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When injected into genetically modified mice, aggregates of the amyloid-beta (A beta) peptide from the brains of Alzheimers disease (AD) patients or transgenic AD mouse models seed cerebral A beta deposition in a prion-like fashion. Within the brain, A beta exists as a pool of distinct C-terminal variants with lengths ranging from 37 to 43 amino acids, yet the relative contribution of individual C-terminal A beta variants to the seeding behavior of A beta aggregates remains unknown. Here, we have investigated the relative seeding activities of A beta aggregates composed exclusively of recombinant A beta 38, A beta 40, A beta 42, or A beta 43. Cerebral A beta 42 levels were not increased in App(NL-F) knock-in mice injected with A beta 38 or A beta 40 aggregates and were only increased in a subset of mice injected with A beta 42 aggregates. In contrast, significant accumulation of A beta 42 was observed in the brains of all mice inoculated with A beta 43 aggregates, and the extent of A beta 42 induction was comparable to that in mice injected with brain-derived A beta seeds. Mice inoculated with A beta 43 aggregates exhibited a distinct pattern of cerebral A beta pathology compared to mice injected with brain-derived A beta aggregates, suggesting that recombinant A beta 43 may polymerize into a unique strain. Our results indicate that aggregates containing longer A beta C-terminal variants are more potent inducers of cerebral A beta deposition and highlight the potential role of A beta 43 seeds as a crucial factor in the initial stages of A beta pathology in AD. Funding Agencies|Canadian Institutes of Health ResearchCanadian Institutes of Health Research (CIHR) [MOP-136899, PJT-173497]; Alzheimer Society Canada/Brain Canada [16-13]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2016-00748]

The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.

The amyloid cascade hypothesis posits that the initiating event in Alzheimer’s disease (AD) is the aggregation and deposition of the β-amyloid (Aβ) peptide, which is a proteolytic cleavage product of the amyloid precursor protein (APP). Mounting evidence suggests that the formation and spread of prion-like Aβ aggregates during AD may contribute to disease progression. Inoculation of transgenic mice that overexpress APP with pre-formed Aβ aggregates results in the prion-like induction of cerebral Aβ deposition. To determine whether Aβ deposition can also be induced when physiological APP levels are present in the brain, we inoculated AppNL-F mice, a knock-in model of AD that avoids potential artifacts associated with APP overexpression, with Aβ aggregates derived from the brains of AD patients or transgenic mice. In all cases, induced Aβ deposition was apparent in the corpus callosum, olfactory bulb, and meningeal blood vessels of inoculated mice at 130–150 days post-inoculation, whereas uninoculated and buffer-inoculated animals exhibited minimal or no Aβ deposits at these ages. Interestingly, despite being predominantly composed of protease-resistant Aβ42 aggregates, the induced parenchymal Aβ deposits were largely diffuse and were unreactive to an amyloid-binding dye. These results demonstrate that APP overexpression is not a prerequisite for the prion-like induction of cerebral Aβ deposition. Accordingly, spreading of Aβ deposition may contribute to disease progression in AD patients. Electronic supplementary material The online version of this article (10.1186/s40478-018-0529-x) contains supplementary material, which is available to authorized users.

The prion protein (PrP) is best known for its ability to cause fatal neurodegenerative diseases in humans and animals. Here, we revisited its molecular environment in the brain using a well-developed affinity-capture mass spectrometry workflow that offers robust relative quantitation. The analysis confirmed many previously reported interactions. It also pointed toward a profound enrichment of Na,K-ATPases (NKAs) in proximity to cellular PrP (PrPC). Follow-on work validated the interaction, demonstrated partial co-localization of the ATP1A1 and PrPC, and revealed that cells exposed to cardiac glycoside (CG) inhibitors of NKAs exhibit correlated changes to the steady-state levels of both proteins. Moreover, the presence of PrPC was observed to promote the ion uptake activity of NKAs in a human co-culture paradigm of differentiated neurons and glia cells, and in mouse neuroblastoma cells. Consistent with this finding, changes in the expression of 5’-nucleotidase that manifest in wild-type cells in response to CG exposure can also be observed in untreated PrPC-deficient cells. Finally, the endoproteolytic cleavage of the glial fibrillary acidic protein, a hallmark of late-stage prion disease, can also be induced by CGs, raising the prospect that a loss of NKA activity may contribute to the pathobiology of prion diseases.

