Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair - PubMed (original) (raw)
Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair
Jan Kosinski et al. Hum Mutat. 2010 Aug.
Abstract
Missense alterations of the mismatch repair gene MLH1 have been identified in a significant proportion of individuals suspected of having Lynch syndrome, a hereditary syndrome that predisposes for cancer of colon and endometrium. The pathogenicity of many of these alterations, however, is unclear. A number of MLH1 alterations are located in the C-terminal domain (CTD) of MLH1, which is responsible for constitutive dimerization with PMS2. We analyzed which alterations may result in pathogenic effects due to interference with dimerization. We used a structural model of CTD of MLH1-PMS2 heterodimer to select 19 MLH1 alterations located inside and outside two candidate dimerization interfaces in the MLH1-CTD. Three alterations (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) caused decreased coexpression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that these alterations interfere with dimerization. All three alterations are located within the dimerization interface suggested by our model. They also compromised mismatch repair, suggesting that defects in dimerization abrogate repair and confirming that all three alterations are pathogenic. Additionally, we provided biochemical evidence that four alterations with uncertain pathogenicity (p.Ala586Pro, p.Leu636Pro, p.Thr662Pro, and p.Arg755Trp) are deleterious because of poor expression or poor repair efficiency, and confirm the deleterious effect of eight further alterations.
Figures
Figure 1. Analysis of dimerization interface in the CTD of E. coli MutL, MLH1, and PMS2
(A) Molecular surface of MutL/MLH1/PMS2 CTD colored according to sequence conservation with a color gradient from blue (variable) to red (conserved). In (A) and (B) the "new" interface corresponding to the dimerization interface proposed in this and previous work (Kosinski, et al., 2008; Kosinski, et al., 2005) is encircled in black, the "old" interface proposed by others for E. coli MutL (Guarne, et al., 2004) and yMutLα (Cutalo, et al., 2006) encircled in green. (B) Molecular surface of MutL/MLH1/PMS2 CTDs colored according to atom type: carbon atoms colored gray, oxygen – red, nitrogen – blue, and sulfur atoms – orange. (C) Amino acid substitutions related to Lynch syndrome studied in this work mapped on the structural model of MutLα-CTD. Mutations are indicated as spheres corresponding to Cα atoms of corresponding residues, and colored according to their classification (Chao, et al., 2008): deleterious – red, VUS – green, and neutral - blue. Residues deleted in the p.Tyr750X variant (VUS) are indicated with a green rectangle. Structures are shown in cartoon representation; MLH1 is colored dark blue, PMS2 is colored gray. The "new" interface corresponding to the dimerization interface proposed in this and previous work (Kosinski, et al., 2008; Kosinski, et al., 2005) is colored orange, the "old" interface proposed by others for E. coli MutL (Guarne, et al., 2004) and yMutLα (Cutalo, et al., 2006) is colored magenta. The interface residues were defined by PROTORP server (Reynolds, et al., 2009) based on the alternative dimer models.
Figure 2. Expression of MLH1 variants
A. MLH1 wild-type (wt) or MLH1 variants were co-transfected with PMS2 into HEK293T cells. After 24h, extracts were prepared and 50 µg of extract was analyzed by SDS-PAGE and western blotting. β-Actin signals were used as loading controls. B. PMS2 and MLH1 form a stable heterodimer, and MLH1 also forms a stable protein when expressed alone. In contrast, PMS2 alone is quickly degraded. C. Several independent transfections of plasmids encoding wildtype MLH1 or its alterations into HEK293T cells using different transfection techniques were performed. Extracts were prepared and expression of MLH1 and PMS2 was analyzed by SDS-PAGE and western blotting. Expression was quantified as detailed in Materials and Methods, and average expression levels as well as standard deviations (n=4 to 10) were determined. MLH1 and PMS2 expression levels are shown with dark and light gray bars respectively, standard deviations are shown by black lines. Corrected p-values were determined for the expression data of all alterations of MLH1. Statistically significant reductions of expression (p<0.05 after correction for multiple testing) are marked by asterisks.
