Prions and prion proteins

Neurodegenerative diseases of animals and humans including scrapie, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease are caused by unusual infectious pathogens called prions. There is no evidence for a nucleic acid in the prion, but diverse experimental results indicate that a host-derived protein called PrPSc is a component of the infectious particle. Experiments with scrapie-infected cultured cells show that PrPSc is derived from a normal cellular protein called PrPC through an unknown posttranslational process. We have analyzed the amino acid sequence and posttranslational modifications of PrPSc and its proteolytically truncated core PrP 27-30 to identify potential candidate modifications that could distinguish PrPSc from PrPC. The amino acid sequence of PrP 27-30 corresponds to that predicted from the gene and cDNA. Mass spectrometry of peptides derived from PrPSc has revealed numerous modifications including two N-linked carbohydrate moieties, removal of an amino-terminal signal sequence, and alternative COOH termini. Most molecules contain a glycosylinositol phospholipid (GPI) attached at Ser-231 that results in removal of 23 amino acids from the COOH terminus, whereas 15% of the protein molecules are truncated to end at Gly-228. The structure of the GPI from PrPSc has been analyzed and found to be novel, including the presence of sialic acid. Other experiments suggest that the N-linked oligosaccharides are not necessary for PrPSc formation. Although detailed comparison of PrPSc with PrPC is required, there is no obvious way in which any of the modifications might confer upon PrPSc its unusual physical properties and allow it to act as a component of the prion. If no chemical difference is found between PrPC and PrPSc, then the two isoforms of the prion protein may differ only in their conformations or by the presence of bound cellular components.     

Prions and the prion disorders

One of us remembers sitting in a high school biology class in 1977 being taught about scrapie, a naturally occurring disorder of sheep. The teacher had no particular interest in agriculture, but was pointing out some peculiar characteristics of this disease as a biological curiosity on a wet Friday afternoon. The prion disorders captured the imagination of a range of biologists (including that teacher) well before the epidemic of bovine spongiform encephalopathy (BSE) and the appearance of a new variant of the human prion disease, Creutzfeldt Jakob disease (CJD), in the UK, because of their extraordinary biology and the unique properties of the infectious agent. We review the results of studies leading to a convergence of evidence that the causative infectious agent, the `prion', is devoid of nucleic acid and is composed of an abnormal isoform of a host-encoded protein, the prion protein (PrP). Received: 2 March 1998 / Accepted: 2 March 1998     

Prions and prion diseases: fundamentals and mechanistic details

Prion diseases, often called transmissible spongiform encephalopathies (TSEs), are infectious diseases that accompany neurological dysfunctions in many mammalian hosts. Prion diseases include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE, "mad cow disease") in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elks. The cause of these fatal diseases is a proteinaceous pathogen termed prion that lacks functional nucleic acids. As demonstrated in the BSE outbreak and its transmission to humans, the onset of disease is not limited to a certain species but can be transmissible from one host species to another. Such a striking nature ofprions has generated huge concerns in public health and attracted serious attention in the scientific communities. To date, the potential transmission ofprions to humans via foodbome infectiorn and iatrogenic routes has not been alleviated. Rather, the possible transmission of human to human or cervids to human aggravates the terrifying situation across the globe. In this review, basic features about prion diseases including clinical and pathological characteristics, etiology, and transmission of diseases are described. Based on recently accumulated evidences, the molecular and biochemical aspects of prions, with an emphasis on the molecular interactions involved in prion conversion that is critical during prion replication and pathogenesis, are also addressed.     

Amyloid cores in prion domains: Key regulators for prion conformational conversion

Despite the significant efforts devoted to decipher the particular protein features that encode for a prion or prion-like behavior, they are still poorly understood. The well-characterized yeast prions constitute an ideal model system to address this question, because, in these proteins, the prion activity can be univocally assigned to a specific region of their sequence, known as the prion forming domain (PFD). These PFDs are intrinsically disordered, relatively long and, in many cases, of low complexity, being enriched in glutamine/asparagine residues. Computational analyses have identified a significant number of proteins having similar domains in the human proteome. The compositional bias of these regions plays an important role in the transition of the prions to the amyloid state. However, it is difficult to explain how composition alone can account for the formation of specific contacts that position correctly PFDs and provide the enthalpic force to compensate for the large entropic cost of immobilizing these domains in the initial assemblies. We have hypothesized that short, sequence-specific, amyloid cores embedded in PFDs can perform these functions and, accordingly, act as preferential nucleation centers in both spontaneous and seeded aggregation. We have shown that the implementation of this concept in a prediction algorithm allows to score the prion propensities of putative PFDs with high accuracy. Recently, we have provided experimental evidence for the existence of such amyloid cores in the PFDs of Sup35, Ure2, Swi1, and Mot3 yeast prions. The fibrils formed by these short stretches may recognize and promote the aggregation of the complete proteins inside cells, being thus a promising tool for targeted protein inactivation.     