Protein interactions of Tau are of interest in efforts to decipher pathogenesis in Alzheimer’s disease, a subset of frontotemporal dementias, and other tauopathies. We CRISPR-Cas9 edited two human cell lines to generate broadly adaptable models for neurodegeneration research. We applied the system to inducibly express balanced levels of 3-repeat and 4-repeat wild-type or P301L mutant Tau. Following 12-h induction, quantitative mass spectrometry revealed the Parkinson’s disease-causing protein DJ-1 and non-muscle myosins as Tau interactors whose binding to Tau was profoundly influenced by the presence or absence of the P301L mutation. The presence of wild-type Tau stabilized non-muscle myosins at higher steady-state levels. Strikingly, in human differentiated co-cultures of neuronal and glial cells, the preferential interaction of non-muscle myosins to wild-type Tau depended on myosin ATPase activity. Consistently, transgenic P301L Tau mice exhibited reduced phosphorylation of regulatory myosin light chains known to activate this ATPase. The direct link of Tau to non-muscle myosins corroborates independently proposed roles of Tau in maintaining dendritic spines and mitochondrial fission biology, two subcellular niches affected early in tauopathies.

Somatostatin (SST) is a cyclic peptide that is understood to inhibit the release of hormones and neurotransmitters from a variety of cells by binding to one of five canonical G protein-coupled SST receptors (SSTR1 to SSTR5). Recently, SST was also observed to interact with the amyloid beta (Aβ) peptide and affect its aggregation kinetics, raising the possibility that it may bind other brain proteins. Here we report on an SST interactome analysis that made use of human brain extracts as biological source material and incorporated advanced mass spectrometry workflows for the relative quantitation of SST binding proteins. The analysis revealed SST to predominantly bind several members of the P-type family of ATPases. Subsequent validation experiments confirmed an interaction between SST and the sodium-potassium pump (Na+/K+-ATPase) and identified a tryptophan residue within SST as critical for binding. Functional analyses in three different cell lines indicated that SST might negatively modulate the K+ uptake rate of the Na+/K+-ATPase.

The fold of a protein determines its function and its misfolding can result in loss-of-function defects. In addition, for certain proteins their misfolding can lead to gain-of-function toxicities resulting in protein misfolding diseases such as Alzheimer's, Parkinson's, or the prion diseases. In all of these diseases one or more proteins misfold and aggregate into disease-specific assemblies, often in the form of fibrillar amyloid deposits. Most, if not all, protein misfolding diseases share a fundamental molecular mechanism that governs the misfolding and subsequent aggregation. A wide variety of experimental methods have contributed to our knowledge about misfolded protein aggregates, some of which are briefly described in this review. The misfolding mechanism itself is difficult to investigate, as the necessary timescale and resolution of the misfolding events often lie outside of the observable parameter space. Molecular dynamics simulations fill this gap by virtue of their intrinsic, molecular perspective and the step-by-step iterative process that forms the basis of the simulations. This review focuses on molecular dynamics simulations and how they combine with experimental analyses to provide detailed insights into protein misfolding and the ensuing diseases.

