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Formation and persistence of polyglutamine aggregates in mistranslating cells.

ABSTRACT: In neurodegenerative diseases, including pathologies with well-known causative alleles, genetic factors that modify severity or age of onset are not entirely understood. We recently documented the unexpected prevalence of transfer RNA (tRNA) mutants in the human population, including variants that cause amino acid mis-incorporation. We hypothesized that a mistranslating tRNA will exacerbate toxicity and modify the molecular pathology of Huntington's disease-causing alleles. We characterized a tRNAPro mutant that mistranslates proline codons with alanine, and tRNASer mutants, including a tRNASerAGA G35A variant with a phenylalanine anticodon (tRNASerAAA) found in ∼2% of the population. The tRNAPro mutant caused synthetic toxicity with a deleterious huntingtin poly-glutamine (polyQ) allele in neuronal cells. The tRNASerAAA variant showed synthetic toxicity with proteasome inhibition but did not enhance toxicity of the huntingtin allele. Cells mistranslating phenylalanine or proline codons with serine had significantly reduced rates of protein synthesis. Mistranslating cells were slow but effective in forming insoluble polyQ aggregates, defective in protein and aggregate degradation, and resistant to the neuroprotective integrated stress response inhibitor (ISRIB). Our findings identify mistranslating tRNA variants as genetic factors that slow protein aggregation kinetics, inhibit aggregate clearance, and increase drug resistance in cellular models of neurodegenerative disease.

SUBMITTER: Lant JT

PROVIDER: S-EPMC8599886 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Formation and persistence of polyglutamine aggregates in mistranslating cells.

Lant Jeremy T JT Kiri Rashmi R Duennwald Martin L ML O'Donoghue Patrick P

Nucleic acids research 20211101 20

In neurodegenerative diseases, including pathologies with well-known causative alleles, genetic factors that modify severity or age of onset are not entirely understood. We recently documented the unexpected prevalence of transfer RNA (tRNA) mutants in the human population, including variants that cause amino acid mis-incorporation. We hypothesized that a mistranslating tRNA will exacerbate toxicity and modify the molecular pathology of Huntington's disease-causing alleles. We characterized a tR ...[more]

PMID: 34718744

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Project description:BackgroundProtein misfolding and subsequent aggregation are hallmarks of several human diseases. The cell has a variety of mechanisms for coping with misfolded protein stress, including ubiquitin-mediated protein degradation. In fact, the presence of ubiquitin at protein aggregates is a common feature of protein misfolding diseases. Ubiquitin conjugating enzymes (UBCs) are part of the cascade of enzymes responsible for the regulated attachment of ubiquitin to protein substrates. The specific UBC used during ubiquitination can determine the type of polyubiquitin chain linkage, which in turn plays an important role in determining the fate of the ubiquitinated protein. Thus, UBCs may serve an important role in the cellular response to misfolded proteins and the fate of protein aggregates.ResultsThe Q82 strain of C. elegans harbors a transgene encoding an aggregation prone tract of 82 glutamine residues fused to green fluorescent protein (Q82::GFP) that is expressed in the body wall muscle. When measured with time-lapse microscopy in young larvae, the initial formation of individual Q82::GFP aggregates occurs in approximately 58 minutes. This process is largely unaffected by a mutation in the C. elegans E1 ubiquitin activating enzyme. RNAi of ubc-22, a nematode homolog of E2-25K, resulted in higher pre-aggregation levels of Q82::GFP and a faster initial aggregation rate relative to control. Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation. However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate. This result is consistent with previous findings that aggregates in young adult worms are smaller after ubc-13 RNAi. mCherry::ubiquitin becomes localized to Q82::GFP aggregates during the fourth larval (L4) stage of life, a time point long after most aggregates have formed. FLIP and FRAP analysis indicate that mCherry::ubiquitin is considerably more mobile than Q82::GFP within aggregates.ConclusionsThese data indicate that initial formation of Q82::GFP aggregates in C. elegans is not directly dependent on ubiquitination, but is more likely a spontaneous process driven by biophysical properties in the cytosol such as the concentration of the aggregating species. The effect of ubiquitination appears to be most significant in later, secondary aggregate growth.

Project description:Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington's disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms. Polyglutamine (polyQ) proteins encoded by repetitive CAG DNA sequences serve a variety of normal biological functions. Yet some proteins with abnormally expanded polyQ regions cause neurodegeneration through unknown mechanisms. To study how distinct cellular environments modulate polyQ aggregation and toxicity, we expressed CAG-expanded huntingtin fragments in Schizosaccharomyces pombe In stark contrast to many other eukaryotes, S. pombe is uniquely devoid of proteins containing long polyQ tracts. Our results show that S. pombe cells, despite their low content of endogenous polyQ proteins, exhibit striking and unexpected resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the emergence and expansion of polyQ domains in eukaryotic evolution.

Project description:BackgroundAggregation and cytotoxicity of mutant proteins containing an expanded number of polyglutamine (polyQ) repeats is a hallmark of several diseases, including Huntington's disease (HD). Within cells, mutant Huntingtin (mHtt) and other polyglutamine expansion mutant proteins exist as monomers, soluble oligomers, and insoluble inclusion bodies (IBs). Determining which of these forms constitute a toxic species has proven difficult. Recent studies support a role for IBs as a cellular coping mechanism to sequester levels of potentially toxic soluble monomeric and oligomeric species of mHtt.Methodology/principal findingsWhen fused to a fluorescent reporter (GFP) and expressed in cells, the soluble monomeric and oligomeric polyglutamine species are visually indistinguishable. Here, we describe two complementary biophysical fluorescence microscopy techniques to directly detect soluble polyglutamine oligomers (using Htt exon 1 or Htt(ex1)) and monitor their fates in live cells. Photobleaching analyses revealed a significant reduction in the mobilities of mHtt(ex1) variants consistent with their incorporation into soluble microcomplexes. Similarly, when fused to split-GFP constructs, both wildtype and mHtt(ex1) formed oligomers, as evidenced by the formation of a fluorescent reporter. Only the mHtt(ex1) split-GFP oligomers assembled into IBs. Both FRAP and split-GFP approaches confirmed the ability of mHtt(ex1) to bind and incorporate wildtype Htt into soluble oligomers. We exploited the irreversible binding of split-GFP fragments to forcibly increase levels of soluble oligomeric mHtt(ex1). A corresponding increase in the rate of IBs formation and the number formed was observed. Importantly, higher levels of soluble mHtt(ex1) oligomers significantly correlated with increased mutant cytotoxicity, independent of the presence of IBs.Conclusions/significanceOur study describes powerful and sensitive tools for investigating soluble oligomeric forms of expanded polyglutamine proteins, and their impact on cell viability. Moreover, these methods should be applicable for the detection of soluble oligomers of a wide variety of aggregation prone proteins.

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Formation and persistence of polyglutamine aggregates in mistranslating cells.

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Formation and persistence of polyglutamine aggregates in mistranslating cells.

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