Erh-Ting graduated with a B.S. in Biochemical Science and Technology from National Taiwan University in 2012. She is beginning her fourth year as a graduate student at Purdue University in the Hrycyna research group, where she studies the mechanism of ZMPSTE24, a unique protease involved in lamin A maturation. Outside the lab, she enjoys traveling, reading and cooking. Currently, Erh-Ting is positioning herself toward a career in industry.
Elucidating the Proteolytic Activity of Human ZMPSTE24 and Disease Mutations
Department of Chemistry and Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA; Department of Cell Biology, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Department of Chemistry and Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
ZMPSTE24 is a unique intramembrane human zinc metalloprotease that is localized to both the endoplasmic reticulum and inner nuclear membranes. The enzyme catalyzes two distinct cleavage steps in the maturation of prelamin A to lamin A, including the endoproteolytic removal of the C-terminal -aaX residues adjacent to an isoprenylated cysteine residue and a second discrete site-specific upstream N-terminal cleavage.
Improper prelamin A processing caused by mutations in the gene encoding ZMPSTE24 results in laminopathies. In previous studies, we demonstrated that the enzymatic aaX-ing activity of ZMPSTE24 correlates to the severity of the disease state. However, how ZMPSTE24 disease mutations affect the second cleavage in prelamin A processing is still unclear. To elucidate the role of each mutant, we measured the enzymatic activity using a newly established fluorescence-based assay with full-length a-factor containing a 2-aminobenzoic acid (Abz) fluorophore at the N-terminus and a dinitophenol (DNP) quencher at the C-terminus. After cleavage, quantification of the fluorescence from the dequenched cleaved peptides enabled us to directly monitor the proteolytic activity of each ZMPSTE24 mutant. Our data revealed that the disease mutants also demonstrated reduced N-terminal cleavage activity.
To further confirm the proteolytic activity of each mutant, I will optimize and perform the fluorescence-based assay with pure ZMPSTE24. Moreover, in vivo proteolytic activity will be studied in yeast by co-expression of prelamin A and ZMPSTE24. Prelamin A cleavage will be monitored using a mobility shift assay on SDS-PAGE. Used together, these data will provide a greater understanding of the mechanism of ZMPSTE24 and provide further insights into laminopathies.