ABSTRACT
The quest to determine the function of a protein can represent a profound challenge. Although this task is the mandate of countless research groups, a general framework for how it can be ...approached is conspicuously lacking. Moreover, even expectations for when the function of a protein can be considered to be ‘known’ are not well defined. In this review, we begin by introducing concepts pertinent to the challenge of protein function assignments. We then propose a framework for inferring a protein's function from four data categories: ‘inheritance’, ‘distribution’, ‘interactions’ and ‘phenotypes’ (IDIP). We document that the functions of proteins emerge at the intersection of inferences drawn from these data categories and emphasise the benefit of considering them in an evolutionary context. We then apply this approach to the cellular prion protein (PrPC), well known for its central role in prion diseases, whose function continues to be considered elusive by many investigators. We document that available data converge on the conclusion that the function of the prion protein is to control a critical post‐translational modification of the neural cell adhesion molecule in the context of epithelial‐to‐mesenchymal transition and related plasticity programmes. Finally, we argue that this proposed function of PrPC has already passed the test of time and is concordant with the IDIP framework in a way that other functions considered for this protein fail to achieve. We anticipate that the IDIP framework and the concepts analysed herein will aid the investigation of other proteins whose primary functional assignments have thus far been intractable.
The bank vole (BV) prion protein (PrP) can function as a universal acceptor of prions. However, the molecular details of BVPrP's promiscuity for replicating a diverse range of prion strains remain ...obscure. To develop a cultured cell paradigm capable of interrogating the unique properties of BVPrP, we generated monoclonal lines of CAD5 cells lacking endogenous PrP but stably expressing either hamster (Ha), mouse (Mo), or BVPrP (M109 or I109 polymorphic variants) and then challenged them with various strains of mouse or hamster prions. Cells expressing BVPrP were susceptible to both mouse and hamster prions, whereas cells expressing MoPrP or HaPrP could only be infected with species‐matched prions. Propagation of mouse and hamster prions in cells expressing BVPrP resulted in strain adaptation in several instances, as evidenced by alterations in conformational stability, glycosylation, susceptibility to anti‐prion small molecules, and the inability of BVPrP‐adapted mouse prion strains to infect cells expressing MoPrP. Interestingly, cells expressing BVPrP containing the G127V prion gene variant, identified in individuals resistant to kuru, were unable to become infected with prions. Moreover, the G127V polymorphic variant impeded the spontaneous aggregation of recombinant BVPrP. These results demonstrate that BVPrP can facilitate cross‐species prion replication in cultured cells and that a single amino acid change can override the prion‐permissive nature of BVPrP. This cellular paradigm will be useful for dissecting the molecular features of BVPrP that allow it to function as a universal prion acceptor.
CAD5 cells lacking endogenous prion protein (PrP) expression (CAD5‐PrP−/−) were stably transfected with vectors encoding expression of wild‐type bank vole PrP or bank vole PrP containing the G127V substitution. Cells were challenged with either mouse or hamster prions and then passaged several times. Expression of bank vole PrP facilitated cross‐species prion infection, as revealed by the presence of proteinase K‐resistant PrP in cell lysates. In contrast, the addition of the protective G127V polymorphism to bank vole PrP completely blocked the ability of cells to become infected with mouse or hamster prions.
Corruption of cellular prion protein (PrP
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) function(s) at the plasma membrane of neurons is at the root of prion diseases, such as Creutzfeldt-Jakob disease and its variant in humans, and Bovine ...Spongiform Encephalopathies, better known as mad cow disease, in cattle. The roles exerted by PrP
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, however, remain poorly elucidated. With the perspective to grasp the molecular pathways of neurodegeneration occurring in prion diseases, and to identify therapeutic targets, achieving a better understanding of PrP
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roles is a priority. Based on global approaches that compare the proteome and metabolome of the PrP
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expressing 1C11 neuronal stem cell line to those of PrP
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-1C11 cells stably repressed for PrP
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expression, we here unravel that PrP
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contributes to the regulation of the energetic metabolism by orienting cells towards mitochondrial oxidative degradation of glucose. Through its coupling to cAMP/protein kinase A signaling, PrP
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tones down the expression of the pyruvate dehydrogenase kinase 4 (PDK4). Such an event favors the transfer of pyruvate into mitochondria and its conversion into acetyl-CoA by the pyruvate dehydrogenase complex and, thereby, limits fatty acids β-oxidation and subsequent onset of oxidative stress conditions. The corruption of PrP
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metabolic role by pathogenic prions PrP
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causes in the mouse hippocampus an imbalance between glucose oxidative degradation and fatty acids β-oxidation in a PDK4-dependent manner. The inhibition of PDK4 extends the survival of prion-infected mice, supporting that PrP
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-induced deregulation of PDK4 activity and subsequent metabolic derangements contribute to prion diseases. Our study posits PDK4 as a potential therapeutic target to fight against prion diseases.
