Abstract
Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies including cancer. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA replication and repair. Here, we present the results of a large-scale proteome analysis that has been undertaken to determine protein partners of different polymorphic WRN proteins found with relatively high prevalence in the human population. We expressed different fluorescently tagged-WRN (eYFP-WRN) variants in human 293 embryonic kidney cells (HEK293) and used a combination of affinity-purification and mass spectrometry to identify different compositions of WRN-associated protein complexes. We found that a WRN variant containing a phenylalanine residue at position 1074 and an arginine at position 1367 (eYFP-WRN(F-R)) possesses more affinity for DNA-PKc, KU86, KU70, and PARP1 than a variant containing a leucine at position 1074 and a cysteine at position 1367 (eYFP-WRN(L-C)). Such results were confirmed in a WRN-deficient background using WS fibroblasts. Interestingly, the exonuclase activity of WRN recovered from immunoprecipitated eYFP-WRN(L-C) variant was lower than the eYFP-WRN(F-R) in WS cells. Finally, HEK293 cells and WS fibroblasts overexpressing the eYFP-WRN(F-R) variant were more resistant to the benzene metabolite hydroquinone than cells expressing the eYFP-WRN(L-C) variant. These results indicate that the protein-protein interaction landscape of WRN is subject to modulation by polymorphic amino acids, a characteristic associated with distinctive cell survival outcome.
Highlights
Werner Syndrome (WS; MIM number #277700) is a rare autosomal recessive disorder that displays many of the clinical symptoms of aging at an early age
By combining large-scale LC-MS/MS identification of affinity-purified eYFPWRN-associated proteins and bioinformatics-based classification, this study represents the first large-scale proteomic identification of proteins binding onto different polymorphic variants of WRN and provides insights into the pathways that could be modulated by different coding single nucleotide polymorphisms (SNPs) in the WRN gene
We found that the WRN(L-C) polymorphism causes a significant drop in binding affinity to major DNA damage response factors such as DNA-PKc, KU70, KU86 and poly(ADP-ribose) polymerase 1 (PARP1) when expressed in both HEK293 cells and WRN-deficient human fibroblasts derived from a WS subject
Summary
Werner Syndrome (WS; MIM number #277700) is a rare autosomal recessive disorder that displays many of the clinical symptoms of aging at an early age From their second decade of life onward, WS patients develop pathologies that prematurely resemble many traits of normal aging such as osteoporosis, ocular cataracts, graying and loss of hair, diabetes mellitus, arteriosclerosis, and cancer [1,2,3,4]. WS cells are characterized by the presence of deletions and variegated chromosomal translocations [5, 6] Processes such as DNA replication or transcription generate regions of single-stranded DNA, which may inadvertently provide a substrate for the initiation of recombination. A mutation in WRN may lead to an increased frequency of illegitimate recombination during the repair of breaks at transcriptional sites or DNA replication forks, creating small deletions or variegated chromosomal translocations. The most frequent cancers in WS patients are thyroid neoplasms, malignant melanoma, meningioma, soft tissue sarcomas, leukemia and preleukemic conditions of the bone marrow [18]
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