Alzheimer’s disease is associated with the formation of toxic aggregates of amyloid beta (Aβ) peptides. Despite tremendous efforts, our understanding of the molecular mechanisms of aggregation, as well as cofactors that might influence it, remains incomplete. The small cyclic neuropeptide somatostatin-14 (SST14) was recently found to be the most selectively enriched protein in human frontal lobe extracts that binds Aβ42 aggregates. Furthermore, SST14’s presence was also found to promote the formation of toxic Aβ42 oligomers in vitro. In order to elucidate how SST14 influences the onset of Aβ oligomerization, we performed all-atom molecular dynamics simulations of model mixtures of Aβ42 or Aβ40 peptides with SST14 molecules and analyzed the structure and dynamics of early-stage aggregates. For comparison we also analyzed the aggregation of Aβ42 in the presence of arginine vasopressin (AVP), a different cyclic neuropeptide. We observed the formation of self-assembled aggregates containing the Aβ chains and small cyclic peptides in all mixtures of Aβ42–SST14, Aβ42–AVP, and Aβ40–SST14. The Aβ42–SST14 mixtures were found to develop compact, dynamically stable, but small aggregates with the highest exposure of hydrophobic residues to the solvent. Differences in the morphology and dynamics of aggregates that comprise SST14 or AVP appear to reflect distinct (1) regions of the Aβ chains they interact with; (2) propensities to engage in hydrogen bonds with Aβ peptides; and (3) solvent exposures of hydrophilic and hydrophobic groups. The presence of SST14 was found to impede aggregation in the Aβ42–SST14 system despite a high hydrophobicity, producing a stronger “sticky surface” effect in the aggregates at the onset of Aβ42–SST14 oligomerization., Author summary Improper folding of proteins causes disorders known as protein misfolding diseases. Under normal conditions most proteins adopt particular folds, which allow them functioning properly. However, for reasons that are not yet fully understood, proteins may misfold and aggregate, forming deposits known as amyloid fibrils, which accumulate in the brain or other tissues. This process affects functioning of the nervous system, gradually causing loss of cognitive abilities. Alzheimer’s disease is one of the most common diseases from this group. A better understanding of the aggregation of peptides implicated in Alzheimer’s disease, known as amyloid beta (Aβ) peptides, may facilitate the development of treatments that ameliorate or prevent the disease. We use detailed molecular dynamics simulations to investigate the influence of somatostatin-14 (SST14), a cyclic neuropeptide that might be involved in the amyloidogenic aggregation of Aβ, on molecular processes occurring at the onset of Aβ aggregation. Results of these simulations explain how the presence of SST14 might alter pathways of aggregation of Aβ, shedding light upon the possible role of extrinsic factors in the aggregation at a molecular level.

Currently, a new revolutionary genome-editing tool is opening new avenues for gene engineering. It is known as the clustered regularly interspaced short palindromic repeats (CRISPR)11 and the CRISPR-associated (Cas) 9 system. In general, the CRISPR-Cas system has been evolved in archaea and bacteria as part of their adaptive immune mechanisms. Mechanistic aspects of the system can be found in the literature. Among the 3 CRISPR-Cas system types that were found in these organisms, the type II system in Streptococcus pyogenes is the most widely applied. The type II (CRISPR-Cas9) system includes the RNA-guided Cas9 nuclease, which binds to specific DNA sequences (complementary to the RNA-guide sequence) and creates double-stranded breaks on the DNA. The dsDNA breaks can be repaired via homology-directed repair (HDR) or nonhomologous end-joining (NHEJ). Based on this principle, the Cas9 and the guide-RNA were modified in various ways to improve the efficiency and specificity of this system, to expand its potential for different applications. This system can be used for altering specific genetic loci through insertions, deletions, point mutations, and sequence inversions. More recently, the system was modified to act as a genome regulator, by tethering effector domains to the Cas9 or guide-RNA, and as a visualization tool by fusing with marker molecules. This multiplex capacity of engineering CRISPR-Cas9 enabled scientists to apply this system for genome modifications in a variety of organisms, like Arabidopsis, Drosophila, Caenorhabditis elegans, zebrafish, mosquitoes, mice, primates, and humans. Lately, the CRISPR-Cas9 gene editing has been used in human embryos and generated several ethical questions and concerns. In this Q&A, 5 experts from around the world discuss the capabilities of the CRISPR-Cas9 system in editing genomes and discuss the associated ethical concerns. The interest for using the CRISPR-Cas9 system to targeted genome editing is rapidly emerging. The efficiency …

