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Pathogenic polyglutamine expansion length correlates with polarity of the flanking sequences
© Kim; licensee BioMed Central Ltd. 2014
Received: 13 June 2014
Accepted: 23 October 2014
Published: 6 November 2014
Polyglutamine (polyQ) repeat expansion within coding sequence of a soluble protein is responsible for eight autosomal-dominant genetic neurodegenerative disorders. These disorders affect cerebellum, striatum, basal ganglia and other brain regions. The pathogenic polyQ-expansion threshold in these proteins varies from 32Q to 54Q. Understanding the reasons for variability in pathogenic polyQ threshold may provide insights into pathogenic mechanisms responsible for development of these disorders.
Here we established a quantitative correlation between the polarity of the flanking sequences and pathogenic polyQ-expansion threshold in this protein family. We introduced an “edge polarity index” (EPI) to quantify polarity effects of the flanking regions and established a strong correlation between EPI index and critical polyQ expansion length in this protein family. Based on this analysis we subdivided polyQ-expanded proteins into 2 groups – with strong and weak dependence of polyQ threshold on EPI index. The main difference between members of the first and the second group is a polarity profile of these proteins outside of polyQ and flanking regions. PolyQ proteins are known substrates for proteasome and most likely mechanistic explanation for the observed correlation is that proteasome may have an impaired ability to process continuous non-polar regions of proteins.
The proposed hypothesis provides a quantitative explanation for variability in pathogenic threshold among polyQ-expansion disorders, which we established to correlate with polarity of flanking regions. To explain these results we propose that proteasome is not efficient in processing continuous non-polar regions of proteins, resulting in release of undigested and partially digested fragments. If supported experimentally, our hypothesis may have wide implications for further understanding the pathogensis of polyglutamine expansion disorders.
Spinal cord, Brain stem
Cerebellum, Basal ganglia
Cerebellum, Brain stem
Cerebellum, Brain stem
Ventral pons, Substantia nigra
Cerebellum, Brain stem, Spinal cord
Calculation of protein polarity profiles and polarity edge indexes
The Zimmerman Polarity index  for each protein was calculated by using ProtScale software package  with the following options: window size: 9, Relative weight of the window edges compared to the window center: 100%, Weight variation model: linear. The polarity edge index was derived by calculating change in polarity on the edges of polyQ sequence induced by the flanking regions. The amino terminal polarity index ( N PI) and carboxy-terminal polarity index ( C PI) are shown in Table 1. The total edge polarity index ( E PI) was calculated as a sum of N PI and C PI indexes.
Primary protein-sequence comparison of polyQ-expanded protein family
Sequence polarity and proteasomal processing of protein
Polarity of polyQ-flanking regions and pathogenic polyQ threshold
The expanded polyQ stretch corresponds to an extended low polarity sequence. As discussed above, we propose that such a sequence is a poor substrate for proteasomal processing. This argument may explain the accumulation of undigested ubiquitinated fragments of polyQ-expanded proteins in cells [23, 24, 31–36]. We further reasoned that the pathogenic threshold of polyQ expansion may be related to potential influence of flanking regions on proteasomal degradation of naked polyQ sequence. Specifically, if polyQ sequence is surrounded by highly polar flanking regions, then these regions can increase the effective polarity on the edges of polyQ stretch, promoting proteasomal processing. On the other hand, if polyQ region is flanked by low polarity regions, then the polarity of polyQ sequence edges remains low, impairing proteasomal processing. This hypothesis predicts that a polyQ sequence embedded within polar flanking sequences must have longer expansion to reach pathogenic threshold than the polyQ sequence embedded within low polarity flanking sequences.
Pathogenic polyQ threshold and influence of protein context
Interestingly, each member of the first group has been reported to be associated with proteasome in biochemical experiments. In some cases association with proteasome occurred via ubiquitinated form of the protein - such as AR [37, 38], Atxn3 [39, 40], or Htt [41, 42]. In some cases association with proteasome did not require ubiquitination, such as for Atxn7 [43, 44] and for TBP [45–47]. In contrast, members of the second group have not been reported to associate with proteasome in biochemical experiments. The only known interaction is proteasomal association of Atxn1 that is mediated by HSP/CHIP [48, 49] and requires partial unfolding of Atxn1 to be initiated. Although correlative, this argument further suggests that the members of the first group are better substrates for proteasomal degradation than the members of the second group.
Effects of Histidine insertion
A unique clinical case provides an indirect support to our hypothesis. A Japanese SCA1 patient was discovered to have an insertion of 2 His residues within polyQ stretch, resulting in sequence Q45HQHQ10. An expected age of disease onset for a typical SCA1-58Q patient is 22 years of age. In contrast, the SCA1-Q45HQHQ10 patient displayed first symptoms of disease at the age of 50 . In addition, the brain stem atrophy of this patient was much milder than expected for a typical SCA1 patient with similar repeat length . What is an explanation for dramatic protective effects of His insertion? Biophysical studies [51–53] and our own crystallographic experiments  suggested that insertion of His has minimal effect on secondary structure of polyQ region. Thus, it is not likely that insertion of 2 His residues disrupted the “toxic conformation” of the 58Q stretch. However, insertion of 2 His residues is expected to introduce a polarity peak within polyQ sequence. Indeed, the polarity profile of Atxn1-55Q2Н (Q45HQHQ10) contains a significant polar peak (Figure 6D). We propose that such polar insertion enhances proteasomal processing of His-containing protein. As a result, “effective” low polarity polyQ region is shortened to approximately 36Q (Figure 6D), which is consistent with the very mild clinical phenotype of this particular patient .
In this paper we established a quantitative correlation between the polarity of the flanking regions and the pathogenic polyQ expansion threshold for the soluble polyQ-containing proteins. The quantitative analysis enabled us to divide soluble polyQ-expanded proteins into 2 groups – with strong and weak dependence of polyQ threshold on the polarity of the flanking regions. The main difference between members of the first group (Htt, Atxn7, AR, TBP, Atxn3) and the second group (Atxn2, Atxn1, ATN1) is related to polarity profile of remainder of these proteins. All members of the first group composed of regular low-high polarity sequences, whereas members of the second group are composed primarily of low polarity sequence regions. PolyQ proteins are known substrates for proteasomal degradation. We analyzed experiments performed with the model proteasomal substrates and concluded that proteasome has impaired ability to process continuous non-polar regions of proteins. We propose that polarity of flanking regions may have an important modulatory effects on ability of proteasome to process continuous polyQ stretches, resulting in accumulation of polyQ-expanded proteins and partially digested protein fragments in cells. Such proteins can then exert “toxic gain of function” effects by interfering with essential neuronal signaling pathways. These ideas need to be tested experimentally. However, indirect support to our hypothesis is provided by partial protective effects of His insertion within polyQ stretch of SCA1 patient, which has a significant effect on polarity profile of polyQ stretch. We propose that such polar insertion can facilitate proteasomal degradation of polyQ-expanded Ataxin 1, which may explain less severe pathology in these patients.
I thank to Leah Taylor for administrative assistance, members of Bezprozvanny and Kim laboratories for helpful discussions and Drs Ilya Bezprozvanny (I.B.) and George De Martino for comments on the paper. MWK is a Young Investigator of the National Ataxia Foundation and supported by the Hereditary Disease Foundation. This work was also supported by the contract with the Russian Ministry of Science 11.G34.31.0056 (I.B), by the Russian Scientific Fund grant 14-25-00024 (IB), and by the NIH grants R01NS074376 and R01NS056224 (I.B.).
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