1989 — 2007 |
Michaelis, Susan D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Biogenesis and Function of Yeast a-Factor @ Johns Hopkins University
Many intracellular and secreted signaling molecules are synthesized as precursors that undergo elaborate post-translational processing events critical to their normal and pathological functions. To better understand these events, we are studying the biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor, an extracellular signaling peptide that is prenylated and carboxyl methylated. Mature a-factor is derived from a precursor that terminates with a CaaX motif ("C" is Cys, "a" is aliphatic, "X" is any of several residues). The biogenesis of the a-factor precursor is distinctive, involving: (1) C-terminal (CaaX) processing (by the farnesyl transferase Ram1p/Ram2p, the endoproteases Ste24p and Rcelp, and the methyltransferase Ste14p), (2) N-terminal proteolytic cleavage (by the endoproteases Ste24p and Axllp), and (3) a non-classical export mechanism (mediated by the a-factor exporter, Ste6p). We have recently shown that one of these components, Ste24p, is a multispanning membrane protein with intrinsic protease activity that acts in dual steps of a-factor biogenesis, mediating both C- and N-terminal processing. Furthermore, in extending our studies to mammalian systems we discovered a new mammalian Ste24p substrate, prelamin A (the nuclear lamin A precursor). In this proposal we will focus on the activity, topology, and interaction of the membrane-associated a-factor CaaX processing components (Ste24p, Rcelp, Ste14p) and explore the possibility that these proteins (and others) may form a CaaX processing "machine" (Aims 1-3). CaaX processing renders a-factor extremely lipophilic; yet to exit the cell a-factor must transit through the aqueous environment of the cytosol, and thereafter must diffuse through the extracellular milieu. We will examine the trafficking of a-factor, both inside and outside of the cell, and genetically identify novel factors involved in these processes (Aims 4-6). Essentially all of the steps in a-factor biogenesis are mediated by multispanning membrane proteins. Therefore in the long-term, our studies will lead to a better fundamental understanding of the structure, function, catalytic properties, and organization of multispanning membrane proteins that mediate diverse cellular processes, extending beyond a-factor biogenesis. Such processes include the proteolytic processing events that generate the cholesterol biosynthesis regulator (SREBP), the Alzheimer's precursor protein (APP), and mammalian prelamin A (defects in which result in human laminopathies). Our studies also bear on the development of chemotherapeutic agents designed to inhibit the CaaX processing of oncogenic Ras proteins.
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0.958 |
1994 — 1998 |
Michaelis, Susan |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Folding and Trafficking of Ste 6 and Cftr in Yeast @ Johns Hopkins University
CFTR, the protein whose defect results in cystic fibrosis (CF), is a member of a superfamily of structurally related membrane proteins designated the ATP binding cassette (ABC) proteins. ABC proteins are comprised of two homologous halves, each half containing six predicted membrane spanning segments and an ATP nucleotide binding fold (NBF) domain. Another membrane of the ABC superfamily is the STE6 protein of Saccharomyces cerevisiae, a transporter which mediates export of the mating pheromone a-factor. In this project, we will use STE6 as a model for investigating the biogenesis and structure of CFTR; the similar overall design of the two proteins suggests they are likely to require similar cellular components to ensure their proper biogenesis, i.e. membrane insertion, folding, and trafficking to the cell surface. The most prevalent CF allele, deltaF508, appears to cause misfolding of CFTR, resulting in aberrant intracellular trafficking and degradation of the mutant protein. Little is known about factors that aid the biogenesis of complex multispanning membrane proteins such as CFTR. A major goal of this project is to utilize the power of yeast genetics to identify genes encoding cellular components that ensure the proper folding and progression of ABC proteins, in particular STE6 and CFTR, from their site of synthesis to the cell surface. A second goal is to probe intramolecular interactions within STE6 (and STE6-CFTR chimeras) as a means of determining which regions within these proteins interact to promote the proper conformation of the molecule. At third goal is to produce high levels of properly folded CFTR in yeast. We will use genetic, molecular, and biochemical approaches to accomplish the following specific aims: 1) Isolate mutations within STE6 that result in its mislocalization or rapid degradation, 2) identify suppressors of ste6 mutants obtained in aim 1; such suppressors will genetically pinpoint components of the cellular machinery involved in membrane insertion, folding, quality control, and trafficking of STE6, 3) Use STE6-CFTR chimeras to identify additional components of the cellular folding and quality control machinery, 4) Express properly folded CFTR at high levels in S. cerevisiae, and 5) Dissect the intramolecular interactions that govern assembly of STE6, particularly focusing on the role of charged residues in the transmembrane spans. These studies will allow us to identify candidate cellular components that assist CFTR in its biogenesis, and will provide a more detailed view of the structure and folding of CFTR. This information could serve as a basis for devising strategies to revitalize the defective gene product in certain CF patients.
