts were lysed as described previously (Okuda et al., 2004). HR23B protein was detected using either fresh or pre-absorbed rabbit polyclonal antibodies against HR23B. Pre-absorption was performed by incubation of the fresh serum with nitrocellulose paper containing homogenates of HR23B−/− cells overnight at 4°C. Horseradish-peroxidase (HRP)-labeled anti-rabbit IgG serum was used as secondary antibody, allowing chemiluminescence detection with ECL
Specificity of anti-HR23B serum
In order to test whether HR23B is a component of inclusion bodies, we applied an immunohistochemical approach. Our polyclonal anti-HR23B serum showed several cross-reacting bands when used on immunoblots. After multiple usages, these aspecific bands vanished while the specific HR23B band did not (Fig. 1). To increase the specificity of the serum, it was pre-absorbed prior to the actual staining. This pre-absorbed antiserum was used for our further immunohistochemical experiments. Next, the
HR23B is present in neuronal inclusion of some specific neurodegenerative disorders
The exact cause and role of the UPS in the formation of inclusions in many distinct neurodegenerative disorders are not defined yet. Here, we report the accumulation of HR23B (and HR23A) in neuronal inclusion bodies in a mouse model for FXTAS, and in patient material from several but not all neurodegenerative disorders, including FTDP-17, HD, PD, SCA3 and SCA7. Interestingly, HR23B was not detected in NFT in brain tissue from AD. Antibodies recognizing ubiquitin are widely used for a
Acknowledgments
We thank Dr. R. de Vos for providing patient material and Dr. F. van Leeuwen for providing the polyQ-GFP construct. Ruud Koppenol and Tom de Vries Lentsch are acknowledged for their excellent photography. This work was supported by grants of The Netherlands Organization for Scientific Research (NWO: 014-90-001) (SB and HY), 912-03-12/917-46-364, NKB EMCR2002-2703 (WV), NFXF (RW) and Prinses Beatrix fonds (RW). The authors declare that there is no conflict of interest.
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