"Silent" mutations are not always silent

Mutations leading to identical amino acid sequences can change protein folding and function

By | December 21, 2006

A mutation in a human gene that does not change the resulting amino acid can nevertheless change a protein's function, according to an online report from Science. The research marks the first time that the phenomenon has been confirmed in mammals. "The habit we all have of disregarding nucleotide changes that don't change protein sequence may not be a good one," coauthor Michael Gottesman at the National Cancer Institute in Bethesda, Md., told The Scientist. "This may be a generalizable phenomenon that may lead to changes in function we haven't been thinking about." Gottesman and his colleagues investigated MDR1, which encodes P-gp, a human membrane transport protein that helps cells pump out anticancer and other drugs. They focused on the C3435T, a single nucleotide polymorphism (SNP) that is synonymous or silent, encoding for the same amino acid as the gene's wild-type version. The researchers first used viral vectors against cancerous HeLa cells to express wild-type P-gp and a number of versions bearing single polymorphisms at C1236T, G2677T or C3435T. They also experimented with specific haplotypes, or sets of polymorphisms, including combinations of the three single polymorphisms studied. The team next examined how well these cells transported any of a variety of fluorescent molecules and tested how well P-gp inhibitors such as CsA, verapamil and rapamycin affected the protein's function. Gottesman and his colleagues found that wild-type P-gps and variants with just one polymorphism apparently functioned the same. However, the haplotypes with C3435T proved more resistant against inhibition from CsA and verapamil, though not rapamycin, when they also included one or two other polymorphisms. The same experiments were conducted with epithelial cells of African green monkey kidney origin, monkey kidney cells and human T cell lines, yielding similar results. "Observations regarding this polymorphism have been very confusing. Some people report it changes the transport of certain drugs, and other people find it has not much effect," Gottesman explained. "The fact that we need more than a single polymorphism to see this effect could explain some of the confusion." C3435T-bearing haplotypes apparently led to differences in how P-gp folded. Conformation-sensitive antibody UIC2 bound significantly differently to haplotype P-gp than to wild-type, and haplotype P-gp was more susceptible to digestion by the enzyme trypsin. The idea that synonymous mutations might lead to differently folded proteins was proposed by Ian Purvis at the University of Glasgow and his colleagues and, independently, by Anton Komar, now at Cleveland State University in Ohio. "Many cases of silent SNPs and their possible link to diseases should be reexamined," Komar, who did not participate in this study, told The Scientist. "Also, one should be quite careful in constructing artificial genes for the purposes of gene therapy, for example, and pay careful attention to the choice of synonymous codons." Gottesman and his colleagues speculate that synonymous mutations represent rare codons for which translation machinery is not optimized. The resulting interruption of the rate at which mRNAs are translated could affect how a protein is folded, they said. Recent experiments in prokaryotes suggest codon usage is not random. Gottesman noted this idea was inspired in part by conversations with Randall Kincaid at Veritas Labs in Rockville, Md., who noted that replacing rare codons in malarial genes with rare bacterial codons could lead to improved expression of those genes in bacteria by better matching bacterial translation rates. Gottesman readily acknowledged that evidence for the idea has not yet been produced. Still, "it's an interesting proposition," Wolfgang Sadee at Ohio State University in Columbus, who was not a coauthor, told The Scientist. Sadee suggested experiments that measure translation rates in vitro might help. He added that recent work of his own suggests that C3435T might affect mRNA folding, which in turn might affect mRNA translation rate and subsequent protein folding. Charles Q. Choi cchoi@the-scientist.com Links within this article: C. Kimchi-Sarfaty et al. "A 'Silent' Polymorphism in the MDR1 Gene Changes Substrate Specificity." Science, published online ahead of print Dec. 21, 2006. http://www.sciencemag.org Michael Gottesman http://ccr.cancer.gov/staff/staff.asp?profileid=5713 R. Lewis. "Race and the Clinic: Good Science?" The Scientist, Feb. 18, 2002. http://www.the-scientist.com/article/display/12869 D. Secko. "Phase I of HapMap Complete." The Scientist, Oct. 26, 2005. http://www.the-scientist.com/article/display/22811 I.J. Purvis et al. "The efficiency of folding of some proteins is increased by controlled rates of translation in vivo. A hypothesis," J. Mol. Biol. 193: 413-7. January 20, 1987. http://www.the-scientist.com/pubmed/3298659 I.A. Krasheninnikov, A.A. Komar, I.A. Adzhubei. "Role of the rare codon clusters in defining the boundaries of polypeptide chain regions with identical secondary structures in the process of co-translational folding of proteins," Dokl. Akad. Nauk. SSSR 303: 995-9, 1988. http://www.the-scientist.com/pubmed/3250842 Anton Komar http://web.bges.csuohio.edu/faculty/komar.htm F. Supek, K. Vlahovicek. "Comparison of codon usage measures and their applicability in prediction of microbial gene expressivity," BMC Bioinformatics 6: 182, July 2005. http://www.the-scientist.com/pubmed/16029499 Wolfgang Sadee http://medicine.osu.edu/pharmacology/1134.cfm D. Wang et al. "Multidrug resistance polypeptide 1 (MDR1, ABCB1) variant 3435C>T affects mRNA stability," Pharmacogenetics and Genomics 15: 693-704. October 2005. http://www.the-scientist.com/pubmed/16141795


