8 research outputs found
Comparative Analysis of Mutant Huntingtin Binding Partners in Yeast Species.
Huntington\u27s disease is caused by the pathological expansion of a polyglutamine (polyQ) stretch in Huntingtin (Htt), but the molecular mechanisms by which polyQ expansion in Htt causes toxicity in selective neuronal populations remain poorly understood. Interestingly, heterologous expression of expanded polyQ Htt is toxic in Saccharomyces cerevisiae cells, but has no effect in Schizosaccharomyces pombe, a related yeast species possessing very few endogenous polyQ or Q/N-rich proteins. Here, we used a comprehensive and unbiased mass spectrometric approach to identify proteins that bind Htt in a length-dependent manner in both species. Analysis of the expanded polyQ-associated proteins reveals marked enrichment of proteins that are localized to and play functional roles in nucleoli and mitochondria in S. cerevisiae, but not in S. pombe. Moreover, expanded polyQ Htt appears to interact preferentially with endogenous polyQ and Q/N-rich proteins, which are rare in S. pombe, as well as proteins containing coiled-coil motifs in S. cerevisiae. Taken together, these results suggest that polyQ expansion of Htt may cause cellular toxicity in S. cerevisiae by sequestering endogenous polyQ and Q/N-rich proteins, particularly within nucleoli and mitochondria
CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins
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
CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins
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
Prion Nucleation Site Unmasked by Transient Interaction with Phospholipid Cofactor
Infectious mammalian prions can be
formed de novo from purified
recombinant prion protein (PrP) substrate through a pathway that requires
the sequential addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
(POPG) and RNA cofactor molecules. Recent studies show that the initial
interaction between PrP and POPG causes widespread and persistent
conformational changes to form an insoluble intermediate species,
termed PrP<sup>Int1</sup>. Here, we characterize the mechanism and
functional consequences of the interaction between POPG and PrP. Negative-stain
electron microscopy of PrP<sup>Int1</sup> revealed the presence of
amorphous aggregates. Pull-down and photoaffinity label experiments
indicate that POPG induces the formation of a PrP<sup>C</sup> polybasic-domain-binding
neoepitope within PrP<sup>Int1</sup>. The ongoing presence of POPG
is not required to maintain PrP<sup>Int1</sup> structure, as indicated
by the absence of stoichiometric levels of POPG in solid-state NMR
measurements of PrP<sup>Int1</sup>. Together, these results show that
a transient interaction with POPG cofactor unmasks a PrP<sup>C</sup> binding site, leading to PrP<sup>Int1</sup> aggregation