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1 |
1994 — 1997 |
Michaelis, Susan D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Synthesis &Function of the Yeast Abc Transporter, Ste6 @ Johns Hopkins University
A critical aspect of cellular physiology is the selective transport of ions, nutrients, proteins, and signaling molecules across cellular and organellar membranes, mediated by membrane transporter proteins. The Saccharomyces cerevisiae STE6 protein is a member of the ATP binding cassette (ABC) superfamily which includes a large number of membrane proteins that mediate transport and channel functions in eukaryotes and prokaryotes. Clinically important members of the ABC family include MDR, the mammalian multidrug resistance protein, and CFTR, the protein defective in patients with cystic fibrosis. The STE6 transporter in yeast mediates export of the lipopeptide mating pheromone a-factor, .and thus is required for mating by yeast. We will use biochemical, genetic, and molecular approaches to dissect the structure and function of STE6 and to identify cellular components involved in its folding and transit to the membrane. One major focus of the work proposed here is to define in molecular terms how transport of a-factor across the membrane is achieved and to determine how STE6 recognizes its a-factor substrate. Specific aims related to this goal include: 1) development of an in vitro system for STE6-mediated translocation of a-factor that will allow examination of nucleotide hydrolysis and substrate specificity of STE6, 2) identification of residues within STE6 that are critical for substrate recognition by isolation of ste6 suppressor mutants that recognize an altered a-factor, 3) genetic conversion of MDR from a drug transporter into an optimized a- factor transporter, and 4) identification of functional domains of STE6 by isolation of "dominant negative" mutants. These mutant proteins will be powerful reagents for biochemical dissection of STE6 transport in the in vitro system developed in the first aim. The function of a membrane protein depends upon its proper folding and membrane insertion. Little is known about factors that assist these processes for multispanning membrane proteins such as the ABC transporters. A second major focus of this proposal is to identify cellular components involved in the folding and assembly of STE6 by: 5) isolation of ste6 mutants whose loss of function is due to rapid degradation, and 6) subsequent isolation of suppressors which restore stability to an unstable STE6 mutant protein; these suppressors are expected to identify molecular chaperones and foldases which execute folding, assembly, and intracellular transport of STE6. These experiments will provide a high resolution view of the function, folding, and trafficking of STE6, that will also be relevant to other ABC proteins. This information can provide insight into preventing multi-drug resistance to chemotherapeutic agents by MDR, and into treating cystic fibrosis, which results from misfolded or misfunctional CFTR.
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0.958 |
1997 — 2000 |
Michaelis, Susan D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Biogenesis and Function of Yeast a Factor @ Johns Hopkins University
DESCRIPTION: Dr. Michaelis is studying the biogenesis of the S. Cerevisiae mating pheromone a-factor. She will explore the hypothesis that a-factor biogenesis may be carried out by a modular or multicomponent "a-factor biogenesis machine. Her long-term goal is to define at high resolution the dynamic events that occur during a-factor biogenesis and to understand how these events are coordinated and regulated.