The amyloid β peptide (Aβ) is a key player in the etiology of Alzheimer disease (AD), yet a systematic investigation of its molecular interactions has not been reported. Here we identified by quantitative mass spectrometry proteins in human brain extract that bind to oligomeric Aβ1-42 (oAβ1-42) and/or monomeric Aβ1-42 (mAβ1-42) baits. Remarkably, the cyclic neuroendocrine peptide somatostatin-14 (SST14) was observed to be the most selectively enriched oAβ1-42 binder. The binding interface comprises a central tryptophan within SST14 and the N-terminus of Aβ1-42. The presence of SST14 inhibited Aβ aggregation and masked the ability of several antibodies to detect Aβ. Notably, Aβ1-42, but not Aβ1-40, formed in the presence of SST14 oligomeric assemblies of 50 to 60 kDa that were visualized by gel electrophoresis, nanoparticle tracking analysis and electron microscopy. These findings may be relevant for Aβ-directed diagnostics and may signify a role of SST14 in the etiology of AD. DOI: http://dx.doi.org/10.7554/eLife.28401.001, eLife digest Treating Alzheimer’s disease and related dementias is one of the major challenges currently facing healthcare providers worldwide. A hallmark of the disease is the formation of large deposits of a specific molecule, known as amyloid beta (Aβ), in the brain. However, more and more research suggests that smaller and particularly toxic amyloid beta clumps – often referred to as oligomeric Aβ – appear as an early sign of Alzheimer’s disease. To understand how the formation of these smaller amyloid beta clumps triggers other aspects of the disease, it is important to identify molecules in the human brain that oligomeric Aβ binds to. To this end, Wang et al. attached amyloid beta or oligomeric Aβ molecules to microscopically small beads. The beads were then exposed to human brain extracts in a test tube, which allowed molecules in the extracts to bind to the amyloid beta or oligomeric Aβ. The samples were then spun at high speed, meaning that the beads and any other molecules bound to them sunk and formed pellets at the bottom of the tubes. Each pellet was then analyzed to see which molecules it contained. The experiments identified more than a hundred human brain proteins that can bind to amyloid beta. One of them, known as somatostatin, selectively binds to oligomeric Aβ. Wang et al. were able to determine the structural features of somatostatin that control this binding. Finally, in further experiments performed in test tubes, Wang et al. noticed that smaller oligomeric Aβ clumps were more likely to form than larger amyloid beta deposits when somatostatin was present. This could signify a previously unrecognized role of somatostatin in the development of Alzheimer’s disease. Further studies are now needed to confirm whether the presence of somatostatin in the brain favors the formation of smaller, toxic oligomeric Aβ clumps over large innocuous amyloid beta deposits. If so, new treatments could be developed that aim to reduce oligomeric Aβ levels in the brain by preventing somatostatin from interacting with amyloid beta molecules. Wang et al. also suggest that somatostatin could be used in diagnostic tests to detect abnormal levels of oligomeric Aβ in the brain or body fluids of people who have Alzheimer’s disease. DOI: http://dx.doi.org/10.7554/eLife.28401.002

The low resolution structure of a protein can sometimes be inferred from information about existing disulfide bridges or experimentally introduced chemical crosslinks. Frequently, this task involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently linked peptides. To facilitate this task, we developed a method for the enrichment of covalently linked peptides following the chemical cleavage of a protein. The method capitalizes on the availability of homoserine lactone moieties at the C-termini of cyanogen bromide cleavage products which support selective conjugation of affinity tags. The availability of two C-termini within covalently linked peptides allows for the conjugation of two distinct affinity tags and thereby enables subsequent removal of unmodified peptides by tandem affinity chromatography. Here, we demonstrate the stepwise implementation of this method using a polyhistidine tag and a biotin tag for the selective two-step purification of covalently linked cyanogen bromide fragments from increasingly complex protein samples. The method is independent of the nature of the covalent bond, is adaptable to fully denaturing conditions, and requires only low picomole quantities of starting material.

