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Errors in Translation Cause Selective Neurodegeneration Jean-Christophe Rochet* Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Heine Pharmacy Building, 575 Stadium Mall Drive, West Lafayette, Indiana 47907-2091
A B S T R A C T The 3D structure of a protein is determined by the unique sequence of amino acid residues comprising the polypeptide chain. Sequence changes can cause protein misfolding, a potentially toxic phenomenon implicated in various neurodegenerative disorders. In a recent paper, translational misincorporation is proposed to be a new biochemical mechanism for generating mutant proteins that misfold and kill neurons.
*Corresponding author,
[email protected].
Published online October 20, 2006 10.1012/cb6004068 CCC: $33.50 © 2006 by American Chemical Society
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typical globular protein adopts a compact structure with buried hydrophobic residues (1). This protein fold is stabilized by a network of intermolecular contacts (including hydrophobic, electrostatic, and hydrogenbonding interactions) involving residues throughout the polypeptide chain. Modifications to the amino acid sequence can disrupt these stabilizing interactions and result in protein misfolding. In turn, misfolded polypeptides have a high propensity to form aggregates via interactions among exposed hydrophobic domains. Several proteins have been found to misfold and aggregate in neurodegenerative disorders (NDDs) (2). In general, these proteins undergo misfolding because of sequence changes originating from gene mutations or post-translational modifications (PTMs). For example, mutant forms of Cu2⫹/Zn2⫹ superoxide dismutase produce aggregates in familial amyotrophic lateral sclerosis (ALS) (3), and oxidative modifications stabilize aggregated forms of ␣-synuclein in Parkinson’s disease (PD) (4, 5). Now in a recent paper published in Nature, Susan Ackerman, Paul Schimmel, and colleagues (6) report that translational misincorporation, a process in which the wrong amino acid is incorporated into the growing polypeptide chain, is another mechanism by which misfolded, neurotoxic proteins are generated. Normally, misfolded proteins in the cytosol are refolded by chaperones (including heat shock proteins (Hsps) upregulated
in response to protein unfolding) or degraded by the ubiquitin proteasome pathway (UPP) (7). However, in cells with a high degree of protein unfolding, the capacity of these “quality-control” systems is exceeded, and misfolded proteins form aggregates. These aggregates are then recruited to perinuclear inclusion bodies named “aggresomes”, where they are targeted for destruction via autophagy (8–10). Autophagy is a cellular process involving sequestration of cellular material into a vesicular structure termed an “autophagosome” (9, 10). The autophagosome ultimately delivers its contents to the lysosome for degradation. In addition to triggering aggresome formation, a buildup of cytosolic protein aggregates elicits protein misfolding in the endoplasmic reticulum (ER); the result is ER stress and induction of the unfolded protein response (UPR) (11). The UPR is a cellular program involving upregulation of ER chaperones, increased degradation of misfolded polypeptides, translational suppression, and under severe conditions of extreme ER stress, apoptotic signaling (11, 12). From the above discussion, it is clear that amino acid substitutions resulting in protein misfolding can have profoundly disruptive effects on cellular homeostasis. One mechanism by which substitutions are avoided is via faithful transmission of the genetic code from DNA to protein. Aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) play a critical role in this process by ensuring that each www.acschemicalbiology.org
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VIEW tRNA is only charged with its cognate amino acid (13). Each of the 20 aaRS catalyzes a two-part reaction: activation of the amino acid with ATP, yielding enzyme-bound aminoacyl-AMP with release of PPi; and ligation of the amino acid to the 3= end of the tRNA, yielding aminoacyl-tRNA (aa-tRNA) with release of AMP. The specificity of the reaction for the cognate amino acid is largely determined by the exclusion of amino acids that are too bulky or lack key functional groups to interact properly with the active site. However, because smaller amino acids can escape these constraints, aaRSs have evolved a second “editing” domain that catalyzes hydrolysis of incorrect aa-tRNAs (the correct aa-tRNA is sterically excluded from the editing