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The Gerace Laboratory

Our laboratory is working to obtain a molecular understanding of basic questions in cell biology and how these relate to function in animals at the organismal level. A main focus of our research involves the nuclear envelope (NE), a specialized domain of the endoplasmic reticulum (ER) that forms the compartmental boundary of the nucleus.  
 
In one research topic, we are studying the functions of the nuclear lamina, a protein meshwork that lines the nucleoplasmic surface of the NE. Mutations in lamina components have been linked to host of human genetic diseases ranging from muscular dystrophies to premature aging. Current projects involve the study of mechanisms by which lamina-associated transmembrane proteins regulate signaling and differentiation, with particular focus on the biology of muscle, a tissue that is frequently affected by lamina mutations. 
 
In a second research area, we are investigating mechanisms for nucleocytoplasmic trafficking of proteins and RNA and the related topic of posttranscriptional gene regulation, which is closely intertwined with nuclear export of mRNA.  As a model system for this work, we are investigating host cell proteins that interact with the HIV Rev protein. Rev is key to the virus life cycle, since it mediates trafficking and expression of unspliced HIV-1 mRNA, which encodes the viral genome and structural proteins. 

Nuclear lamina and regulation of signaling
 
The nuclear lamina serves as an attachment site at the NE both for the cytoplasmic cytoskeleton and for chromatin, and has a well-recognized role in the mechanical integrity of the nucleus.  Recently, it has become clear that that lamina also is a platform for regulation of signaling within the nucleus. research image The lamina negatively regulates a multitude of signaling pathways, including those involving MAP kinases, TFG-b/Smad, Wnt/b catenin, and mTOR.  The major emphasis of our recent work has involved transmembrane proteins concentrated at the inner nuclear membrane and associated with the lamina, which are emerging as key signaling regulators. We identified over 50 novel NE transmembrane proteins (NETs) in a proteomics screen, and recently have characterized three of these NETs that are highly
expressed in muscle and that have essential functions in cultured myoblast differentiation. We are dissecting the functions of these proteins in myogenic cells and in mouse models in the context of muscle differentiation and homeostasis.
 
We found that one of these NETs, Lem2, attenuates ERK1/2 signaling at the onset of differentiation in cultured myoblasts.  Moreover, we determined that Lem2 has an overlapping function with emerin, a structurally unrelated inner nuclear membrane protein implicated in Emery-Dreifuss muscular dystrophy that also is implicated in ERK regulation.  We found that disruption of the Lem2 gene in mouse leads to embryonic lethality at the stage of heart formation, supporting the possibility that Lem2 has an essential function in heart as well as in skeletal muscle. A second protein we have analyzed, Net37, has a luminal glycosidase domain whose activity is required for myoblast differentiation. Cells in which Net37 is silenced do not secrete IGF-II, an essential autocrine factor for myoblast differentiation. Net37 is associated with pro-IGF-II in cells, and we postulate that it serves as a folding chaperone essential for the maturation of this fundamental hormone.  The NE-restricted localization of Net37 contrasts with the uniform ER localization seen for all other ER folding chaperones in the secretory pathway.  This raises the intriguing possibility that Net37 is regulated by transmembrane signaling from the nucleoplasm to the ER lumen.  A third protein we characterized recently, Net39, binds mTOR and negatively regulates its activity.  The properties of Net39 suggest a role as part of a negative feedback loop to attenuate the transcription of muscle-specific genes following terminal differentiation. 
 
Nucleocytoplasmic transport and posttranscriptional gene regulation
 
Nuclear import and export of proteins and RNAs occurs through nuclear pore complexes 
(NPCs), %7e100 MDa protein assemblies that provide transport channels across the NE. research image 2Transport of protein cargoes and many RNAs is mediated by nuclear transport receptors of the karyopherin family. The binding of cargoes to karyopherins is regulated by the small GTPase Ran, which in its GTP-bound state dissociates cargoes from import
receptors and promotes cargo binding to export receptors. Whereas the proteins of the NPC and mobile components of the nuclear transport machinery have been characterized in considerable detail and X-ray structures have been obtained for representative components, the conformational changes that dictate discrete steps of transport are still poorly understood.  As a chemical biology strategy to this problem, we have identified small molecule inhibitors of importin b-mediated import.  One of these, karyostatin 1A, selectively inhibits the importin b pathway in living cells by blocking the interaction of importin b to RanGTP, and is a promising tool for future mechanistic studies of nuclear transport.
 
In a related project, as a component of the CHEETAH Center for analysis of host cell factors involved in the biology of HIV-1, we are analyzing the mechanisms by which the HIV Rev protein performs its essential late functions in HIV replication. Rev promotes export and translation of unspliced HIV-1 transcripts, which encode the viral structural proteins, and also enhances genome packaging.  r3Rev mediates it functions by binding and oligomerizing along the 350 nt Rev Response Element (RRE), to recruit the classical export receptor Crm1 and other factors to RRE-containing transcripts.  In an effort to characterize the broader set of Rev functions related to HIV RNA, we carried out a proteomics screen to identify host cell proteins that bind to Rev in complex with the RRE.  This revealed ~100 novel candidate Rev cofactors with specific binding, including 5 RNA helicases and 1 ubiquitin E3 ligase. We have shown that a number of these novel factors are required for HIV replication using RNAi, and are in the process of analyzing the posttranscriptional networks in which they function.

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