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(The fitness cost of, and response to, misfolded proteins)
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==Key Questions==
 
#What proportion of newly synthesized proteins fail to fold, and why?
 
#How do cells decide when a misfolded should be degraded rather than given another chance to fold?
 
#What is the cost of producing a protein that misfolds, compared to the cost if that protein folds properly?
 
#Why are misfolded proteins costly?
 
#How does inaccuracy in the translational apparatus (ribosomes, aa-tRNA synthetases, etc.) shape the evolution of coding sequences and proteins?
 
  
==The fitness cost of, and cellular response to, misfolded proteins==
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[[Image:Synthesis-to-misfolding.png]]
  
Evolving lineages face a constant intracellular threat: most new coding-sequence
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What are the spectrum, frequency and consequences of errors in protein synthesis? How do cells sense and respond to misfolded proteins, particularly in the eukaryotic cytosol? How does stochasticity in protein synthesis alter the composition and stability of the proteome? What benefits do errors confer? Does error-induced protein misfolding influence the progression of neurodegenerative diseases such as ALS?
mutations destabilize the folding of the encoded protein. Misfolded proteins form
 
insoluble aggregates and are hypothesized to be intrinsically cytotoxic. Here,
 
we experimentally isolate a fitness cost caused by toxicity of misfolded proteins. We
 
exclude other costs of protein misfolding, such as loss of functional protein or attenuation
 
of growth-limiting protein-synthesis resources, by comparing growth rates of budding
 
yeast expressing folded or misfolded variants of a gratuitous protein, yellow fluorescent
 
protein (YFP), at equal levels. We quantify a fitness cost that increases with misfolded
 
protein abundance, up to as much as a 3.2% growth-rate reduction when misfolded YFP
 
represents less than 0.1% of total cellular protein. Comparable experiments on variants of
 
the yeast gene URA3 produce similar results. Quantitative proteomic measurements
 
reveal that within the cell, misfolded YFP induces coordinated synthesis of interacting
 
cytosolic chaperone proteins in the absence of a wider stress response, providing
 
evidence for an evolved modular response to misfolded proteins in the cytosol. These
 
results underscore the distinct and evolutionarily relevant molecular threat of protein
 
misfolding, independent of protein function. Assuming most misfolded proteins impose
 
similar costs, yeast cells express almost all proteins at steady-state levels sufficient to
 
expose their encoding genes to selection against misfolding, lending credibility to the
 
recent suggestion that such selection imposes a global constraint on molecular evolution.
 
  
[[Drummond/MisfoldingCost2010|supporting website here]].
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We are pursuing mechanistic answers to these questions, taking a biochemical and genetic approach, with an emphasis on developing high-resolution, high-mass-accuracy mass spectrometric techniques for proteome-scale quantitation. From a theoretical standpoint, we are interested in understanding the imprints that natural selection on fidelity and misfolding leave on evolving genes and genomes.
  
==Mistranslation-induced misfolding and gene evolution==
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==Spectrum, frequency, and consequences of errors in protein synthesis==
Strikingly consistent correlations between rates of coding-sequence evolution and gene expression levels are apparent across taxa, but the biological causes behind the selective pressures on coding-sequence evolution remain controversial. Here we demonstrate conserved patterns of simple covariation between sequence evolution, codon usage, and mRNA level in E. coli, yeast, worm, fly, mouse, and human that suggest that all observed trends stem largely from a unified underlying selective pressure. In metazoans, these trends are strongest in tissues composed of neurons, whose structure and lifetime confer extreme sensitivity to protein misfolding. We propose, and demonstrate using a molecular-level evolutionary simulation, that selection against toxicity of misfolded proteins generated by ribosome errors suffices to create all the observed covariation. The mechanistic model of molecular evolution which emerges yields testable biochemical predictions, calls into question use of nonsynonymous-to-synonymous substitution ratios (Ka/Ks) to detect functional selection, and suggests how mistranslation may contribute to neurodegenerative disease.
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At the canonical translational misreading error rate of 1 error per 2000 amino acids translated, <em>one in five</em> average-length proteins will contain at least one amino acid not encoded in the messenger RNA. Single amino-acid substitutions frequently destabilize proteins, sometimes disrupting protein folding. Historical measurements of translational error focus on single codons, often in heterologous reporter constructs, leaving open basic questions, such as: how does the frequency of translation errors vary between proteins and along a single protein sequence? Do proteins differ in their tolerance for errors? Are error rates predictable from codons alone, or do other features of transcriptions or nascent polypeptides modulate error rates?
  
