Vishwanath Rao Lingappa, MD PhD - US grants

This proposal combined recombinant DNA technology with the use of reconstituted cell-free systems towards the analysis of key problems in protein topogenesis (the processes by which newly synthesized proteins are transported to their correct subcellular locations). Using recombinant expression plasmids containing restriction endonuclease directed deletions in genes for eukaryotic secretory proteins as a source of mRNA we shall use a transcription linked, translation coupled translocation system to define rigorously several currently debated concepts involving the location and structure of signal sequences. By constructing expression plasmids of non-secretory proteins into which sections of signal sequence encoding DNA have been inserted (either intact or altered by site directed mutagenesis) we will determine the functional domains within wignal sequences and try to elucidate the mechanisms by which they function. If these experiments uphold our working hypothesis that discrete polypeptide sequences are primary determinants of protein localization, we shall initiate the construction of a 'topogeneic library', containing DNA segments whose insertion into the coding region of the gene for any protein will alter that protein's topogenic fate on expression in vivo or in vitro. Through this work, a more precise molecular understanding of topogenic sequences will emerge: 1) the functional domains within topogenic sequences will be identified; 2) the rules by which gene recombination may confer new locations (topogenic fates) to proteins will be determined; 3) a variety of currently debated issues in the field of protein topogenesis will be resolved. These studies would be an invaluable adjunct to future attempts to selectively (re)direct any particular newly synthesized protein to any chosen intracellular compartment by reprogramming its complement of topogenic sequences.

DESCRIPTION: (Adapted from the Investigator's abstract): The investigators propose to investigate apolipoprotein B (apo B) biogenesis and apo B-containing lipoprotein particle formation. Apo B plays a major role in cholesterol transport. Hypercholesterolemia is a risk factor for atherosclerosis and coronary artery disease. Some patients with elevated serum cholesterol have a known defect in apo B structure. Many others have uncharacterized aberrations in metabolism of apo B containing lipoproteins, which may be related to defective assembly. An understanding of how these diverse lesions cause hyperlipoproteinemia will require detailed knowledge of the mechanism by which a normal lipoprotein particle is assembled and secreted. Yet, important features of lipoprotein particle assembly are poorly understood and not easily explained by current value of protein biogenesis and trafficking. Recently, the investigators have discovered unusual intermediates in the early events of apo B translocation across the endoplasmic reticulum membrane (see Preliminary Studies) by expression of cloned cDNAs encoding domains of apo B in both membrane-supplemented cell-free systems and in living cells. These intermediates will be characterized and related to stages in apo B-containing lipoprotein particle assembly, regulation and secretion. This analysis will be extended to later events in apo B biogenesis. Through these experiments a better understanding of apo B biogenesis and its relationship to lipoprotein particle formation will emerge. New insight into the mechanisms by which apo B may be post-translationally regulated under physiologic conditions and in hypercholesterolemic lipoprotein disorders of humans may follow.

In recent years, progress has been made in dissecting the molecular events of prion protein (PrP) biogenesis. A novel topogenic sequence, termed the Stop Transfer Effector (STE), which directs nascent PrP in cell-free systems to either a doubly transmembrane or a secretory topology, has been identified. The choice between these topologic fates was shown to depend on the presence of a cytosolic factor. An intermediate with features of both topologic forms has been identified in vivo. Pathways by which alternate topologic fates and rapid ER degradation may occur have been identified. However, the relationship of these events to scrapie remains unknown. We propose to explore the role of this novel topogenic sequence in scrapie pathogenesis. The STE sequence will be mutagenized and the effects of mutations on PrP biogenesis and scrapie pathogenesis investigated. Mutants which alter steps in PrP biogenesis will be selected by cell-free transcription- linked translation and Xenopus oocyte microinjection. Some of these will be used to identify receptors and molecular chaperones with which nascent PrP interacts. Selected mutants will be studied in transgenic mice in order to determine if a relationship exists between unusual events in prion biogenesis and the pathogenesis of scrapie. Finally mutants transfected into N2a cells will be used to probe the effect of molecular chaperones on PrP biogenesis and parameters of scrapie infection.

DESCRIPTION (Applicant's abstract): Prison diseases encompass a heterogeneous group of transmissible and spontaneous disorders. Altered forms of the prison protein (PrP) appear to play a central role in their pathogenesis. In this transmissible prison disorders, a protease-resistant form termed PrPSC accumulates in the brain and appears to be a component of the agent of the disease. However, in the generic prison disorders, PrPSC is often not detected. Instead, new data suggests that a transmembrane form of PrP (termed ctmPrP) is involved in triggering neurodegeneration in genetic prison diseases, and that a protective factor in brain normally prevents ctmPrP expression. The applicant proposes to establish a defined framework, using transgenic mice and derived cell lines, to study the role of both ctmPrP and host genes with which it interacts. The applicant will test the hypotheses that ctmPrP is involved at an early step in the pathway of cellular pathology of all prison diseases, and that transmission of disease and neuronal cell death are independent features of prison disorders. The applicant will attempt to develop new biochemical assays for the molecular mechanisms involved in one or more of the classical features of selected prison diseases, such as incubation time, and the nature, extent and location of neurodegeneration. Through this work, a novel paradigm of prison disease pathogenesis will be established and specific hypotheses on the role of host genes that influence ctmPrP will be tested. The proposed studies set the stage for the future manipulation of host factors in ways that may protect cells from prison disease.

