Prelamin A is a CAAX-box protein which undergoes the canonical sequential post-translational cysteine modifications: farnesylation, AAX endopeptidase and carboxymethylation. Uniquely among known mammalian farnesylated proteins it also undergoes an upstream endoproteolytic cleavage to form the nuclear lamin protein: lamin A. Our laboratory has demonstrated, in vitro, that this reaction is mediated by the endoprotease Zmpste24. There is substantial genetic evidence that catalysis of this reaction is unique to Zmpste24. The progerias Hutchinson-Gilford Progeria Syndrome and Restrictive Dermopathy arise from defects in the prelamin A cleavage site and Zmpste24 respectively. Although these diseases manifest the pathological consequences of a defect in the prelamin A processing pathway, the functional significance of prelamin A processing in the normal events of cell division is unknown. However, it is noteworthy that patients with HGPS usually die prematurely from atherosclerotic cardiovascular disease, suggesting that whatever physiological events are mediated by prelamin A might be particularly important in the cardiovascular system. In this proposal we present preliminary evidence that Zmpste24 is down-regulated in G0/G1 arrested cultured cells. Therefore, we hypothesize that the resultant farnesylated and carboxymethylated prelamin A plays a functional role in cellular quiescence. We propose to explore and test this hypothesis, and in particular to test the hypothesis that FC-prelamin A affects gene expression in quiescence. We also propose to determine the mechanism by which Zmpste24 is down-regulated during quiescence.
The nuclear lamin proteins, A, B and C, form a meshwork in the nucleus of cells that has several functions including regulation of gene expression. Our laboratory has reported that lamin A undergoes a unique chemical event in its formation in which after synthesis as a precursor derivatized with the lipid farnesyl, called prelamin A, it is clipped by an enzyme called Zmpste24 to form the final lamin A molecule. The physiological function of this protein maturation process has remained a mystery, but in the last few years it has been found that patients suffering from two different severe congenital diseases have defects in this processing pathway. In preliminary studies described in this proposal, we have found that the maturation of lamin A is blocked in non-proliferating cells resulting in the accumulation of prelamin A. This is a common condition of cells in adult organisms, referred to as quiescence. We also have found that the reason that prelamin A accumulates is that its processing enzyme, Zmpste24, is not active in quiescent cells. Given these observations, we have hypothesized that the normal function of prelamin A is to facilitate quiescence, which we further hypothesize occurs through the regulation of genes which are associated with quiescence. In this proposal we describe experiments to test whether these hypotheses are correct. We also propose to determine the genes regulated by prelamin A and the mechanisms by which prelamin A regulates these specific genes. Finally we propose to determine the mechanisms by which Zmpste24 activity is suppressed during quiescence. ? ? ?
| Singh, Sanjay K; Thirumalai, Avinash; Hammond Jr, David J et al. (2012) Exposing a hidden functional site of C-reactive protein by site-directed mutagenesis. J Biol Chem 287:3550-8 |