Figure 3. Co-immunoprecipitation of MutLα
MLH1 was precipitated from extracts with an antibody binding the N-terminus of MLH1 as detailed in Materials and Methods. The amount of co-precipitated PMS2 was determined in relation to wild-type (100%) (average; standard deviation when more than one experiment was performed): Gln542Leu (54; 26); Asn551Thr (87; 7); Leu559Arg (68; 25); Ala586Pro (17; 16); Asp601Gly (112; 7); Lys618Ala (109; 3); Leu622His (73; 8); Leu636Pro (78; 1); Pro648Leu (68); Pro654Leu (51); Arg659Leu (77); Arg659Gln (126); Thr662Pro (24); Glu663Gly (96); Leu749Pro (30; 24); Tyr750X (23;33); Arg755Ser (134;9); Arg755Trp (82; 37). One variant (p.Arg659Pro) was omitted from the analysis due to low expression.
Figure 4. MMR activity of MLH1 variants
MMR activity of MutLα heterodimer variants was assessed in vitro in parallel to wild-type MutLα as detailed in Materials and Methods. The mismatch is formed by the third thymine of an AseI restriction sequence (ATTAAT) within a 2 kbp plasmid. The unrecognizable mismatched AseI restriction site will be restored when subjected to a MMR reaction. The plasmid contains a second AseI restriction site, therefore unrepaired plasmids will be linearized by AseI ("lin."), while repaired plasmids will be cut into two fragments (1200 bp and 800 bp, "dig."). Numerical values of 4 independent measurements of the individual alterations were (mean and standard deviation): Gln542Leu, 44(18); Ala586Pro 24(27); Asp601Gly 96(5); Lys618Ala 92(8); Leu636Pro 77(9); Arg659Gln 97(4); Thr662Pro 89(11); Glu663Gly 92(7); Leu749Pro 33(34); Tyr750X 16(22); Arg755Trp 7(8).
Figure 5. Mapping of amino acid substitutions onto the structural model of MutLα-CTD with their effect on protein expression and dimerization
Substitutions are indicated as spheres corresponding to Cα atoms of corresponding residues, and colored according to their effect on protein expression and dimerization (red: interfering with dimerization, blue: not affecting dimerization and with good expression, green: significantly decreasing expression). Substitutions resulting in only moderately compromised expression and having no effect on PMS2 dimerization (p.Arg659Gln and p.Arg755Trp) are indicated as dashed green-blue spheres. Residues corresponding to p.Tyr750X variant are indicated as red rectangle. Structures are shown in cartoon representation; MLH1 is colored dark blue, PMS2 is colored gray. The "new" interface corresponding to the dimerization interface proposed in this and previous work (Kosinski, et al., 2008; Kosinski, et al., 2005) is colored orange, the "old" interface proposed by others for E. coli MutL (Guarne, et al., 2004) and yMutLα (Cutalo, et al., 2006) is colored magenta.
Similar articles
- Multivariate analysis of MLH1 c.1664T>C (p.Leu555Pro) mismatch repair gene variant demonstrates its pathogenicity.
Farrell MP, Hughes DJ, Drost M, Wallace AJ, Cummins RJ, Fletcher TA, Meany MA, Kay EW, de Wind N, Power DG, Andrews EJ, Green AJ, Gallagher DJ. Farrell MP, et al. Fam Cancer. 2013 Dec;12(4):741-7. doi: 10.1007/s10689-013-9652-9. Fam Cancer. 2013. PMID: 23712482 - Conservation of functional asymmetry in the mammalian MutLα ATPase.
Johnson JR, Erdeniz N, Nguyen M, Dudley S, Liskay RM. Johnson JR, et al. DNA Repair (Amst). 2010 Nov 10;9(11):1209-13. doi: 10.1016/j.dnarep.2010.08.006. DNA Repair (Amst). 2010. PMID: 20864418 Free PMC article. - Epigenetic mechanisms in the pathogenesis of Lynch syndrome.
Peltomäki P. Peltomäki P. Clin Genet. 2014 May;85(5):403-12. doi: 10.1111/cge.12349. Epub 2014 Feb 17. Clin Genet. 2014. PMID: 24443998 Review. - Mismatch repair gene deficiency and genetic anticipation in Lynch syndrome: myth or reality?