Prions: on the enzymatic function of PrPc from mammalian and prion "families"

This minireview deals with some approaches and results of experiments which enabled to discover SOD-like activity of the mammal PrP protein. This activity required the unchanged region of the repeated octapeptide and Cu2+ binding to the appropriate sites of the PrP molecule. It was shown that an infectious prion isoform could bind the normal isoform. This leads to the loss of PrP SOD-like function accompanied by Cu2+ release from the molecule. Also, the problem of sowing the protein seeds of prion propagation is briefly summarized and the first evidence of abnormal protein conformation induced in vivo using yeast cell and in vitro formed Sup 35 prion seed is described.     

Scrambled prion domains form prions and amyloid

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The amino-terminal prion domain of Ure2p is necessary and sufficient for prion formation and has a high glutamine (Q) and asparagine (N) content. Such Q/N-rich domains are found in two other yeast prion proteins, Sup35p and Rnq1p, although none of the many other yeast Q/N-rich domain proteins have yet been found to be prions. To examine the role of amino acid sequence composition in prion formation, we used Ure2p as a model system and generated five Ure2p variants in which the order of the amino acids in the prion domain was randomly shuffled while keeping the amino acid composition and C-terminal domain unchanged. Surprisingly, all five formed prions in vivo, with a range of frequencies and stabilities, and the prion domains of all five readily formed amyloid fibers in vitro. Although it is unclear whether other amyloid-forming proteins would be equally resistant to scrambling, this result demonstrates that [URE3] formation is driven primarily by amino acid composition, largely independent of primary sequence.     

Prions

Prions were originally defined as infectious agents of protein nature, which caused neurodegenerative diseases in animals and humans. The prion concept implies that the infectious agent is a protein in special conformation that can be transmitted to the normal molecules of the same protein through protein-protein interactions. Until the 1990s, the prion phenomenon was associated with the single protein named PrP. Discovery of prions in lower eukaryotes, the yeast Saccharomyces cerevisiae and fungus Podospora anserina, suggests that prions have wider significance. Prions of lower eukaryotes are not related to diseases; their propagation caused by aggregation of prion-like proteins underlies the inheritance of phenotypic traits and most likely has adaptive significance. This review covers prions of mammals and lower eukaryotes, mechanisms of their appearance de novo and maintenance, structure of prion particles, and prospects for the treatment of prion diseases. Recent data concerning the search for new prion-like proteins is included. The paper focuses on the [PSI+] prion of S. cerevisiae, since at present it is the most investigated one. The biological significance of prions is discussed.     

Prions and proteinaceous proteinase inhibitors: structural analogs and their consequences. II. Dynamics of prion diseases

The assumption about pathogenic prions as the proteins supplying the extracellular proteinases transport into intracellular space permits to bring the pathogenesis of prion diseases to order of the known and partially proved process regarding the case of prion diseases. We present the mathematical model of the dynamics of prion pathogenesis explaining the existence of the minimal infectious dose and small influence of its exceeding on the duration of long-term latent period of the disease. According to the model proposed the transformation of the neuronal cell into PrPSc breeder is the result of proteolytic damage of shaperoning system caused by accumulation in the cell of some crucial amount of proteinase-transporting prions. Such an accumulation is considered as the result of successive and centripheral lay-by-lay transformation of compact cellular locus from higher affinity to prions to normal one. The formation in the moveable frontier lays of the wave with high prion consisting and its closing into the locus center leads to dramatic splash of prion concentration even at moderate difference between higher and normal affinity levels. The final concentration of prions depends mainly on the correlation between these affinities whilst on exceeding of some value the dimension of the locus is of no importance.     

Prions

The discovery of infectious proteins, denoted prions, was unexpected. After much debate over the chemical basis of heredity, resolution of this issue began with the discovery that DNA, not protein, from pneumococcus was capable of genetically transforming bacteria (Avery et al. 1944). Four decades later, the discovery that a protein could mimic viral and bacterial pathogens with respect to the transmission of some nervous system diseases (Prusiner 1982) met with great resistance. Overwhelming evidence now shows that Creutzfeldt-Jakob disease (CJD) and related disorders are caused by prions. The prion diseases are characterized by neurodegeneration and lethality. In mammals, prions reproduce by recruiting the normal, cellular isoform of the prion protein (PrP(C)) and stimulating its conversion into the disease-causing isoform (PrP(Sc)). PrP(C) and PrP(Sc) have distinct conformations: PrP(C) is rich in α-helical content and has little β-sheet structure, whereas PrP(Sc) has less α-helical content and is rich in β-sheet structure (Pan et al. 1993). The conformational conversion of PrP(C) to PrP(Sc) is the fundamental event underlying prion diseases. In this article, we provide an introduction to prions and the diseases they cause.     