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0.958 |
1999 — 2002 |
Michaelis, Susan D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Folding and Activity of Abc Proteins in Yeast @ Johns Hopkins University
DESCRIPTION (taken from the application) Mammalian CFTR and the Saccharomyces cerevisiae a mating pheromone transporter Ste6p are both members of the ATP binding cassettes (ABC) superfamily. In this project, we will use Ste6p as a model for investigation the biogenesis structure, and activity of CFTR. Because these two proteins share a common overall design, they are likely to require similar cellular machinery to ensure their proper membrane insertion, folding, and activity. The most prevalent CF allele, delta F508, causes misfolding of the mutant CFTR protein, resulting in its recognition by the ER quality control machinery, ER retention, and subsequent degradation by the ubiquitin- proteasome system. Little is known in the folding and biogenesis of secretory and membrane proteins or that recognize when they are not properly folded. One long-term goal of this project is to utilize the power of yeast genetics to identify cellular components that assist in and monitor the proper folding of ABC proteins. A second long-term goal is to purify and reconstitute Ste6p in active form to determine its biochemical properties. We will use genetic, molecular, and biochemical approaches to accomplish the following specific aims: 1) Isolate suppressors that stabilize an ER-retained, rapidly degraded mutant form of Ste6p identified in the previous project period; such suppressors will genetically pinpoint components of the cellular machinery involved in the membrane insertion, folding, and ER quality control of an ABC protein. 2) Determine how the ER quality control machinery distinguishes folded and unfolded cytosolic subdomains of a membrane protein, suing a C-terminal. Step6p truncation series and a "designer chimera". 3) Purify and functionally reconstitute His-tagged Ste6p into phospholipid vesicles and examine ATPase activity, substrate specificity, and transport activity. The biochemical properties of purified Ste6p, CFTR, and MDR, will be compared in collaboration with P. Maloney, and colleagues. 4) To establish a second yeast CFTR model (ER-retained Ycf1p) that can be used in addition to Ste6p to dissect the ER quality control pathway. We are optimistic that much of the basic knowledge we acquired about yeast Ste6p will apply to human CFTR, and in the long-term will provide a firm foundation for developing directed chemical and physiological strategies to revitalize the defective gene product in CF patients.
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0.958 |
2000 — 2003 |
Michaelis, Susan D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Synthesis and Function of the Yeast Abc Transporter Ste6 @ Johns Hopkins University
DESCRIPTION (from applicant's Abstract) The proper intracellular folding and trafficking of membrane proteins, including transporters, channels, and receptors are key events that contribuute to their function. Ste6p, the mating pheromone a-factor transporter in yeast, is being used as a model protein for dissecting the trafficking steps of mulitspanning membrane proteins. Ste6p is a member of the ATP binding cassette (ABC) superfamily that includes the cystic fibrosis protein CFTR, multidrug resistance transporters, and related proteins important in human health and disease. Studies proposed here focus on two aspects of Ste6p trafficking that represent key regulatory points for a plasma membrane protein: 1) ER quality control, a process intertwined with ER exit, and 2) endocytosis. The applicant cites several advances in the previous project period: a) The identification of distinct classes of ER-retained ste6 mutants, which revealed considerable complexity in ER quality control; b) Discovery of a prominent novel structure, the ER-associated body (ERAB) that is formed in response to certain ste6 mutants and may provide clues about ER quality control; c) Demonstration of a role for ubiquitin both in ER quality control and endocytosis of Ste6p. These findings set the stage for the present goal of identifying cellular components involved in "decision making" aspects of ER exit and endocytosis. The renewal lists the following aims: 1) Isolate chromosomal suppressors and enhancers of ER-retained ste6 mutants to identify cellular components that participate in ER quality control and/or ER exit. 2) Identify cellular components enriched within ERABs or induced upon their formation, by biochemical, microscopic, and microarray analysis. 3) Establish by mutant studies an ordered pathway of events (phosphorylation -> ubiquitination -> endocytosis (PUE pathway) required for Ste6p internalization, and genetically identify novel Ste6p endocytosis specficity factors. 4) Compare the requirements for ubqiuitination leading to ER-associated degradation (ERAD) of mutant Ste6p versus endocytosis of WT Ste6p. Human membrane protein "trafficking diseases" are numerous. The applicant hopes that these efforts to identify cellular components involved in trafficking will provide targets for rational drug design for these and related diseases.