December 21, 2006

Although the study is very interesting, the conclusion is novel only to those not familiar with the field of "proteins" or their translation or conformational maturation. Many molecular and developmental biologists are only now beginning to appreciate the importance of proteins, even though it has always been accepted that folded protein, not the gene, directly transmits inherited information into biological activity. To me, the finding is reminiscent of the so-called novel discovery by neuroscientists in the '90's that diseases could be caused by protein toxicity. Again, the notion was not novel, but estabished about 100 years earlier. If one reads the literature, he/she will be surprised by the numerous discoveries aready made in many in others fields. At least the referenced article might help continue the demise of the genomics era so we can return to the study of disease, and not merely focus on the inheritance of stored information. \n \n\n

December 22, 2006

In two congress presentations:\n[a]Anogianakis G, Anogeianaki A, Papaliagkas V. Do Synonymous Codons Point Towards a Thermodynamic Theory of Tissue Differentiation? Lecture Series on Computer and Computational Sciences, VSP International Science Publishers, Volume 1, 2004, pp. 1-3, The Netherlands and \n[b] AnogianakisG, KapritsosE, Makris Ch, MpaltasN, Perdikuri K, Themelis K, Tsakalidis A. Identification of protein patterns in nucleic acid sequences and exploration of synonymous codons in tissue differentiation. The 9th World Congress on CIRCUITS - SYSTEMS - COMMUNICATIONS and COMPUTERS. CSCC 2005. October 11-13, 2004, Athens, Greece.\nWe deduced on theoretical grounds that there is reason to the madness of DNA code redundancy and proposed an algorithm to search for potential candidates of what you describe as silent mutations in genomic datasets.\n\nWe , then, underscored the fact that linking tissue differentiation with thermodynamic constraints, in synonymous codon expression, has profound philosophical and even aesthetic implications: It reduces intellectual dependence on models of molecular evolution where extremely detailed designs in outcome have to be ?designed? by blind evolutionary forces and introduces a view whereby some of the most basic and best understood natural principles (those of thermodynamics) undertake to assist evolution in shaping the biological order at a more fundamental level. At the same time, its aesthetic implications sprout from the fact that it introduces a vision of differentiation very similar to that observed in a garden at spring. \n \nApart from its philosophical significance, however, the answer to the problem of whether the existence (and expression characteristics) of synonymous codons, also implies a thermodynamic theory of tissue differentiation, will have a significant impact on cancer research. Indeed a central issue of cancer research has to do with the mechanisms which cancer cells dedifferentiate, lose the ability to perform the normal functions of the normal cell type from which they mutated and often resemble embryonic cells. It is plausible, therefore, that in case cancer development can be associated with usage of the wrong codon synonyms, that gene therapies can be devised in a much more logical fashion. \n\nGeorge Anogianakis\nProfessor of Physiology\nAristotle University Faculty of Medicine\n54124 Thessaloniki Greece
Avatar of: Andy Roche

Andy Roche

Posts: 1

December 22, 2006

Very interesting study if for no other reason than it serves to highlight how a publication in 2002 by Cortazzo et al. in prokaryotes, has been ignored despite the significance of the studies detailed within. \n\n\n\n
Avatar of: Alan Carter

Alan Carter

Posts: 1

December 22, 2006

Great article. From Wes Hatfield's earlier work on codon pair usage (PNAS 1989;86;3699-3703), we are finding corellations between silent SNPs and the introduction or deletion of translational pause events at the ribosomal level.\n\nMuch work needs to be done on whether these pauses are key to a chaperone binding, secretion, folding and/or post-translational modification events but we know they exist.\n\nAlan Carter\nCODA Genomics

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