Alternative Prion Proteins
2012-07-31

I read with interest the article entitled, “An overlapping reading frame in the PRNP gene encodes a novel polypeptide distinct from the prion protein,” by Vanderperre et al. (1). The paper describes the discovery of a polypeptide that is synthesized when translation is initiated at an AUG codon in the +3 reading frame downstream from the normal +1 open-reading frame (ORF) initiation site of the gene, PRNP, which encodes the prion protein (PrP), the main component of the etiological agent of transmissible spongiform encephalopathy (TSE). This alternative PrP (AltPrP) spans a region of PRNP that encodes a series of octapeptide repeats in the normal ORF, and it is comprised of 60–80 aa in most species known to be susceptible to TSE, the exact length depending on the number of encoded octa-repeats and the location of the next downstream stop codon in the +3 frame. AltPrP is coexpressed with the normal PrP, at least in humans, cattle, sheep, and deer, and is localized in the outer mitochondrial membrane. The possibility that AltPrP might participate in the pathogenesis of TSEs is discussed in the paper (1), but there is no mention of prior consideration given to the role of PrP molecules containing AltPrP sequence elements as the main etiological agents of these diseases (2,3). What may be the most significant feature of the new findings has been overlooked. Although still the subject of occasional dispute, the infectious agent active in TSEs appears to be a variant of PrP. The difference between the normal cellular form of the PrP (PrPC) and the “scrapie associated” form (PrPSc) is widely thought to come down to nothing more than differences in the way the amino acid chain is folded—PrPSc replicates by inducing molecules of PrPC to refold into the PrPSc form. However, there has never been an investigation precise enough to exclude the possibility that a small minority of prion molecules containing frameshifted segments is an obligatory component of the etiological agents of most TSEs, serving as nuclei for the aggregation of normal-sequence, misfolded PrP that merely obscures the presence of full-length PrPs containing AltPrP elements. Stochastic variation occurs in all molecular biological processes, and ribosomal framekeeping is no exception. Frameshifting errors occur at an average rate of ∼10−5 /codon assignment. Thus, it is to be expected that during synthesis of AltPrP, the normal reading frame will occasionally be restored, before the stop codon in the +3 frame is encountered, resulting in the production of hybrid AltPrP-PrP molecules. The C-terminal bulk of these molecules will be identical to the corresponding part of normal PrP, and the hybrids will be only 8 aa shorter than normal PrP, which has a 22-aa N-terminal signal peptide removed. These hybrid molecules would be expected to copurify with PrPSc when it is extracted from cells, and they would form an occult component of infectious preparations, including those derived from recombinant microorganisms through “protein misfolding cyclic amplification” (PMCA). This procedure is generally very efficient as far as the refolding of PrPC into PrPSc is concerned but often fails to produce high specific infectivity (4), suggesting that the production of infectivity may arise from the selective concentration of small numbers of lower solubility species of AltPrP sequence-containing prion molecules with highly hydrophobic N-termini. The orthodox “induced misfolding model” of prion replication is encountering difficulties, as it cannot define the TSE disease threshold in terms of the amount and form of PrPSc needed for infectivity. There are cases in which misfolding of PrP is transmissible but innocuous and others in which disease is transmitted, but misfolded protein cannot be detected. On the other hand, hybrid AltPrP-PrP molecules could only account for infectivity if they were capable of stimulating, above some critical threshold, the abnormal processes that led to their production during cellular synthesis of PrP. The primary process to be stimulated would be translation in the +3 frame of the N-terminal region of the PRNP gene, either through initiation at the appropriate AUG codon (1) or —1 ribosomal frameshifting at the nearby site just upstream from a conserved pseudo-knot in the mRNA (3,5). The extremely rare, condensed tryptophan triplet elements in the resulting AltPrP peptides could easily be effectors of eitherribosomal process. Once amplified translation in the +3 frame was established, a compensating downstream +1 frameshift could occur simply as a result of ribosome slippage at a UGG “hungry codon” (6). Local depletion of Trp-tRNA is very likely during amplified synthesis in the +3 frame of the octarepeat region of PRNP, as it contains 32.5% of UGG codons compared with a proteome-wide average of only 1.3%. Thus, hybrid AltPrP-PrP could be self-replicating through a combination of — 1 and +1 frameshifting processes. Minimal units of the most infectious preparations of the scrapie agent, in the form of brain homogenate, contain some 106 molecules of protease-resistant PrPSc, dispersed as soluble aggregates consisting of 14–28 molecules of PrPSc (7). It is not clear why enormous numbers of soluble aggregates are needed to infect a brain when aggregates of this size represent the most efficient means of propagating infectivity, unless of course most aggregates do not contain the etiological agent. This would be the case if infectious preparations contained a small proportion, perhaps up to 100 ppm, of truly active AltPrP sequence-containing molecules of PrP. Normal brain material and recombinant microorganisms that synthesize PrP would be expected to contain only a tiny proportion of the active species (∼0.01 ppm; one molecule/10 cells of healthy brain material), produced as a result of normal but infrequent errors in ribosomal framekeeping and retrievable through selective precipitation during the PMCA process. The hypothesis that the etiological agents active in TSEs are sequence variants of the PrPC was first proposed one-quarter century ago (8). Now that AltPrP has been discovered (1), and the orthodox model of prion replication cannot produce a quantitatively consistent interpretation of the available data, searching for odd protein molecules with AltPrP sequence elements in infectious preparations has renewed purpose.