site). Ackerman, Schimmel, and colleagues (6) discovered a link between errors in translation and protein misfolding in their studies of mice with the “sticky” (sti) mutation. These mice (so named because of the unkempt appearance of their fur) were found to have motor deficits, including trembling and ataxia. The mutant mice also exhibited age-dependent, apoptotic neuronal death in the Purkinje cell layer of the cerebellum. The authors mapped the sti mutation to the gene encoding alanyl-tRNA synthetase (AlaRS) and found that it resulted in the substitution of Ala734 with a glutamate residue (A734E). Expression of a transgene encoding wild-type AlaRS suppressed Purkinje cell loss and ataxia in the sti mutant mice, confirming that the A734E substitution is responsible for the neurodegenerative phenotype. Ala734 is a conserved residue in the putative editing domain of AlaRS. Mutations in this domain of the Escherichia coli enzyme lead to increased levels of misacylated Ser- or Gly-tRNAAla, and cells expressing mutant AlaRS have decreased viability in media supplemented with high amounts of serine or glycine (14). Presumably, these elevated noncognate amino acids act as an environmental “stressor” that elicits toxicity www.acschemicalbiology.org
via increased tRNAAla misacylation and, therefore, decreased translational fidelity. To address whether misacylation is involved in the sti phenotype, Ackerman, Schimmel, and colleagues (6) compared wild-type and mutant mouse embryonic fibroblasts (MEFs) in terms of their sensitivity to elevated amino acids in the cell-culture medium. Homozygous (sti/sti) and heterozygous (sti/⫹) mutant MEFs were markedly more sensitive to elevated serine than were wildtype fibroblasts, and this suggests that inefficient editing of Ser-tRNAAla by mutant AlaRS may contribute to neurodegeneration in the mutant mice. To characterize the editing function of AlaRS-A734E more directly, Ackerman, Schimmel, and colleagues (6) conducted acylation and deacylation assays with the recombinant enzyme. Wild-type and mutant AlaRS exhibited similar kinetics of tRNAAla acylation with alanine, and neither enzyme catalyzed Ala-tRNAAla deacylation above background levels. In contrast, A734E catalyzed deacylation of Ser-tRNAAla less efficiently than wild-type AlaRS (although the rate of deacylation by the mutant enzyme was still well above background). The A734E mutant also catalyzed more rapid misacylation of tRNAAla with serine compared with the wild-type enzyme. These biochemical data provided strong evidence that the sti mutation causes a specific defect in AlaRS editing of Ser-tRNAAla. The authors predicted that the editing defect of mutant AlaRS would lead to errors in translation and, therefore, a buildup of misfolded proteins with amino acid substitutions (Figure 1, panel a). Many of these misfolded polypeptides should be ubiquitylated, reflecting abortive attempts by the UPP to eliminate these potentially toxic species (see above). In support of this idea, the authors found that ubiquitylated proteins were more abundant in sti/sti MEFs and in mutant Purkinje cells than in the corresponding wild-type cells. Strikingly, perinuclear inclusions and autophagosome-
like structures were detected in cerebellar neurons of sti/sti mice. The mutant Purkinje neurons also contained increased levels of cytosolic chaperones, including Hsp72, Hsc70, and Hsp40, and components of the UPR, including the ER chaperone immunoglubulin heavy-chain binding protein (BiP) and the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). These data are consistent with a model in which mutant AlaRS causes increased protein misfolding and aggregation in sti mutant mice and results in cerebellar neurodegeneration (Figure 1, panel b). Defects in tRNA acylation have been shown to play a role in some human NDDs. Two groups reported that overaccumulation of p38, a cofactor involved in the assembly of the multi-aaRS complex, contributes to the death of dopaminergic neurons in PD (15, 16). In addition, mutations in the gene encoding glycyl- or tyrosyl-tRNA synthetase were linked to motor neuropathy in CharcotMarie-Tooth disease (17, 18). The study by Ackerman, Schimmel, and colleagues is the first to show that impaired aaRS proofreading causes neurodegeneration. The finding that the sti mutation targets the editing domain of AlaRS is highly significant because it suggests that amino acid substitutions will be introduced throughout the proteome. Accordingly, this mutation is predicted to cause more widespread misfolding and aggregation than do monogenic lesions that elicit destabilization of a single protein. Protein aggregation in sti mutant mice may trigger cell death via a gain of function involving toxic effects of oligomeric species (e.g., membrane permeabilization) (19, 20) or via a loss of function involving the inactivation of proteins that are recruited into the aggregates. Although the data argue convincingly that Purkinje cell death in sti mutant mice involves a buildup of toxic protein aggregates, the cerebellar phenotype may also result from the disruption of protein activities required for neuronal survival, indepenVOL.1 NO.9 • 562–566 • 2006