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0.958 |
2005 — 2010 |
Michaelis, Susan D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Trafficking of Abc Proteins in Yeast @ Johns Hopkins University
DESCRIPTION (provided by applicant): ER "quality control" (ERQC) is a fundamental and conserved cellular process that prevents the exit of misfolded secretory and membrane proteins from the ER. ERQC consists of two sequential processes: 1) the unfolded protein response (UPR), which refers to the transcriptional upregulation of genes such as chaperones that enable the cell to cope with misfolded proteins, followed by 2) ER-associated degradation (ERAD), whereby misfolded ER-retained proteins are degraded by the ubiquitin-proteasome system. Recent studies suggest that for membrane proteins there are two classes of ERAD substrates, based on the topological location of their misfolded lesion, either luminal (L) or cytosolic (C). In the present project, we propose an extension of this view, namely that cells employ two mechanistically distinct branches of ER quality control: ERQC-L (comprising UPR-L and ERAD-L) and ERQC-C (comprising UPR-C and ERAD-C), to cope with substrates whose domains are luminal or cytosolic, respectively. The long-term goal of this project is to identify and mechanistically dissect the components and workings of the ERQC-C pathway in Saccharomyces cerevisiae, and determine how ERQC-C differs from ERQC-L. Evidence for distinct branches of ERQC is based on our studies of mutant forms of the yeast ATP-binding cassette (ABC) transporters Ste6p and Ycflp, that are subject to ERQC. Using these as model ERQC-C substrates, we have made significant advances in this project that include defining a prominent ER compartment (the ERAC) as a marker for UPR-C, defining differences in machinery between the ERAD-C and ERAD-L, and gaining an initial glimpse into differences in the transcriptional induction profiles of UPR-C and UPR-L. These findings set the stage for the present proposal. Here, we will apply traditional and high-throughput yeast genetic, molecular, and cell biological methodologies to accomplish the following aims: 1) To elucidate the circuitry of the UPR-C signaling pathway by defining the key regulators, upregulated genes, and cytoprotective mechanisms evoked by a UPR-C stress; 2) To define the machinery, steps, and mechanism of the ERAD-C pathway by probing the substrate specificity of E3 ubiquitin ligases and identifying novel ERAD components; and 3) To further develop MRP proteins as model ERQC substrates, in particular to gain new insights into the relationship between ERQC and ER exit. Our studies are expected to shed light on a diverse array of membrane protein trafficking diseases, best exemplified by cystic fibrosis, which most commonly results from the ER-retention and degradation of CFTR-deltaF508.
