Hutchinson–Gilford progeria syndrome (HGPS) is a genetic disease that results in premature aging because of a de novo point mutation (1824C→T) in the LMNA gene (Goldman et al., 2002; De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003). The mutation results in sporadic activation of a cryptic splice donor site in exon 11 of the prelamin A pre-mRNA, leading to production of a farnesylated and carboxyl-methylated lamin A mutant protein (progerin). Progerin causes nuclear envelope dysfunction, DNA double-strand breaks (DSBs), activation of DNA damage responses, early replicative arrest, and, ultimately, accelerated aging (Bridger & Kill, 2004; Capell & Collins, 2006; Liu et al., 2006). Progerin also occurs in the cells of healthy individuals and is responsible for the HGPS-like nuclear defects in the cells of normal aging individuals (Scaffidi & Misteli, 2006; Cao et al., 2011). The level of progerin (Olive et al., 2010) or wild-type prelamin A (Ragnauth et al., 2010) also increases with the age of healthy individuals and can be correlated with aging-related cardiovascular diseases.
Despite these findings, the molecular mechanism by which progerin causes HGPS or premature aging is largely unknown. Because early replicative arrest is a hallmark phenotype of HGPS and DSBs can form from collapsed replication forks, we reasoned that the replication machinery in HGPS could be dysfunctional. Indeed, analysis of the nuclear extracts of HGPS cells demonstrated that the integrity of replication factor C (RFC) was compromised in HGPS cells (Fig. 1A). Specifically, the large subunit of RFC (RFC1, 128 kDa) was found to be degraded to a ∼ 75-kDa C-terminal fragment (RFC1-75), suggesting that a proteolytic cleavage of RFC1 occurred in HGPS cells. The Western blot results were confirmed using three different RFC1 C-terminus-specific antibodies (data not shown). Also, this RFC1-75 band was absent in blots probed with N-terminal-specific antibodies (data not shown). In addition, RFC1 siRNA efficiently knocked down the expression of both intact and truncated RFC1 in HGPS (Fig. 1B), further confirming that the truncated fragment was from RFC1. Also, the level of RFC1-75 increased as the growth rate decreased with passage number of the cells (Fig. 1A). This RFC1 cleavage is a common event in HGPS because the RFC1-75 fragment was seen in cells from five different patients with HGPS (Fig. 1C). To determine whether RFC1 degradation is a result of DNA damage known to occur in HGPS cells, HeLa cells were treated with UV-C irradiation or camptothecin to induce bulky DNA damage or DSBs, respectively. The treatment resulted in no cleavage of RFC1 even though a significant amount of DSBs were induced (Fig. 1D), suggesting that the cleavage is independent of DNA damage but an event unique to HGPS.
RFC1 is the large subunit of the RFC complex consisting of RFC1, RFC2, RFC3, RFC4, and RFC5. RFC1 contains the major DNA-binding domains of RFC and is directly involved in RFC–proliferating cell nuclear antigen (PCNA) interaction. RFC, widely known as the DNA clamp loader, plays a crucial role in replication: RFC binds to the 3′ end of the primed nascent DNA strand and loads PCNA and DNA polymerase δ or ε onto the replication forks. It is highly possible that the truncation of RFC1 may disrupt the assembly of replication machinery and stall the forks, ultimately leading to replication fork collapse and formation of DSBs. Indeed, recruitment of PCNA and pol δ to chromatin was inhibited as the cleaved RFC1 accumulated in HGPS cells (Fig. 1E), suggesting that RFC1-75 is functionally defective in recruiting PCNA and pol δ to replication forks. This is consistent with the fact that HGPS cells are characterized by early replicative arrest.
The finding that RFC1 is unexpectedly truncated in HGPS could have a significant and broad implication in addressing the mechanisms of replicative senescence and aging because of the close relationship between replication and cell proliferation and to the relevance of progerin to aging. This is partially supported by the correlation between RFC1-75 accumulation and the growth rate of HGPS cells in which the cells rapidly reached replicative senescence around passage number 21 (Fig. 1A) (Liu et al., 2006). In addition, our preliminary results suggest that the same correlation also may be true for normal human fibroblasts in an aging- and passage-dependent manner (data not shown).
To determine whether inhibition of progerin farnesylation could block RFC1 truncation, HGPS cells were treated with pravastatin and/or zoledronic acid, which have been shown to inhibit farnesylation and alternative prenylation (Varela et al., 2008). Indeed, the treatment efficiently inhibited the truncation (Fig. 2A). Also, it is of great interest to identify the protease(s) that cleaved RFC1 in HGPS cells because the identification may lead to a therapeutic treatment of HGPS. Thus, inhibitors targeting different types of proteases were tested. Pepstain, a potent inhibitor of aspartyl proteases, and E64, which inhibits cysteine peptidases, showed no inhibition of RFC1 cleavage (Fig. 2B). In contrast, AEBSF, a potent serine protease inhibitor, efficiently inhibited the cleavage in a dose-dependent manner. We further examined the effect of the proteasome inhibitor MG132 which showed no substantial effect on RFC1 cleavage (Fig. 2C). Taken together, our results suggest that the protease responsible for the RFC1 cleavage is likely a serine or serine-like protease. Obviously, future effort to identify the protease would be very helpful not only to understand the mechanisms of HGPS disease progression but also to provide new strategies for treatment of the HGPS disease.