At the endoplasmic reticulum (ER) membrane, nascent prion protein (PrP) can be synthesized in both fully translocated and transmembrane forms, as a result of the action of Translocation Accessory Factors (TrAFs). The relevance of these observations has recently become apparent with the demonstration of spontaneous neurodegenerative disease in both mice and humans expressing PrP mutations that favor synthesis and export of transmembrane PrP from the ER. Some evidence suggests that the expression of neurodegeneration -associated transmembrane PrP may be a default outcome in the absence of TrAF activity. The investigation of topological regulation during PrP biogenesis is crucial for an understanding the molecular pathogenesis of these genetic prion diseases. Here we propose to dissect the mechanisms by which PrP biogenesis affects PrP topology disease. Here we propose to dissect the mechanism by which PrP biogenesis affects PrP topology, resulting in an transmembrane form that leads to neurodegeneration. Mutations in PrP structural domains will be systematically correlated to effects on topological regulation. Trafficking of selected mutants will be studied in transfected cells. Transgenic mouse model systems will be developed for those mutants with the most interesting phenotypes. Protein-protein interactions between the translocation machinery and nascent PrP will be identified and correlated with the pathway of neurodegeneration. Through this work, the pathway of PrP biogenesis, as it relates to a newly recognized pathway of neurodegeneration, will be better understood in molecular terms. In the long run these studies should make possible novel approaches to manipulation of PrP topology and may contribute to prevention or treatment of prion diseases and other neurodegenerative disease.

[unreadable] DESCRIPTION (provided by applicant): The prion protein (PrP) is a conserved glycoprotein of vertebrates that is involved in a group of spongiform neurodegenerative disorders, collectively termed prion diseases, some of which pose a substantial and currently unmet public health hazard around the globe. Hence a better understanding of PrP's role in prion disease pathogenesis is paramount. [unreadable] [unreadable] Prion diseases include overlapping sets of disorders that vary as to whether, and to what degree, they are inherited (genetic), infectious (transmissible), or seemingly spontaneous (sporadic) in etiology. These diverse manifestations suggest that important relationships between PrP and other genes involved in the development, maintenance, or function of the nervous system remain to be elucidated. Thus, the study of prion disease pathogenesis may be a powerful probe of otherwise currently intractable dimensions of neurobiology. [unreadable] [unreadable] PrP has also been of interest because of its remarkable biogenesis. Initially homogeneous nascent PrP chains are synthesized as distinctive forms, termed conformers that are identical in primary amino acid sequence but different in both transmembrane topology and intrinsic folding. A complex machinery has been implicated in the genesis of two of the PrP conformers, termed SecPrP and CtmPrP, and a surprising role for signal sequences in directing this process has been identified. [unreadable] [unreadable] Independent lines of inquiry converged with the demonstration that the unusual features of PrP biogenesis are central to its role in genetic prion disease. From studies carried out over the past 4 years with support of the present grant, major progress has been made in understanding the conformer termed PrP, and its role in prion disease pathogenesis. In particular it has been established that CtmPrP also plays a crucial role in infectious as well as genetic prion disease and brings about neurodegeneration by triggering a pathway of apoptosis. Furthermore, powerful model systems, including cell culture and Tg mice that reproduce key features of prion disorders, have been developed and are beginning to be used for dissection of the disease-associated apoptotic pathway involving CtmPrP. [unreadable] [unreadable] In part as a result of the progress in the present grant period, important new questions regarding CtmPrP and other PrP conformers have emerged. In this renewal application we propose to build on our previous studies, utilizing the full range of tools and reagents that we have developed over the past four years, for the study of PrP conformers in disease. Based on our recent demonstration of their utility in detection of individual conformers, we will also generate conformer-specific mAbs optimized for immunocytochemistry. Using these tools we will: i) corroborate the Ctm index in other model systems of prion disease and explore the possibility of variations on this theme in triggering or suppressing apoptosis; ii) elucidate the precise interactions by which CtmPrP triggers apoptosis; iii) address the role of posttranslational modifications of PrP conformers in prion disease, including the putative role of cleavage of the signal peptide as a regulator of conformer production/export, iv) attempt to detect NtmPrP and explicate its role, if any, in prion disease, and v) identify the signaling pathway(s) by which PrPSc appears to trigger CtmPrP production. [unreadable] [unreadable] Through these studies, an ongoing fruitful line of investigation with regards to PrP-mediated neurodegeneration will be extended, providing a greater degree of clarity and precision with regards to the role of CtmPrP, SecPrP, and NtmPrP in prion disease. In addition, exciting new directions regarding the regulation of PrP conformers will be explored, setting the stage for the development of potential therapeutic modalities based on enhanced or suppressed production and maturation of PrP conformers. [unreadable] [unreadable]

