Inference of functional regions in proteins by quantification of evolutionary constraints

Simon, Alexander L.; Stone, Eric A.; Sidow, Arend

doi:10.1073/pnas.042692299

Cited by 67 publications

(60 citation statements)

References 35 publications

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“…As noted by several previous studies, frequency of a variant in the p53 somatic mutation database (Olivier et al 2002) is a proxy for the likelihood that the variant will allow tumor formation, and does not primarily reflect variation in mutation rate (Walker et al 1999;Simon et al 2002). We stratified variants according to their count in the somatic mutation database as appearing one to four times (663 variants, 1293 mutations), five to nine times (206 variants, 1346 mutations), or greater than nine times (248 variants, 10,870 mutations).…”

Section: Human Diseasementioning

confidence: 99%

Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity

Stone¹,

Sidow²

2005

Genome Res.

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347

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We find that the degree of impairment of protein function by missense variants is predictable by comparative sequence analysis alone. The applicable range of impairment is not confined to binary predictions that distinguish normal from deleterious variants, but extends continuously from mild to severe effects. The accuracy of predictions is strongly dependent on sequence variation and is highest when diverse orthologs are available. High predictive accuracy is achieved by quantification of the physicochemical characteristics in each position of the protein, based on observed evolutionary variation. The strong relationship between physicochemical characteristics of a missense variant and impairment of protein function extends to human disease. By using four diverse proteins for which sufficient comparative sequence data are available, we show that grades of disease, or likelihood of developing cancer, correlate strongly with physicochemical constraint violation by causative amino acid variants.[Supplemental material is available online at www.genome.org. A Java executable of MAPP and documentation are freely available for download at http://mendel.stanford.edu/supplementarydata/stone_MAPP_2005.]Missense mutations that impair protein function may result in disease. For diseases caused by such deleterious mutations, a simple but plausible model presents itself: The type of disease is dependent on when and where the protein's function is required in the organism. Given the type of disease, its severity is likely determined by at least three parameters: (1) the degree to which the function of the protein is impaired by the missense mutation; (2) variants of other genes that modulate the effect of the major locus, also referred to as genetic background; and (3) the environment. We present here a predictive statistical framework for the first of these parameters, impairment of protein function by missense mutations.Our study was motivated by several observations relevant to protein structure, function, and evolution, and by previous studies that addressed the relationship between the nature of missense variants, impairment of protein function, and resulting disease ( In aggregate, these studies suggest that mutations in evolutionarily conserved sites tend to impair protein function and lead to disease. There is also weak evidence that protein impairment and disease severity are somewhat correlated with the physicochemical difference between the original amino acid and the missense variant. However, no transparent, quantitative relationship between evolutionary constraint, functional impairment, and disease severity has been described. We believe that there has been a methodological barrier to uncovering such a relationship.Our analysis rests on two complementary ideas: (1) that differences in standard physicochemical properties between the "wild-type" amino acid and the missense variant are the root cause of functional impairment; and (2) that evolutionary variation among orthologs in the affected position is a sample ...

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Section: Human Diseasementioning

confidence: 99%

Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity

Stone¹,

Sidow²

2005

Genome Res.

Self Cite

347

319

View full text Add to dashboard Cite

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“…In addition, within a given protein, the rates of evolution of individual amino acids vary greatly, largely as a result of the structure-function requirements for a given amino acid at a particular position within that protein. For example, active sites of enzymes, DNA-binding domains of transcription factors, and residues important for structural maintenance evolve slowly, as substitutions in these residues are particularly deleterious (Suckow et al 1996;Simon et al 2002).…”

Section: Sequence Function and Evolutionary Ratementioning

confidence: 99%

Qualifying the relationship between sequence conservation and molecular function

Cooper¹,

Brown²

2008

Genome Res.