Prions

The prion hypothesis^1-3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^{4, 5} Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^{6, 7} Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and – most probably - a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.     

Cooperative binding of dominant-negative prion protein to kringle domains

Conversion of the cellular prion protein (PrP(C)) to the pathogenic isoform (PrP(Sc)) is a major biochemical alteration in the progression of prion disease. This conversion process is thought to require interaction between PrP(C) and an as yet unidentified auxiliary factor, provisionally designated protein X. In searching for protein X, we screened a phage display cDNA expression library constructed from prion-infected neuroblastoma (ScN2a) cells and identified a kringle protein domain using full-length recombinant mouse PrP (recMoPrP(23-231), hereafter recMoPrP) expressing a dominant-negative mutation at codon 218 (recMoPrP(Q218K)). In vitro binding analysis using ELISA verified specific interaction of recMoPrP to kringle domains (K(1+2+3)) with higher binding by recMoPrP(Q218K) than by full-length recMoPrP without the mutation. This interaction was confirmed by competitive binding analysis, in which the addition of either a specific anti-kringle antibody or L-lysine abolished the interaction. Biochemical studies of the interactions between K(1+2+3) and various concentrations of both recMoPrP molecules demonstrated binding in a dose-dependent manner. A Hill plot analysis of the data indicates positive cooperative binding of both recMoPrP(Q218K) and recMoPrP to K(1+2+3) with stronger binding by recMoPrP(Q218K). Using full-length and an N-terminally truncated MoPrP(89-231), we demonstrate that N-terminal sequences enable PrP to bind strongly to K(1+2+3). Further characterization with truncated MoPrP(89-231) refolded in different conformations revealed that both alpha-helical and beta-sheet conformations bind to K(1+2+3). Our data demonstrate specific, high-affinity binding of a dominant-negative PrP as well as binding of other PrPs to K(1+2+3). The relevance of such interactions during prion pathogenesis remains to be established.     

Yeast Prions

Prions (infectious proteins) analogous to the scrapie agent have been identified in Saccharomyces cerevisiae and Podospora anserina based on their special genetic characteristics. Each is a protein acting as a gene, much like nucleic acids have been shown to act as enzymes. The [URE3], [PSI+], [PIN+] and [Het-s] prions are self-propagating amyloids of Ure2p, Sup35p, Rnq1p and the HET-s protein, respectively. The [b] and [C] prions are enzymes whose precursor activation requires their own active form. [URE3] and [PSI+] are clearly diseases, while [Het-s] and [b] carry out normal cell functions. Surprisingly, the prion domains of Ure2p and Sup35p can be randomized without loss of ability to become a prion. Thus amino acid content and not sequence determine these prions. Shuffleability also suggests amyloids with a parallel in-register b-sheet structure.     

Yeast Prions Compared to Functional Prions and Amyloids

Saccharomyces cerevisiae is an occasional host to an array of prions, most based on self-propagating, self-templating amyloid filaments of a normally soluble protein. [URE3] is a prion of Ure2p, a regulator of nitrogen catabolism, while [PSI +] is a prion of Sup35p, a subunit of the translation termination factor Sup35p. In contrast to the functional prions, [Het-s] of Podospora anserina and [BETA] of yeast, the amyloid-based yeast prions are rare in wild strains, arise sporadically, have an array of prion variants for a single prion protein sequence, have a folded in-register parallel β-sheet amyloid architecture, are detrimental to their hosts, arouse a stress response in the host, and are subject to curing by various host anti-prion systems. These characteristics allow a logical basis for distinction between functional amyloids/prions and prion diseases. These infectious yeast amyloidoses are outstanding models for the many common human amyloid-based diseases that are increasingly found to have some infectious characteristics.     

Multiple Gln/Asn-rich prion domains confer susceptibility to induction of the yeast [PSI(+)] prion

The yeast prion [PSI(+)] results from self-propagating aggregates of Sup35p. De novo formation of [PSI(+)] requires an additional non-Mendelian trait, thought to result from a prion form of one or more unknown proteins. We find that the Gln/Asn-rich prion domains of two proteins, New1p and Rnq1p, can control susceptibility to [PSI(+)] induction as well as enhance aggregation of a human glutamine expansion disease protein. [PSI(+)] inducibility results from gain-of-function properties of New1p and Rnq1p aggregates rather than from inactivation of the normal proteins. These studies suggest a molecular basis for the epigenetic control of [PSI(+)] inducibility and may reveal a broader role for this phenomenon in the physiology of protein aggregation.