Replication Factor C1, the Large Subunit of Replication Factor C, Is Proteolytically Truncated in Hutchinson-Gilford Progeria Syndrome

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder due to a LMNA gene mutation which produces a mutant lamin A protein (progerin). Progerin also has been correlated to physiological aging and related diseases. However, how progerin causes the progeria remains unknown. Here we report that the large subunit (RFC1) of replication factor C is cleaved in HGPS cells, leading to the production of a truncated RFC1 of ~75 kDa which appears to be defective in loading PCNA and pol δ onto DNA for replication. Interestingly, the cleavage can be inhibited by a serine protease inhibitor, suggesting that RFC1 is cleaved by a serine protease. Due to the crucial role of RFC in DNA replication our findings provide a mechanistic interpretation for the observed replicative arrest and premature aging phenotypes of HPGS, and may lead to novel strategies in HGPS treatment. Furthermore, this unique truncated form of RFC1 may serve as a potential marker for HGPS.

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease that results in premature aging due to a de novo point mutation (1824C→T) in the LMNA gene (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003; Goldman et al., 2002). 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 and Kill, 2004; Capell and 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 (Cao et al., 2011; Scaffidi and Misteli, 2006). 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. Since 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, 140 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 increased passage number of the cells (Fig. 1A). This RFC1 cleavage is a common event in HGPS since the RFC1–75 fragment was seen in cells from five different HGPS patients (Fig. 1C). To determine if RFC1 degradation is a result of DNA damage known to occur in HGPS cells, HeLa cells were treated with UV-C irradiation or camptothecin (CPT) to induce bulky DNA damage or DSBs, respectively. The treatment resulted in no cleavage of RFC1 even though significant amount DSBs were induced (Fig. 1D), suggesting that the cleavage is independent of DNA damage but an event unique to HGPS.

Fig. 1. Proteolytic cleavage of RFC1 in HGPS cells.

(A) HGPS fibroblasts were grown in culture to various passage numbers. Nuclear extracts were analyzed by Western blotting with anti-RFC1 antibody. HeLa cells were used as a control cell line and histone 3 as a loading control. The relative level of the truncated RFC1 was quantified as the function of cell growth rate (PD/day). PD stands for population doubling. (B) RFC1 siRNAs were transfected into HGPS cells for 72 hrs. A scrambled siRNA was used as a control. (C) Cells from five different HGPS patients were lysed and analyzed by Western blotting. BJ and WI38 are human normal fibroblasts used as control cells. (D) HeLa cells were treated or mock-treated with 20 J/m² UV or 10 uM CPT. The cell lysates were analyzed using anti-RFC1 antibodies. The γ–H2AX bands indicate formation of DSBs induced by the treatments. (E) Chromatin association assays were performed for HGPS cells with different passage numbers. A549 cells served as a control.

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-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 due to 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 of 21 (Fig. 1A) (Liu et al., 2006). In addition, our preliminary results suggest that the same correlation may also be true for normal human fibroblasts in an aging- and passage-dependent manner (data not shown).

To determine if 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 since 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 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.

Fig. 2. Degradation of RFC1 in HGPS cells is prevented by prenylation and protease inhibitors.

(A) HGPS cells were treated with pravastatin and/or zoledronic acid were analyzed by Western blotting. The accumulation of pre-lamin A indicates the efficiency of deprenylation of the inhibitors. (B) HGPS cells were treated with the indicated concentrations of protease inhibitor. Cell lysates were analyzed by Western blotting. “C” indicates the HGPS cells that were mock treated. (C) HGPS cells were treated with the proteasome inhibitor MG132 and analyzed as in (B).