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Quantification of evolutionary constraints via sequence conservation can be leveraged to annotate genomic functional sequences. Recent efforts addressing the converse of this relationship have identified many sites in metazoan genomes with molecular function but without detectable conservation between related species. Here, we discuss explanations and implications for these results considering both practical and theoretical issues. In particular, phylogenetic scope influences the relationship between sequence conservation and function. Comparisons of distantly related species can detect constraint with high specificity due to the loss of conserved neutral sequence, but sensitivity is sacrificed as a result of functional changes related to lineage-specific biology. The strength of natural selection operating on functional sequence is also important. Mutations to functional sequences that result in small fitness effects are subject to weaker constraints. Therefore, particularly when comparing highly divergent species, functional sequences that are degenerate or biologically redundant will be prone to turnover, wherein functional sequences are replaced by effectively equivalent, but nonorthologous counterparts. Finally, considering the size and complexity of metazoan genomes and the fact that many nonconserved sequences are associated with sequence-degenerate, low-level molecular functions, we find it likely that there exist many biochemically functional sequences that are not under constraint. This hypothesis does not lead to the conclusion that huge amounts of vertebrate genomes are functionally important, but rather that such "functionality" represents molecular noise that has weak or no effect on organismal phenotypes.

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“…Local evolutionary rates over the primary sequence of NPC2 protein were estimated by using the Evolution Structure Function method (13). Briefly, a multiple sequence alignment of vertebrate NPC2 homologs was made with CLUSTALW (14), a phylogenetic tree was built by using the program SEMPHY (15) and, holding the branching pattern of this tree constant, SEMPHY was used to calculate the number of substitutions per site in each window of 11 amino acid residues over the entire alignment.…”

Section: Purification Of Npc2mentioning

confidence: 99%

“…To aid in designing mutants that disrupt NPC2 function, we identified evolutionarily constrained regions (ECRs) of the protein (13) and developed a homology model of the NPC2 protein structure. The ECR procedure assigns sequence conservation scores by comparing sliding windows of 11 aa.…”

Section: Rationale For Site-directed Mutagenesis: Evolutionary Analysmentioning

confidence: 99%

The integrity of a cholesterol-binding pocket in Niemann–Pick C2 protein is necessary to control lysosome cholesterol levels

Binkley

Sidow

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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181

196

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The neurodegenerative disease Niemann-Pick Type C2 (NPC2) results from mutations in the NPC2 (HE1) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (K d ‫؍‬ 30 -50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.point mutants ͉ secretion ͉ protein evolution N iemann-Pick Type C (NPC) is an autosomal-recessive disorder characterized by progressive ataxia, dystonia, and dementia (1). Substantial cell death, particularly in the cerebellum, accounts for some of the symptoms. At the cellular level, mutant cells accumulate cholesterol and other lipids in aberrant compartments with features of late endosomes and lysosomes, and the normal homeostatic response to this excess cholesterol is abolished (2-4).Our understanding of the molecular basis of this disease has progressed substantially over the last several years, because the two genes damaged by NPC mutations, NPC1 and NPC2, have been identified (5-7). NPC1 encodes a multiple-membranespanning protein containing sequences similar to the sterol cleavage activating protein (SCAP) regulator of cholesterol metabolism and the receptor Patched, a transducer of the Hedgehog family of protein signals. Mutations in NPC1 are responsible for Ϸ95% of the NPC cases (8). The exact role that NPC1 plays in maintaining normal levels of cholesterol in late endosomes and lysosomes remains mysterious. Even less is known about the NPC2 [human epididymis (HE1)] protein, the loss of which is responsible for 5% of NPC cases.NPC2 is a small secreted glycoprotein originally identified as a transcript enriched in the HE1 (9). The porcine HE1 homolog was reported to bind cholesterol with micromolar affinity (K d ϭ 2.3 M; ref. 10), information that was crucial in identifying HE1 as a candidate npc2 gene product (7). However, the similarity between the reported K d and the solubility of cholesterol in aqueous solution (5 M, Merck Index) warrants reexamination of the strength and specificity of this interaction. If the binding is meaningful, NPC2 activity could be altered when cholesterol is bound, thus serving as a sensor of cholesterol levels, accessibility, or location. Alternatively, NPC2 could process or transport cholesterol. Resolving these questions is crucial for determining why cholesterol accumulates in people lacking functional NPC2 protein and for designing rational treatment strategies.In this report, we combine in vitro binding experiments, genetic analysis, a quantitative cell-based ...

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Inference of functional regions in proteins by quantification of evolutionary constraints

Cited by 67 publications

References 35 publications

Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity

Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity

Qualifying the relationship between sequence conservation and molecular function

The integrity of a cholesterol-binding pocket in Niemann–Pick C2 protein is necessary to control lysosome cholesterol levels

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