A distinctive feature of prion protein (PrP) biogenesis, which appears central to an understanding of prion disease pathophysiology, is the observation that nascent PrP is synthesized in the form of at least three distinctive conformers that also differ in transmembrane topology. A growing body of evidence suggests that one of these, termed (Ctm)PrP, triggers a final common pathway of apoptosis occurring in response to both genetic and infectious prion disease, while another conformer, termed (Sec)PrP, is anti-apoptotic and neuroprotective. Work over the last decade has revealed a number of means by which the mix of conformers synthesized from an initially homogeneous population of nascent PrP chains can be altered. In particular, the signal sequence, and a charged region termed the Stop Transfer Effector (STE) sequence immediately preceding the transmembrane region, have been demonstrated to be sequence determinants of PrP topology. More recent data suggests that: i) other regions besides the signal sequence and STE sequence may contribute to regulation of PrP transmembrane topology and conformation, ii) interaction of distinct domains within PrP are involved in the protein's topogenesis, and iii) mapping of protein-protein interactions during translocation may provide useful information for conformer manipulation. Here we propose studies designed to i) explore new topogenic sequences and their interactions in cis, and ii) identify the partner proteins with which nascent PrP is involved in trans. We will then use this information to better understand the mechanism by which individual PrP conformers are generated, and the relationship of those mechanisms to signaling in infectious scrapie. In the long run, this work may also make possible the development of novel approaches to treatment of prion disease through conformer manipulation.

[unreadable] DESCRIPTION (provided by applicant): The prion protein (PrP) is a conserved glycoprotein of vertebrates that is involved in a group of spongiform neurodegenerative disorders, collectively termed prion diseases, some of which pose a substantial and currently unmet public health hazard around the globe. Hence a better understanding of PrP's role in prion disease pathogenesis is paramount. [unreadable] [unreadable] Prion diseases include overlapping sets of disorders that vary as to whether, and to what degree, they are inherited (genetic), infectious (transmissible), or seemingly spontaneous (sporadic) in etiology. These diverse manifestations suggest that important relationships between PrP and other genes involved in the development, maintenance, or function of the nervous system remain to be elucidated. Thus, the study of prion disease pathogenesis may be a powerful probe of otherwise currently intractable dimensions of neurobiology. [unreadable] [unreadable] PrP has also been of interest because of its remarkable biogenesis. Initially homogeneous nascent PrP chains are synthesized as distinctive forms, termed conformers that are identical in primary amino acid sequence but different in both transmembrane topology and intrinsic folding. A complex machinery has been implicated in the genesis of two of the PrP conformers, termed SecPrP and CtmPrP, and a surprising role for signal sequences in directing this process has been identified. [unreadable] [unreadable] Independent lines of inquiry converged with the demonstration that the unusual features of PrP biogenesis are central to its role in genetic prion disease. From studies carried out over the past 4 years with support of the present grant, major progress has been made in understanding the conformer termed PrP, and its role in prion disease pathogenesis. In particular it has been established that CtmPrP also plays a crucial role in infectious as well as genetic prion disease and brings about neurodegeneration by triggering a pathway of apoptosis. Furthermore, powerful model systems, including cell culture and Tg mice that reproduce key features of prion disorders, have been developed and are beginning to be used for dissection of the disease-associated apoptotic pathway involving CtmPrP. [unreadable] [unreadable] In part as a result of the progress in the present grant period, important new questions regarding CtmPrP and other PrP conformers have emerged. In this renewal application we propose to build on our previous studies, utilizing the full range of tools and reagents that we have developed over the past four years, for the study of PrP conformers in disease. Based on our recent demonstration of their utility in detection of individual conformers, we will also generate conformer-specific mAbs optimized for immunocytochemistry. Using these tools we will: i) corroborate the Ctm index in other model systems of prion disease and explore the possibility of variations on this theme in triggering or suppressing apoptosis; ii) elucidate the precise interactions by which CtmPrP triggers apoptosis; iii) address the role of posttranslational modifications of PrP conformers in prion disease, including the putative role of cleavage of the signal peptide as a regulator of conformer production/export, iv) attempt to detect NtmPrP and explicate its role, if any, in prion disease, and v) identify the signaling pathway(s) by which PrPSc appears to trigger CtmPrP production. [unreadable] [unreadable] Through these studies, an ongoing fruitful line of investigation with regards to PrP-mediated neurodegeneration will be extended, providing a greater degree of clarity and precision with regards to the role of CtmPrP, SecPrP, and NtmPrP in prion disease. In addition, exciting new directions regarding the regulation of PrP conformers will be explored, setting the stage for the development of potential therapeutic modalities based on enhanced or suppressed production and maturation of PrP conformers. [unreadable] [unreadable]