===Citation===
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We have assembled evidence that high-expression proteins are under selection to reduce errors (increased translational accuracy), and to fold properly whether error-free or, particularly, despite the errors that do occur (increased translational robustness). We are using high-resolution, high-mass-accuracy mass spectrometry to survey the actual sequences of proteins <i>in vivo</i>, and to address these basic questions about variability in error rates and consequences across the proteome.
<biblio>
 
#Drummond-Cell-2008 pmid=18662548
 
</biblio>
 
  
Download an author's [[Media:Drummond08-MisfoldingPreprint.pdf|preprint]] and [[Media:Drummond08-MisfoldingPreprintSuppl.pdf|supplementary materials]].
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==Response to misfolded proteins==
 
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When proteins misfold, they become aggregation-prone, toxic species that must be identified and processed by the cell. We study the cytosolic unfolded protein response (UPR<sup>cyto</sup>), the analogue of the well-known unfolded protein response in the endoplasmic reticulum (UPR<sup>ER</sup>). In budding yeast, the appearance of a defined unfolded protein in the cytosol at normal growth temperature induces ~25 interacting proteins involved in cytosolic protein quality control. Taking a biochemical and genetic approach, we are studying the regulation of the UPR<sup>cyto</sup>, asking questions such as: how are unfolded proteins recognized mechanistically? How is this signal propagated into the nucleus to induce gene expression? How does the response return to baseline after the intracellular threat has been dispatched?
===Data===
 
 
 
====Evolution and expression data====
 
These tab-delimited files include gene and ortholog identifiers, dN, dS, ts/tv ratio, expression level, Fop, and (for the multicellular organisms) intronic guanine/cytosine (GC) content.
 
*[[Media:DrummondWilkeCell2008Data.zip|Evolution and expression data]] [ZIP archive, ~800K]
 
 
 
====Coding sequence alignments====
 
Alignments are in FASTA format, ZIP-compressed.
 
*[[Media:Ecoli-styphimurium-alignments.zip|<i>E. coli</i> vs. <i>S. typhimurium</i>]] (~1.8MB)
 
*[[Media:Scer-spar-alignments.zip|<i>S. cerevisiae</i> vs. <i>S. paradoxus</i>]] (~3.8MB)
 
*[[Media:Cele-cbri-alignments.zip|<i>C. elegans</i> vs. <i>C. briggsae</i>]] (~4.7MB)
 
*[[Media:Dmel-dyak-alignments.zip|<i>D. melanogaster</i> vs. <i>D. yakuba</i>]] (~5.3MB)
 
*[[Media:Mouse-rat-alignments.zip|<i>M. musculus</i> vs. <i>R. norvegicus</i>]] (~8.7MB)
 
*[[Media:Human-dog-alignments.zip|<i>H. sapiens</i> vs. <i>C. familiaris</i>]] (~8.3MB)
 
  
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Latest revision as of 19:37, 20 January 2013

Synthesis-to-misfolding.png

What are the spectrum, frequency and consequences of errors in protein synthesis? How do cells sense and respond to misfolded proteins, particularly in the eukaryotic cytosol? How does stochasticity in protein synthesis alter the composition and stability of the proteome? What benefits do errors confer? Does error-induced protein misfolding influence the progression of neurodegenerative diseases such as ALS?

We are pursuing mechanistic answers to these questions, taking a biochemical and genetic approach, with an emphasis on developing high-resolution, high-mass-accuracy mass spectrometric techniques for proteome-scale quantitation. From a theoretical standpoint, we are interested in understanding the imprints that natural selection on fidelity and misfolding leave on evolving genes and genomes.

Spectrum, frequency, and consequences of errors in protein synthesis

At the canonical translational misreading error rate of 1 error per 2000 amino acids translated, one in five average-length proteins will contain at least one amino acid not encoded in the messenger RNA. Single amino-acid substitutions frequently destabilize proteins, sometimes disrupting protein folding. Historical measurements of translational error focus on single codons, often in heterologous reporter constructs, leaving open basic questions, such as: how does the frequency of translation errors vary between proteins and along a single protein sequence? Do proteins differ in their tolerance for errors? Are error rates predictable from codons alone, or do other features of transcriptions or nascent polypeptides modulate error rates?

We have assembled evidence that high-expression proteins are under selection to reduce errors (increased translational accuracy), and to fold properly whether error-free or, particularly, despite the errors that do occur (increased translational robustness). We are using high-resolution, high-mass-accuracy mass spectrometry to survey the actual sequences of proteins in vivo, and to address these basic questions about variability in error rates and consequences across the proteome.

Response to misfolded proteins

When proteins misfold, they become aggregation-prone, toxic species that must be identified and processed by the cell. We study the cytosolic unfolded protein response (UPRcyto), the analogue of the well-known unfolded protein response in the endoplasmic reticulum (UPRER). In budding yeast, the appearance of a defined unfolded protein in the cytosol at normal growth temperature induces ~25 interacting proteins involved in cytosolic protein quality control. Taking a biochemical and genetic approach, we are studying the regulation of the UPRcyto, asking questions such as: how are unfolded proteins recognized mechanistically? How is this signal propagated into the nucleus to induce gene expression? How does the response return to baseline after the intracellular threat has been dispatched?