There is growing evidence that zinc and its transporters are involved in cell migration during development and in cancer. In the present study, we show that zinc transporter ZIP10 (SLC39A10) ...stimulates cell motility and proliferation, both in mammalian cells and in the zebrafish embryo. This is associated with inactivation of GSK (glycogen synthase kinase)-3α and -3β and down-regulation of E-cadherin (CDH1). Morpholino-mediated knockdown of zip10 causes delayed epiboly and deformities of the head, eye, heart and tail. Furthermore, zip10 deficiency results in overexpression of cdh1, zip6 and stat3, the latter gene product driving transcription of both zip6 and zip10 The non-redundant requirement of Zip6 and Zip10 for epithelial to mesenchymal transition (EMT) is consistent with our finding that they exist as a heteromer. We postulate that a subset of ZIPs carrying prion protein (PrP)-like ectodomains, including ZIP6 and ZIP10, are integral to cellular pathways and plasticity programmes, such as EMT.
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.
Traditional drug development for Alzheimer's disease (AD) is costly, time consuming and burdened by a very low success rate. An alternative strategy is drug repositioning, redirecting existing drugs ...for another disease. The large amount of biological data accumulated to date warrants a comprehensive investigation to better understand AD pathogenesis and facilitate the process of anti-AD drug repositioning. Hence, we generated a list of anti-AD protein targets by analyzing the most recent publically available 'omics' data, including genomics, epigenomics, proteomics and metabolomics data. The information related to AD pathogenesis was obtained from the OMIM and PubMed databases. Drug-target data was extracted from the DrugBank and Therapeutic Target Database. We generated a list of 524 AD-related proteins, 18 of which are targets for 75 existing drugs-novel candidates for repurposing as anti-AD treatments. We developed a ranking algorithm to prioritize the anti-AD targets, which revealed CD33 and MIF as the strongest candidates with seven existing drugs. We also found 7 drugs inhibiting a known anti-AD target (acetylcholinesterase) that may be repurposed for treating the cognitive symptoms of AD. The CAD protein and 8 proteins implicated by two 'omics' approaches (ABCA7, APOE, BIN1, PICALM, CELF1, INPP5D, SPON1, and SOD3) might also be promising targets for anti-AD drug development. Our systematic 'omics' mining suggested drugs with novel anti-AD indications, including drugs modulating the immune system or reducing neuroinflammation that are particularly promising for AD intervention. Furthermore, the list of 524 AD-related proteins could be useful not only as potential anti-AD targets but also considered for AD biomarker development.
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.
Mycobacteria use a unique system for covalently modifying proteins based on the conjugation of a small protein, referred to as prokaryotic ubiquitin‐like protein (PUP). In this study, we report a ...proteome‐wide analysis of endogenous pupylation targets in the model organism Mycobacterium smegmatis. On affinity capture, a total of 243 candidate pupylation targets were identified by two complementary proteomics approaches. For 41 of these protein targets, direct evidence for a total of 48 lysine‐mediated pupylation acceptor sites was obtained by collision‐induced dissociation spectra. For the majority of these pupylation targets (38 of 41), orthologous genes are found in the M. tuberculosis genome. Interestingly, approximately half of these proteins are involved in intermediary metabolism and respiration pathways. A considerable fraction of the remaining targets are involved in lipid metabolism, information pathways, and virulence, detoxification and adaptation. Approximately one‐third of the genes encoding these targets are located in seven gene clusters, indicating functional linkages of mycobacterial pupylation targets. A comparison of the pupylome under different cell culture conditions indicates that substrate targeting for pupylation is rather dynamic.
The tau protein is central to the etiology of several neurodegenerative diseases, including Alzheimer's disease, a subset of frontotemporal dementias, progressive supranuclear palsy and dementia ...following traumatic brain injury, yet the proteins it interacts with have not been studied using a systematic discovery approach. Here we employed mild in vivo crosslinking, isobaric labeling, and tandem mass spectrometry to characterize molecular interactions of human tau in a neuroblastoma cell model. The study revealed a robust association of tau with the ribonucleoproteome, including major protein complexes involved in RNA processing and translation, and documented binding of tau to several heat shock proteins, the proteasome and microtubule-associated proteins. Follow-up experiments determined the relative contribution of cellular RNA to the tau interactome and mapped interactions to N- or C-terminal tau domains. We further document that expression of P301L mutant tau disrupts interactions of the C-terminal half of tau with heat shock proteins and the proteasome. The data are consistent with a model whereby a higher propensity of P301L mutant tau to aggregate may reflect a perturbation of its chaperone-assisted stabilization and proteasome-dependent degradation. Finally, using a global proteomics approach, we show that heterologous expression of a tau construct that lacks the C-terminal domain, including the microtubule binding domain, does not cause a discernible shift of the proteome except for a significant direct correlation of steady-state levels of tau and cystatin B.
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.
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