Ponti G, Ruini C, Tomasi A. Ponti G, et al. Dis Colon Rectum. 2015 Jan;58(1):141-2. doi: 10.1097/DCR.0000000000000275. Dis Colon Rectum. 2015. PMID: 25489705 Review. No abstract available.
Cited by
- MRE11A: a novel negative regulator of human DNA mismatch repair.
Du D, Yang Y, Zhang Y, Wang G, Chen L, Guan X, Rasmussen LJ, Liu D. Du D, et al. Cell Mol Biol Lett. 2024 Mar 14;29(1):37. doi: 10.1186/s11658-024-00547-z. Cell Mol Biol Lett. 2024. PMID: 38486171 Free PMC article. - A conserved motif in the disordered linker of human MLH1 is vital for DNA mismatch repair and its function is diminished by a cancer family mutation.
Wolf K, Kosinski J, Gibson TJ, Wesch N, Dötsch V, Genuardi M, Cordisco EL, Zeuzem S, Brieger A, Plotz G. Wolf K, et al. Nucleic Acids Res. 2023 Jul 7;51(12):6307-6320. doi: 10.1093/nar/gkad418. Nucleic Acids Res. 2023. PMID: 37224528 Free PMC article. - Functional characterization of MLH1 missense variants unveils mechanisms of pathogenicity and clarifies role in cancer.
Mahdouani M, Ben Ahmed S, Hmila F, Rais H, Ben Sghaier R, Saad H, Ben Said M, Masmoudi S, Hmida D, Brieger A, Zeuzem S, Saad A, Gribaa M, Plotz G. Mahdouani M, et al. PLoS One. 2022 Dec 1;17(12):e0278283. doi: 10.1371/journal.pone.0278283. eCollection 2022. PLoS One. 2022. PMID: 36454741 Free PMC article. - Inhibition of ABL1 by tyrosine kinase inhibitors leads to a downregulation of MLH1 by Hsp70-mediated lysosomal protein degradation.
Daniels HG, Knicely BG, Miller AK, Thompson A, Plattner R, Goellner EM. Daniels HG, et al. Front Genet. 2022 Oct 20;13:940073. doi: 10.3389/fgene.2022.940073. eCollection 2022. Front Genet. 2022. PMID: 36338985 Free PMC article. - Site of analysis matters - Ongoing complete response to Nivolumab in a patient with HIV/HPV related metastatic anal cancer and MLH1 mutation.
Demes M, Pession U, Jeroch J, Schulze F, Eichler K, Martin D, Wild P, Waidmann O. Demes M, et al. Oncotarget. 2022 Sep 14;13:1034-1042. doi: 10.18632/oncotarget.28274. eCollection 2022. Oncotarget. 2022. PMID: 36128324 Free PMC article.
References
- Brieger A, Plotz G, Raedle J, Weber N, Baum W, Caspary WF, Zeuzem S, Trojan J. Characterization of the nuclear import of human MutLalpha. Mol.Carcinog. 2005;43(1):51–58. - PubMed
- Burt R, Neklason DW. Genetic testing for inherited colon cancer. Gastroenterology. 2005;128(6):1696–1716. - PubMed
- Chang DK, Ricciardiello L, Goel A, Chang CL, Boland CR. Steady-state regulation of the human DNA mismatch repair system. J.Biol.Chem. 2000;275(24):18424–18431. - PubMed
- Chao EC, Velasquez JL, Witherspoon MS, Rozek LS, Peel D, Ng P, Gruber SB, Watson P, Rennert G, Anton-Culver H, et al. Accurate classification of MLH1/MSH2 missense variants with multivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR) Hum Mutat. 2008;29(6):852–860. - PubMed
- Chen-Shtoyerman R, Theodor L, Harmati E, Friedman E, Dacka S, Kopelman Y, Sternberg A, Zarivach R, Bar-Meir S, Fireman Z. Genetic analysis of familial colorectal cancer in Israeli Arabs. Hum Mutat. 2003;21(4):446–447. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases