Characterization of prelamin a tail cleavage activity of ZMPSTE24

Shengfeng Xu, Purdue University

Abstract

Post-translational modifications are critical for the function, stability, localizations, and protein-protein interactions of a wide spectrum of proteins, which often cannot be achieved with unmodified forms. Proteins ending with CAAX motif (in which the "C" is cysteine, "A" is often an aliphatic amino acid, and "X" is one of several different aminio aicds), undergo a series of sequential modifications. First, the cysteine of CAAX motif is prenylated with either a farnesyl or geranylgeranyl group. Second, the AAX tripeptide is endoproteolytically released by one of endoproteases, RCE1 or ZMPSTE24. Finally, the newly exposed farnesylcysteine is methylated by an isoprenylcysteine carboxyl methyltransferase (ICMT). When "X" is one among M, S, and Q (as well as A, T, and C to a lesser extent), CAAX proteins are modified by a farnesyl isoprenoid. The 72 kDa nuclear lamina protein, lamin A, is synthesized as a 74 kDa precursor, with CSIM motif at the C-terminus. Conversion of this precursor to mature lamin A requires an additional ZMPSTE24 mediated upstream endoproteolysis step subsequent to the canonical CAAX processing. Lamin A is a critical component of lamina, which provides structural support for nuclear envelop and an anchoring site at the nuclear periphery for interphase chromatin. Proper localization of fully processed lamin A is related to human health and longevity. The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a mutation in the lamin A gene (LMNA), which activates the cryptic splice donor site and results in a defective prelamin A in lack of the upstream cleavage site recognized by ZMPSTE24. Likewise, mutations in ZMPSTE24, which impair the enzymatic activities of the metalloprotease, lead to a wide array of progeroid diseases varying in severity, from the neonatal lethal disorder restiictive dermopathy (RD), atypical premature aging disorder ("HGPS") to relatively milder madibuloacral dysplasia type B (MAD-B). Several progeroid disorders related mutations in ZMPSTE24 has been identified and their remaining activities in AAX'ing reaction and upstream endoproteolytic event were tested with couple proteolysis methyltransfease assay and the newly developed in vitro reconstitution of prelamin A endoproteolysis, respectively. The disease severity has been found to be reversely proportional to the retained partial activity of ZMPSTE24 encoded by two alleles of a patient. Most of the mutations discovered are concentrated in loop 5 and C-terminal portion of ZMPSTE24 in its predicted topology model, and the importance of these two parts can be extended to Ste24p. As the yeast homolog of ZMPSTE24, Ste24p has similar predicted hydropathy plots and 2D topological structure. Ste24p also plays a dual role in maturation of its substrate a-factor, which is synthesized as 36 amino acids precursor ending with CVIS motif. Ste24p is functionally redundant with Rce1p in removing AAX amino acids from the farnesylated cysteine of CAAX motif, while this metalloprotese is uniquely required for the first step of two N-terminal cleavage events. Despite the facts that a-factor is not the homolog of prelmin A, and yeast does not express prelaminA, the maturation pathways of a-factor and prelamin A are in parallel. ZMPSTE24 can complement the generation of bioactive a-factor in yeast lacking Ste24p and Rce1p. Conversely, prelamin A can be converted into mature lamin A by enzymes implicated in a-factor maturation in S. cerevisiae. Ste24p has been identified to be the enzyme required to mediate the prelamin A tail cleavage event in vivo and in vitro prelamin A tail cleavage assay.

Degree

Ph.D.

Advisors

Hrycyna, Purdue University.

Subject Area

Biochemistry

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