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Figure 1. Defects in the editing function of aaRSs cause protein misfolding and aggregation. a) Wild-type AlaRS and the A734E mutant catalyze the acylation of tRNAAla with the cognate amino acid, alanine, resulting in error-free translation and correct protein folding (top). A734E also catalyzes the acylation of tRNAAla with the noncognate amino acid, serine, resulting in translational misincorporation and protein misfolding (bottom). The large green oval represents the AlaRS aminoacylation domain, and the smaller blue oval corresponds to the editing domain. b) Protein misfolding is induced by (i) PTMs, for example, oxidative damage, (ii) gene mutations, or (iii) errors in translation due to defective AlaRS editing. (iv) Under normal conditions, misfolded proteins are eliminated by the UPP. However, at sufficiently high concentrations, misfolded polypeptides form aggregates, shown here as (v) early oligomers and (vi) mature inclusions. Protein misfolding induces (vii) the UPR, including upregulation of BiP and CHOP, and (viii) increased expression of cytosolic chaperones, including Hsp70 and Hsp40. (ix) The cell attempts to eliminate aggregated proteins via autophagy. (x) Failure to eliminate toxic protein aggregates ultimately leads to apoptosis, in part regulated by UPR signaling. tRNA image courtesy of Neil Voss, Wikipedia.
dent of aggregation. It is also possible that the sti mutation triggers cell death by eliminating noncanonical functions of AlaRS distinct from aa-tRNA synthesis or editing (21). Additional evidence in support of a role for protein aggregation rather than these alternative mechanisms might be obtained by testing whether the overexpression of chaperones such as Hsp70 suppresses inclusion formation and neurodegeneration in sti mutant mice. 564
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The extent to which the sti mutation disrupts translational fidelity in vivo has yet to be determined. Because recombinant A734E has significant (albeit reduced) SertRNAAla deacylation activity, only a fraction of alanine residues in the proteome are likely to be replaced with serine. The data obtained by Ackerman, Schimmel, and colleagues suggest that this fraction may vary from tissue to tissue because of differences in serine content. Even a low overall percentROCHET
age of alanine-to-serine substitutions would be expected to have a pronounced impact on cellular function, given that all of the proteins in the cell would be targeted. Moreover, small amounts of substituted, misfolded polypeptides may increase their toxic effect by “seeding” the aggregation of more abundant wild-type isoforms (22, 23). Because translational proofreading is an essential part of protein synthesis in all cell types, it is remarkable that impaired AlaRS editing results in a specific neurodegenerative phenotype in sti mutant mice. The observation that protein misfolding specifically targets the central nervous system in these mice is consistent with the involvement of protein aggregation in other NDDs, including Alzheimer’s disease, PD, ALS, Huntington’s disease, and the spinocerebellar ataxias (2, 24). One reason for the sensitivity of terminally differentiated neurons to protein misfolding is that these cells are unable to dilute out toxic protein aggregates via mitosis (6). In addition, postmitotic neurons are highly dependent on the UPP to avoid cell-cycle re-entry, a phenomenon that can trigger neuronal apoptosis (25). Accordingly, these cells may be more vulnerable than other cell types to mechanisms that cause impairment of the UPP, including protein misfolding and aggregation. Although one can rationalize why terminally differentiated neurons are sensitive to protein misfolding, it is unclear why Purkinje cells are especially vulnerable in sti mutant mice. Purkinje neurons are selectively targeted in other protein aggregation disorders, including spinocerebellar ataxias (26); this suggests that these cells have inherently inefficient quality-control systems (6). Nevertheless, brain regions other than the Purkinje cell layer (e.g., the substantia nigra) are sensitive to conditions that induce protein misfolding (27–29), and these would also be expected to undergo neurodegeneration as a consequence of impaired translational editing in sti mutant mice. A rationale for the selective degenerative phewww.acschemicalbiology.org