High resolution peptide separation is pivotal for successful shot-gun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3 – 10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides) while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88 % of all peptides binned to a single fraction, as compared to 61 % of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.

Despite intense research efforts, the physiological function and molecular environment of the amyloid precursor protein has remained enigmatic. Here we describe the application of time-controlled transcardiac perfusion cross-linking, a method for the in vivo mapping of protein interactions in intact tissue, to study the interactome of the amyloid precursor protein (APP). To gain insights into the specificity of reported protein interactions the study was extended to the mammalian amyloid precursor-like proteins (APLP1 and APLP2). To rule out sampling bias as an explanation for differences in the individual datasets, a small scale quantitative iTRAQ (isobaric tags for relative and absolute quantitation)-based comparison of APP, APLP1, and APLP2 interactomes was carried out. An interactome map was derived that confirmed eight previously reported interactions of APP and revealed the identity of more than 30 additional proteins that reside in spatial proximity to APP in the brain. Subsequent validation studies confirmed a physiological interaction between APP and leucine-rich repeat and Ig domain-containing protein 1, demonstrated a strong influence of Ig domain-containing protein 1 on the proteolytic processing of APP, and consolidated similarities in the biology of APP and p75.

A few well-characterized protein assemblies aside, little is known about the topology and interfaces of multiconstituent protein complexes. Here we report on a novel indirect strategy for low-resolution topology mapping of protein complexes. Following crosslinking, purified protein complexes are subjected to chemical cleavage with cyanogen bromide (CNBr) and the resulting fragments are resolved by 2-D electrophoresis. The side-by-side comparison of a thus generated and a 2-D CNBr fragment map obtained from uncrosslinked material reveals candidate gel spots harboring crosslinked CNBr fragments. In-gel trypsinization and MALDI MS analysis of these informative spots identify the underlying crosslinked CNBr fragments based on unmodified tryptic peptides. Matching the cumulative theoretical molecular mass and predicted pI of these crosslinked CNBr fragments with original gel spot coordinates is required for confident crosslink assignment. The above strategy was successfully validated with the Escherichia coli RNA polymerase (RNAP) core complex and subsequently applied to query the quaternary structure of components of the yeast Skp1-Cdc53/Cullin-F box (SCF) ubiquitin ligase complex. This protocol requires low picomole sample quantities, can be applied to multisubunit protein complexes, and does not rely on specialized data mining software.

The cellular prion protein, PrP(C), is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP ('DeltaPrP'). This paradigm has facilitated mapping of activity determinants in PrP(C) and implicated a cryptic PrP(C)-like protein, 'pi'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrP(C), but is low in cerebellar granular neurons (CGNs) containing PrP(C) and high in PrP(C)-deficient dendritic processes. In Prnp(0/0) CGNs, Sho transgenes were PrP(C)-like in their ability to counteract neurotoxic effects of either Doppel or DeltaPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for pi, and since it engenders a PrP(C)-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis.

The cellular prion protein, notorious for its causative role in a range of fatal neurodegenerative diseases, evolved from a Zrt-/Irt-like Protein (ZIP) zinc transporter approximately 500 million years ago. Whilst atomic structures for recombinant prion protein (PrP) from various species have been available for some time, and are believed to stand for the structure of PrPC, the first structure of a ZIP zinc transporter ectodomain was reported only recently. Here, we compare this ectodomain structure to structures of recombinant PrP. A shared feature of both is a membrane-adjacent helix-turn-helix fold that is coded by a separate exon in the respective ZIP transporters and is stabilized by a disulfide bridge. A ‘CPALL’ amino acid motif within this cysteine-flanked core domain appears to be critical for dimerization and has undergone stepwise regression in fish and mammalian prion proteins. These insights are intriguing in the context of repeated observations of PrP dimers. Other structural elements of ZIP transporters and PrP are discussed with a view to distilling shared versus divergent biological functions.

