This project focuses on ZMPSTE24, a fascinating integral membrane zinc metalloprotease important for human health and longevity. ZMPSTE24 plays a critical role in the proteolytic processing of the farnesylated CAAX protein prelamin A, precursor of the nuclear scaffold component lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that impede prelamin A cleavage cause the devastating premature aging disorder Hutchinson-Gilford Progeria syndrome (HGPS) and a set of related progeroid diseases, due to the presence of the uncleaved and persistently farnesylated form of prelamin A. Notably, growing evidence suggests that diminished ZMPSTE24 processing of prelamin A is also a factor in normal physiological aging. My laboratory pioneered the study of ZMPSTE24 in early cycles of this project. We were the first to report its discovery in yeast, where it is calle Ste24p, and demonstrate that it has two distinct proteolytic activities. We went on to show that mammalian ZMPSTE24 mediates prelamin A processing. In the current project period we defined several features of the prelamin A substrate that are important for ZMPSTE24 cleavage and we showed that "mild" ZMPSTE24 disease alleles retain low but residual biochemical function, using a humanized yeast system. Through our sustained body of work on ZMPSTE24, we have developed a full arsenal of assays and tools that will facilitate the studies proposed here. The present proposal has been galvanized by the recently published structure of human ZMPSTE24 (and that of the nearly superimposable yeast Ste24p) which reveal a novel and surprising structure, never heretofore seen. The seven transmembrane spans of ZMPSTE24 form a voluminous, enclosed, water-filled intramembrane chamber that is capped at both ends. Notably, the ZMPSTE24 metalloprotease domain faces the chamber interior, so that substrate access is restricted. Prelamin A must be threaded into the chamber through one of several side portals. Here we propose mechanistic studies of human ZMPSTE24 (Aim 1) and its substrate prelamin A (Aim 2) to define the residues that mediate substrate access and activity, to identify interactors, and to precisely define the step(s) at which ZMPSTE24 disease alleles malfunction. These studies will provide the framework to understand the basis of premature aging disorders and to ensure optimal functioning of ZMPSTE24 as we age.
In Aim 1 we will determine how ZMPSTE24 mediates proteolysis of prelamin A inside of an intramembrane chamber, using structure-guided mutagenesis and photo affinity probes to identify residues important for substrate entry, catalysis, binding, and product release.
In Aim 2, we will use mutagenesis to define key features of the prelamin A substrate required for cleavage by ZMPSTE24, to better understand enzyme function. We will also identify ZMPSTE24/Ste24p binding partners that could contribute to substrate delivery or regulation of proteolytic activity. Together, our studies will reveal fundamental mechanistic principles relevant to nuclear membrane protein biology, premature aging disorders, and normal aging.

Public Health Relevance

The enzyme ZMPSTE24 is a zinc metalloprotease important for healthy aging;mutations that alter this enzyme or its substrate, the nuclear scaffold protein lamin A, result in a premature aging disease called progeria. This project capitalizes on an exciting new advance by our collaborator that provides the first-ever glimpse into the inner workings of ZMPSTE24 at the atomic level. By combining this new blueprint with the many biochemical and cell biological tools we have developed over the years since we discovered this enzyme, we are now poised to gain a precise view of how ZMPSTE24 works, which in turn is expected to provide insights important for healthy aging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM041223-20
Application #
8817086
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Ainsztein, Alexandra M
Project Start
1988-12-01
Project End
2018-04-30
Budget Start
2014-09-15
Budget End
2015-04-30
Support Year
20
Fiscal Year
2014
Total Cost
$412,233
Indirect Cost
$157,768
Name
Johns Hopkins University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Kane, Megan S; Lindsay, Mark E; Judge, Daniel P et al. (2013) LMNA-associated cardiocutaneous progeria: an inherited autosomal dominant premature aging syndrome with late onset. Am J Med Genet A 161A:1599-611
Michaelis, Susan; Hrycyna, Christine A (2013) Biochemistry. A protease for the ages. Science 339:1529-30
Barrowman, Jemima; Hamblet, Corinne; Kane, Megan S et al. (2012) Requirements for efficient proteolytic cleavage of prelamin A by ZMPSTE24. PLoS One 7:e32120
Barrowman, Jemima; Bhandari, Deepali; Reinisch, Karin et al. (2010) TRAPP complexes in membrane traffic: convergence through a common Rab. Nat Rev Mol Cell Biol 11:759-63
Barrowman, Jemima; Michaelis, Susan (2009) ZMPSTE24, an integral membrane zinc metalloprotease with a connection to progeroid disorders. Biol Chem 390:761-73
Hudon, Sarah E; Coffinier, Catherine; Michaelis, Susan et al. (2008) HIV-protease inhibitors block the enzymatic activity of purified Ste24p. Biochem Biophys Res Commun 374:365-8
Barrowman, Jemima; Hamblet, Corinne; George, Carolyn M et al. (2008) Analysis of prelamin A biogenesis reveals the nucleus to be a CaaX processing compartment. Mol Biol Cell 19:5398-408
Gelb, Michael H; Brunsveld, Lucas; Hrycyna, Christine A et al. (2006) Therapeutic intervention based on protein prenylation and associated modifications. Nat Chem Biol 2:518-28
Huyer, Gregory; Kistler, Amy; Nouvet, Franklin J et al. (2006) Saccharomyces cerevisiae a-factor mutants reveal residues critical for processing, activity, and export. Eukaryot Cell 5:1560-70
Young, Stephen G; Fong, Loren G; Michaelis, Susan (2005) Thematic review series: Lipid posttranslational modifications. Prelamin A, Zmpste24, misshapen cell nuclei, and progeria--new evidence suggesting that protein farnesylation could be important for disease pathogenesis. J Lipid Res 46:2531-58

Showing the most recent 10 out of 30 publications