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Title: Interactome Analyses Identify Ties of PrPC and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones}
Article:
Abstract:
The physiological environment which hosts the conformational conversion of the cellular prion protein( PrPC) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrPC interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel( Prnd) and Shadoo( Sprn), two mammalian PrPC paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrPC and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases( PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrPC with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrPSc. A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrPC organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.
Introduction:
Prions are the causative agents underlying a range of rare but invariably fatal neurodegenerative diseases in humans and other mammals. In disease, the cellular prion protein( Prnp; herein referred to as PrPC) converts to a disease-associated conformer( PrPSc) with different physicochemical properties[1]. PrPC is a relatively small protein which assembles into an unstructured N-terminal domain and a globular C-terminal half characterized by the presence of an internal disulfide bridge, up to two N-linked glycans and a glycosylphosphatidylinositol( GPI) anchor for insertion into the cellular plasma membrane. Multiple lines of investigation have led to the conclusion that mature PrPC is embedded in specialized membrane domains, so-called raft-like domains, rich in cholesterol and sphingolipids[2]. It has been suggested that these raft-like domains host the self-perpetuated accumulation of PrPSc which subsequently triggers a poorly-understood cascade of events that ultimately leads to cell death. Whereas significant progress has been made in the past few years in defining the minimal requirements for PrPC conversion in vitro[3], the molecular environment which hosts the earliest steps in prion disease manifestation in neuronal cells remains enigmatic. This shortcoming is not due to a lack of proteins proposed to interact directly with PrPC. In fact, more than three dozen proteins have been suggested to reside in spatial proximity to PrPC using multiple experimental paradigms[4]. Additional candidate interactors have been proposed to bind preferentially to PrPSc[5],[6],[7]. In surveying this body of literature, however, it is apparent that very few of the candidate interactors have been independently verified by multiple investigators and, overall, little agreement exists as to their relative importance for prion protein biology. Notable exceptions may represent the 37-kDa/67-kDa laminin receptor precursor( herein referred to as LRP; also known as ribosomal protein SA( Rpsa), and not to be confused with Lrp1, the low density lipoprotein receptor-related protein 1)[8], one of the first proteins identified to bind to the prion protein in a yeast two-hybrid( Y2H) screen[9]; the neural cell adhesion molecule( Ncam1; herein referred to as NCAM), which was initially identified in a cellular crosslinking study based on a co-immunoprecipitation methodology[10]; and heparin sulfate proteoglycans( HSPGs)[11],[12]. Since its original discovery, LRP has been proposed to act as a cell surface receptor for PrPC that may play a role in prion propagation[13]. In the case of NCAM, binding to PrP has been shown to play a role in neuritic outgrowth possibly mediated through an interaction with Fyn tyrosine kinase[14],[15]. In studying the molecular environment of membrane proteins, a limitation exists in that the proteins must be solubilized by the addition of detergents in a manner that does not disrupt the protein-protein interactions under investigation. A solution to this obstacle constitutes the covalent stabilization of interactions by chemical crosslinking prior to the disruption of cellular integrity. In this regard, we previously reported on a large-scale investigation of the molecular PrPC neighborhood in mice following limited in vivo crosslinking by time-controlled transcardiac perfusion( tcTPC)-based delivery of formaldehyde( FA) to the brain[16]. A conspicuous feature of this PrPC interactome dataset was the relative abundance of membrane proteins which harbor immunoglobulin( Ig)-like folding motifs in their extracellular domains confirming previous data describing a binding domain of PrPC within these folds[10]. Given the large diversity of cell types present in the brain, we questioned whether an equivalent concentration of Ig-like domain-harboring proteins would have been found if the investigation of the PrPC interactome had been restricted to a single cell type. A particular concern was that the relative abundance of Ig-like domain-harboring proteins in the previous dataset may have masked the ability to identify biologically important interactions of PrPC that are more transient in nature or involve less abundant proteins. We therefore chose to reinvestigate the PrPC interactome in mouse neuroblastoma cells( N2a), which are by far the best-characterized cell model for the study of the biology and conversion of PrPC to PrPSc[17],[18]. To facilitate the discrimination of unspecific binders from specific interactors in this study, we incorporated quantitative mass spectrometry based on isotopic labeling and extended our investigation to the mammalian PrPC paralogs Doppel( encoded by the Prnd gene; protein product herein referred to as Dpl) and Shadoo( encoded by the Sprn gene; protein product herein referred to as Sho). To avoid the use of non-identical affinity chromatography steps, we equipped the three bait proteins with the same N-terminal FLAG epitope[19], with the awareness that a similar PrPC expression construct had in prior investigations neither interfered with the posttranslational processing nor the conversion of PrPC in cell or animal models[18]. We now present data which suggest that members of the mammalian prion protein family may populate highly similar molecular environments when expressed in the neuroblastoma cell model system. We further document that a subset of endoplasmic reticulum( ER) chaperones which interact strongly with PrPC escape from the ER to reside in spatial proximity to PrPC at the plasma membrane. Pharmacological inhibition of these chaperones could increase PrPSc levels in prion-infected N2a sublines, suggesting a protective role for the chaperones in this paradigm. Finally, our data consolidate multiple previously controversial interactions and suggest a scenario whereby PrPC may organize its molecular environment by its ability to recognize a specialized subset of cell adhesion molecules which recruit membrane proteins carrying high-mannose glycans into spatial proximity with PrPC.
Discussion:
Taken together, this work suggests that members of the prion protein family may be embedded in specialized membrane domains characterized by an extended molecular network which supports functions in adhesion control, lactate metabolism and cell fusion events. Our investigation leads to the conclusion that biochemical interactions among the members of the mammalian prion protein family exist. It confirms multiple candidate interactors of PrPC in a unified paradigm including an independent verification of the laminin receptor precursor( LRP) as an authentic PrP interactor. It thereby not only reconciles a fragmented literature, but should facilitate future efforts to further define the relative contribution of these interactors to the biology of PrPC. Novel interactors uncovered in this study paint a coherent picture based on which an argument for the spatial proximity of NCAM, basigin, Na/K ATPase and LRP can be made, even independent of their ability to bind to PrPC. Moreover, data presented here raise many new questions as to( i) whether biological outputs of PrPC implied by the “guilt-by-association” logic will be confirmed,( ii) the importance of oligomannosidic N-glycans for the structural integrity and assembly of the molecular environment of PrPC,( iii) the role which a subset of cellular chaperones may play in PrP biology and disease, and( iv) the exact environment which hosts the conversion of PrPC. It is hoped that this work will stimulate investigations which will not only resolve these questions, but lead to insights from which a therapeutic strategy for addressing prion diseases can be derived.
Prions underlie rare but invariably fatal neurodegenerative diseases in humans and other mammals . Public awareness of these diseases has grown with the occurrence of cases of BSE ( also known as “Mad Cow Disease” ) and the realization that this disease can , in rare instances , be transmitted to humans . The normal cellular prion protein is found in most cell types within the body . In disease , this protein acquires a different shape which tends to aggregate and poison nearby cells . The disease-associated conversion of the prion protein appears to require its localization to specialized cellular membrane regions rich in cholesterol; however , the precise molecular environment which hosts this event has remained elusive . We used a cell-based disease model to identify proteins which reside in close proximity to the prion protein and two closely related mammalian proteins . We demonstrate that these three proteins may not only populate highly similar environments but may also interact with each other . We further identified an extended molecular network in proximity of these proteins which supports functions in adhesion control , lactate metabolism and cell fusion events . It is anticipated that these insights will contribute to efforts to rationally interfere with aberrant protein-protein interactions underlying these diseases .