The molecular function of the cellular prion protein (PrPC) and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer's disease are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate guide RNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of>3000 proteins (data deposited to ProteomeXchange Consortium) were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ∼ 120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton.

To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.

Cellular prion protein (PrP(C)) misfolds to form infectivity-associated scrapie prion protein and generates C-terminal fragments C1 and C2 in healthy and prion-infected animals. C1 cleavage occurs N-terminally of PrP(C)'s hydrophobic domain, whereas the larger C2 fragment is generated by cleavage at the end of the octarepeat region. As the PrP-like proteins Doppel and Shadoo (Sho) have been reported to inhabit similar membrane environments as PrP(C), we investigated endoproteolysis by using a panel of mutant alleles. Doppel undergoes efficient in vivo cleavage at a C1 site mapped to the start of the globular domain, which is a structurally similar cleavage site to that in PrP(C). Sho is processed to C1 and C2 fragments, and proved refractory to mutagenesis to inactivate C1 cleavage. As a reciprocal product of C1 cleavage, Sho also engenders a metabolically stable N1 fragment with a C-terminus after its hydrophobic domain, an observation that may account for N1's association with membrane and/or cellular fractions in vitro and in vivo. Our data indicate that glycosylation status and yet to be identified proteases modulate internal C1 and C2 proteolysis events within the mammalian prion protein family.

The time-controlled transcardiac perfusion crosslinking (tcTPC) method differs from conventional perfusion fixation in that the crosslinking reagent is administered throughout the circulatory system for only a relatively short period of time, thereby allowing limited crosslinking to occur. Bait protein complexes are isolated by affinity capture (AC) under stringent conditions and are recovered from the AC matrix by acidic elution. Affinity-purified proteins are reduced, alkylated, and digested with a specific endoproteinase, such as trypsin. Subsequently, peptides are isotopically labeled, separated by reversed-phase chromatography and analyzed by quantitative tandem mass spectrometry (MS/MS). The proteins crosslinked to the bait protein during tcTPC are identified by database searches with conventional protein identification software. The tcTPC strategy offers unique advantages over alternative approaches for studying a subset of protein complexes which require a particular environment for their structural integrity, such as membrane protein complexes that are notorious for their tendency to dissociate upon detergent solubilization. The sensitivity and utility of this method are influenced by the spatial distribution of chemical groups within the bait protein complexes that can engage in productive crosslinks.

During prion infections of the central nervous system (CNS) the cellular prion protein, PrPC, is templated to a conformationally distinct form, PrPSc. Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrPC and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp a and Prnp b mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrPSc and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrPSc. Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrPSc accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis., Author Summary In prion infections of the nervous system the cellular prion protein, PrPC, changes to a distinct form, PrPSc. Recent studies have demonstrated that another glycoprotein Shadoo (Sho), which occupies a similar membrane environment as PrPC, is reduced in the brains of rodents with terminal prion disease. Our analyses of prion-infected mice revealed that reduction of Sho protein was not due to reductions in the corresponding messenger RNA. Reduction in Sho was clearly evident upon propagation of a variety of prion strains, but was not seen in mice with other types of neurodegenerative disease. Also, as prion-infected mice with only one copy of the PrP gene exhibited both accumulation of PrPSc and a reduction of Sho protein hundreds of days prior to onset of neurologic symptoms, the drop in Sho protein level can be excluded as an important trigger for clinical disease, or a non-specific consequence of brain cell damage. Instead, our studies define a effect restricted to prion disease and we hypothesize that Sho protein is a “bystander” for degradative processes aimed at destroying PrPSc.