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0.958 |
2008 — 2011 |
Michaelis, Susan D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Lamin a Biogenesis, Processing and Progeria @ Johns Hopkins University
DESCRIPTION (provided by applicant): This project focuses on the biogenesis of Lamin A, a critical structural component of the nuclear envelope. Surprisingly, recent studies have revealed that mutations in the gene encoding Lamin A result in a wide range of diseases called the "laminopathies" (encompassing cardiomyopathy, muscular dystrophy, lipodystrophy, and progeroid disorders), leading to a resurgence of interest in the biology of Lamin A. The Lamin A precursor, prelamin A, undergoes a series of post-translational processing events, including: 1) C- terminal CaaX modification (prenylation, proteolysis, and carboxyl methylation), followed by 2) an endoproteolytic cleavage event, mediated by the zinc metalloprotease ZmpSte24, that removes the CaaX- modified C-terminus to yield mature Lamin A. In previous years of this project, our laboratory discovered ZmpSte24 as a key enzyme in the biogenesis of yeast a-factor. From these findings this project has evolved to study the biogenesis of Lamin A in mammalian cells. The most severe laminopathy is the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS). Strikingly, there appears to be a direct link between defective Zmpste24-mediated endoproteolytic processing of prelamin A and progeroid diseases based on the findings that: 1) HGPS results from a mutation in which the ZmpSte24 cleavage site within Lamin A is deleted, and 2) the progeroid disorders mandibuloacral dysplasia (MAD) and restrictive dermopathy (RD) map to ZmpSte24. Thus, it appears that the persistently prenylated form of Lamin A that is present in HGPS or zmpste24-/- cells leads to accelerated aging pathologies. In this proposal we will define key cell biological aspects of Lamin A processing and address the role that a lack of processing plays in disease mechanisms. We will use molecular, cell biological, genetic, and biochemical approaches to address the following aims: Aim 1) determine the cellular location of Lamin A processing (nucleoplasmic vs. the cytosolic face of the ER);Aim 2) determine the fate of the cleaved C- terminal tail;Aim 3) determine the recognition sequences within prelamin A important for ZmpSte24 cleavage, and define how Zmpste24 cleaves prelamin A using purified enzyme;and Aim 4) investigate the molecular mechanisms by which failure to cleave the prelamin A tail leads to cellular and disease phenotypes. In particular we will test whether methylation may contribute to the toxicity of Lamin A in HGPS. The intriguing finding that progeroid diseases are caused by incomplete processing of prelamin A has underscored the importance of a comprehensive understanding of the entire processing pathway, which we address in this proposal. Our studies will provide insight into therapeutic options for progeroid disorders. The significance of this research is heightened by recent findings that inhibition of ZmpSte24-mediated processing of prelamin A may contribute to HIV therapy-induced side effects, and possibly to the mechanisms of normal aging. PUBLIC HEALTH RELEVANCE: Mutations in the nuclear structural protein Lamin A cause the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) and a spectrum of diseases known as "laminopathies". This project addresses fundamental unanswered questions about Lamin A biology, including how it is processed within the cell and how abnormal processing can cause disease, as in HGPS. Our studies will provide insight into potential therapeutic options for premature aging disorders, and may also shed light on the mechanisms underlying the normal aging process.
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0.958 |
2014 — 2017 |
Michaelis, Susan D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Integral Membrane Protease Zmpste24, Lamin a Processing, and Progeria @ Johns Hopkins University
DESCRIPTION (provided by applicant): 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.
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0.958 |
2018 — 2019 |
Michaelis, Susan D. Worman, Howard J. (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Role of Permanently Farnesylated Prelamin in the Cardiovascular Disease of Aging @ Johns Hopkins University
PROJECT SUMMARY The most important determinant of cardiovascular health is a person's age, as the risk of cardiovascular disease (CVD) increases significantly as we grow old. In this project, we will generate a new mutant Lmna mouse, as a model to probe the role of permanently farnesylated prelamin A in driving the CVD of physiological aging. LMNA encodes prelamin A, the precursor of the nuclear scaffold protein lamin A. Normally, prelamin A undergoes farnesylation and subsequent proteolytic cleavage by the protease ZMPTE24 that removes the farnesylated C-terminal portion of the protein. In the premature aging disorder Hutchinson- Gilford Progeria Syndrome (HGPS), an internally deleted (?50aa) form of prelamin A called progerin that remains permanently farnesylated causes disease phenotypes, including CVD. However, HGPS mouse models may not effectively model the CVD of physiological aging in unaffected individuals, because