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Figure 2. Model illustrating various possible outcomes of the AlaRS proofreading defect. (i) Alanine-to-serine substitutions may be introduced in proteins with relatively few buried alanine residues at sites that cannot accommodate a serine residue. (ii) This pattern of replacement (represented by a green asterisk) causes little disruption of the protein fold. (iii) Some proteins with these substitutions may undergo additional “hits” in the form of PTMs, represented by a yellow “burst”. (iv) The combined effects of the alanine-to-serine replacements and PTMs may weaken the protein structure and thus trigger misfolding. (v) Some misfolded polypeptides may be inherently unstable (e.g., because of the presence of a PEST sequence) and therefore will be rapidly degraded. (vi) Longer-lived misfolded proteins may accumulate and form aggregates, depicted as -sheet-rich oligomers. (vii) Alanine-to-serine substitutions may also be introduced in proteins with a relatively large number of buried alanine residues. This pattern of substitution (represented by red asterisks) leads to protein misfolding. (viii) Some of the misfolded polypeptides may be rapidly degraded, whereas others (ix) with longer half-lives may accumulate to form aggregates.
notype of these mice is implied by the predicted biochemical consequences of the AlaRS editing defect. Although impaired proofreading should trigger translational misincorporation throughout the proteome, only a fraction of substituted polypeptides are expected to undergo misfolding and aggregation (Figure 2). Some proteins may be resistant to the potentially destabilizing effects of alanine-to-serine substitutions because they have a relatively low proportion of buried alanine residues at sites that cannot accommodate a serine residue (30). Other proteins that unfold in response to alanine-to-serine replacements may be targeted for rapid degradation before they can accumulate (e.g., proteins with a proline, www.acschemicalbiology.org
glutamic acid, serine, and threonine (PEST) sequence (31)). Conversely, proteins with long half-lives and large numbers of buried alanine residues are more likely to form aggregates in response to impaired AlaRS editing. An abundance of these proteins in the Purkinje cell layer could explain why this region of the brain is specifically targeted in sti mutant mice. Moreover, some proteins with alanine-to-serine substitutions may only misfold and aggregate after undergoing PTMs that further destabilize the protein fold (Figure 2). If these PTMs occur frequently in Purkinje neurons, then this “multi-hit” phenomenon could also account for the selective nature of the sti phenotype.
In summary, the findings reported by Ackerman, Schimmel, and colleagues suggest a novel mechanism for protein misfolding and aggregation in postmitotic neurons and provide intriguing insight into potential biochemical changes underlying selective neurodegeneration. To better understand the molecular basis for the sti phenotype, it will be important to determine which proteins misfold and aggregate in response to defective translational editing. This problem can be addressed via comparative proteomics, to identify proteins aggregated in mutant but not wild-type cerebellar tissue, and via computational modeling, to identify proteins with a high likelihood of misfolding in response to alanine-to-serine substitutions. It would also be of interest to determine whether defects in the editing function of aaRSs other than AlaRS cause neurodegeneration and whether the patterns of cell loss in these cases differ from the selective pathology in sti mutant mice. Finally, a high priority will be to determine whether mutations in aaRS genes are involved in human NDDs. Presumably, such mutations could only cause a moderate impairment of translational proofreading, with neurodegeneration occurring after the onset of reproductive age; otherwise, they would not be retained in the human population. Evidence of a role for defective editing in human NDDs could have important implications for drug discovery: it would suggest that improving translational fidelity may be a useful therapeutic strategy in the treatment of these disorders.
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