The cellular prion protein PrP(C) refolds into a beta-sheet enriched, infectivity-associated form called PrP(Sc). Shadoo (Sho) is a newly discovered glycoprotein that is also expressed in the adult brain. Wild type (wt) mouse Sho consists of an arginine-rich region, a hydrophobic central domain of five tandem A/LAAG amino acid repeats R1-R5 with similarity to the hydrophobic domain of PrP(C), and a C-terminal domain with one N-linked carbohydrate. As some alanine-rich proteins and PrP with a shortened C-terminal domain form amyloid we investigated conformational properties of wt Sho and polymorphic variants with insertion/deletions centered on R3. Recombinant mouse and sheep Sho converted to an amyloid-like form without recourse to chemical denaturation or acidification. For wt proteins this transition was marked by increased thioflavin T binding, Congo red staining, presence of fibrillar structures by electron microscopy, formation of sodium dodecyl sulfate-resistant complexes and the generation of a C-terminal proteinase K resistant core of 5-8 kDa. Variant Sho proteins differing within the R1-R5 region exhibited most but not all of these properties. Our studies define a proteinase K -resistant signature fragment for the amyloid fold of Sho and raise the question of a physiological role for this form of the wt protein.

TMP21 has been shown to be associated with the gamma-secretase complex and can specifically regulate gamma-cleavage without affecting epsilon-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of gamma-secretase activity was investigated independent of epsilon-cleavage using an amyloid-beta precursor proteinepsilon (APPepsilon) construct which lacks the amyloid intracellular domain domain. The APPepsilon construct behaves similarly to the full-length precursor protein with respect to alpha- and beta-cleavages and is able to undergo normal gamma-processing. Co-expression of APPepsilon and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Abeta-positive fragments, consistent with an inhibition of gamma-secretase cleavage. The APPepsilon system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the gamma-secretase complex and could decrease gamma-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Abeta production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased gamma-cleavage activity.

Publisher Summary This chapter reviews the methodological advancements and applications aimed at the study of interactomes and protein complex topologies in cells and tissues. The choice of crosslinking reagent can strongly influence the methodology and outcome of any crosslinking experiment. A choice needs to be made with respect to the tether length of the reagent and the nature of the reactive groups that mediate the intended covalent crosslink. For more specialized applications, it may be helpful for the crosslinking reagent to be equipped with built-in functionalities that facilitate the enrichment of crosslinked products, allow reversal of crosslinks downstream in the sample purification procedure, and provide a means to quantify crosslinks. A variety of strategies are currently available for mapping protein interactions. Techniques range from genetic approaches—such as the two-hybrid system (THS), synthetic lethal screens, fluorescence resonance energy transfer (FRET), and the quantitative genetic interaction mapping method—to biochemical protocols that reveal the composition of a protein complex by immunoprecipitation or affinity-tag purification and mass spectrometry (MS). Crosslinking-based interactome studies are well suited to complement some of the purely genetic interaction mapping approaches that may point at functional interactions without establishing direct contacts of proteins.

The study of protein-protein interactions involving endogenous proteins frequently relies on the immunoaffinity capture of a protein of interest followed by mass spectrometry-based identification of co-purifying interactors. A notorious problem with this approach is the difficulty of distinguishing physiological interactors from unspecific binders. Additional challenges pose the need to employ a strategy that is compatible with downstream mass spectrometry and minimizes sample losses during handling steps. Finally, the complexity of data sets demands solutions for data filtering. Here we present an update on co-immunoprecipitation procedures for sensitive interactome mapping applications. We define the relevant terminology, review methodological advances that reduce sample losses, and discuss experimental strategies that facilitate recognition of candidate interactors through a combination of informative controls and data filtering. Finally, we provide starting points for initial validation experiments and propose conventions for manuscripts which report on co-immunoprecipitation work.

Presenilin 1 or presenilin 2, nicastrin, APH-1, and PEN-2 form high molecular weight complexes that play a pivotal role in the cleavage of various Type I transmembrane proteins, including the beta-amyloid precursor protein. The specific function of PEN-2 is unclear. To explore its function and intermolecular interactions, we conducted deletion and mutagenesis studies on a series of conserved residues at the C terminus of PEN-2. These studies suggest that: 1) both the presence and amino acid sequence of the conserved DYLSF domain at the C terminus of PEN-2 (residues 90-94) is critical for binding PEN-2 to other components in the presenilin complex and 2) the overall length of the exposed C terminus is critical for functional gamma-secretase activity.