progerin is produced in vanishingly small amounts and the ?50aa deletion may impart novel binding properties to this prelamin A variant. On the other hand, compelling recent studies provide evidence that prelamin A, normally a transiently expressed farnesylated precursor that is rapidly converted to mature lamin A, accumulates in vascular smooth muscle cells of aged, but not young, individuals and in atherosclerotic lesions. Although the observed accumulation of farnesylated prelamin A in the vasculature of old human subjects is intriguing, there is no mouse model to directly assess experimentally the role of this protein in the development of CVD. The existing Zmpste24-/- mouse is not ideal for such studies because this enzyme has another critical cellular function besides prelamin A processing, which may confound analysis. Instead, to test the hypothesis that permanently farnesylated prelamin A promotes accelerated CVD, we propose to generate mice with a LmnaL648R mutation. The L648R amino acid substitution abolishes the ZMPSTE24 cleavage recognition site, leading to the accumulation of permanently farnesylated full-length prelamin A, essentially the same species seen in aging vessels. In Aim 1, we will generate knock-in LmnaL648R mice and analyze them for multiple organismal and cellular phenotypes associated with progeria and physiological aging. In Aim 2, we will characterize the development of vascular pathology in heterozygous and homozygous LmnaL648R mice by performing a longitudinal study of vascular stiffness using non-invasive pulse-wave velocity testing over their lifetimes. We will also perform interventional and ex vivo assessments of vascular mechanics, vasoreactivity, vasomechanics and vascular pathology. This R21 proposal involves development ? generation of a novel mouse model to study the potential role of prelamin A in CVD of aging ? and exploration ? studying these mice for the progression of CVD as they age. The potential for reward is huge, as these studies could pave the way for a paradigm-shifting understanding of CVD, the most common cause of morbidity and mortality for old Americans.
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0.958 |
2018 — 2021 |
Michaelis, Susan D. |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
The Integral Membrane Protease Zmpste24, Lamin a Processing, and the Premature Aging Disease Progeria @ Johns Hopkins University
Project Summary This project focuses on ZMPSTE24, an intriguing 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 block cleavage cause the severe premature aging disorder Hutchinson- Gilford Progeria syndrome (HGPS) and a set of related progeroid diseases in which an aberrant permanently farnesylated form of lamin A is the ?molecular culprit? that promotes aging-related symptoms. Importantly, diminished prelamin A processing by ZMPSTE24 may also be a critical factor in normal physiological aging. My laboratory pioneered the study of ZMPSTE24 in the early years of this project. We discovered this protease in yeast, where it is called Ste24, and showed that it has two distinct proteolytic activities in the biogenesis of the mating pheromone a-factor (cleavage of the CAAX motif and a second upstream cleavage). Importantly, we demonstrated that mammalian ZMPSTE24 performs these same cleavages in prelamin A maturation. Through this sustained body of work, together with a powerful new humanized yeast system for prelamin A cleavage recently developed in my laboratory, and our recent work showing a quality control role for ZMPSTE24 in removing misfolded proteins from ?clogged? translocons, we have at our fingertips a full arsenal of tools, including a variety of biochemical and in vivo assays, cell lines, and disease alleles that will facilitate the proposed studies. The recently published structure of human ZMPSTE24 and the nearly superimposable yeast Ste24 revealed surprising features, defining ZMPSTE24/Ste24p as truly novel class of intramembrane proteases. The seven transmembrane spans of ZMPSTE24/Ste24p form a voluminous water- filled intramembrane chamber with the zinc metalloprotease catalytic site facing the chamber interior, so that substrate access is restricted and must occur through one of several side portals. One major challenge in the field is to establish how this novel intramembrane protease works. We propose to mechanistically dissect human ZMPSTE24 and its substrate prelamin A to define precisely how proteolysis of prelamin A occurs inside of an intramembrane chamber. We will identify features of ZMPSTE24 and prelamin A important for substrate recognition, entry, binding, catalysis, and product release, and will determine the step at which ZMPSTE24 and lamin A disease alleles malfunction. We will also deploy powerful designer screens possible in yeast to identify new Ste24 substrates that may shed light on its mechanism. We will begin an exciting new series of studies aimed at exploring a second major challenge, namely whether and how diminished ZMPSTE24 activity and prelamin A accumulation may drive physiological aging. Together, these studies will reveal fundamental principles relevant to intramembrane protease biology, premature aging, and normal physiological aging. 1
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0.958 |