Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2

Abstract

MSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of more than half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wildtype levels restored mismatch repair, suggesting that overexpression is not always required to regain function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants and identified the primary ubiquitin ligase as San1. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. The unstable variants interacted with San1, whereas wild-type Msh2 did not. Additionally, san1Δ suppressed the mismatch repair defect of unstable variants. Of medical significance, the clinically approved drug Bortezomib partially restored protein levels and mismatch repair function for low-level variants and reversed the resistance to cisplatin, a common chemotherapeutic. Our results provide the foundation for an innovative therapeutic regime for certain mismatch-repair-defective cancers that are refractory to conventional chemotherapies.Lynch syndrome | MutS | mutator | hereditary nonpolyposis clorectal cancer L ynch syndrome, also known as hereditary nonpolyposis colorectal cancer, is a leading cause of inherited cancer mortality in the United States (1). Approximately 2-7% of colorectal cancer cases are the consequence of Lynch syndrome, a dominant and highly penetrant disease afflicting individuals at an early age (1). Although colorectal is the most common form of cancer found in Lynch syndrome families, endometrial, ovarian, stomach, small intestine, liver, gallbladder ducts, upper urinary tract, brain, skin, and prostate cancers are all associated with the syndrome.Lynch syndrome cancers, as well as many sporadic tumors, are a consequence of defects in mismatch repair; for example, ∼17% of all colorectal cancers originate from defects in mismatch repair (2). DNA mismatch repair is a conserved mechanism that significantly contributes to the accurate preservation of genetic material (3). Mismatch recognition is accomplished in eukaryotes by MutS heterodimers in which Msh2 is the invariant partner (4). Mismatch binding initiates subsequent events, including cleavage and excision of the error-containing strand followed by new synthesis and ligation (5). Without mismatch repair, DNA displays microsatellite instability and acquires numerous mutations, some of which are deleterious. Included among the types of harmfu...

Predicting the impact of Lynch syndrome-causing missense mutations from structural calculations

“…Previous studies on yeast MSH2 mutants suggest that a high proportion of missense mutations affect the steady-state protein levels [7,50]. Our studies on human MSH2 variants confirm these findings.…”

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

“…Several genetic diseases corresponding to missense variants that are targeted by the ubiquitin-proteasome pathway, including cystic fibrosis, congenital erythropoietic porphyria, neurofibromatosis, Lynch syndrome, classic late-infantile neuronal ceroid lipofuscinosis, multiple endocrine neoplasia type 1, ␣-1 antitrypsin deficiency, and phenylketonuria, have been described previously (38)(39)(40)(41)(42)(43)(44). In several of the described cases, the destabilized mutant protein may nevertheless retain substantial functional properties.…”

Cytosolic Hsp60 Can Modulate Proteasome Activity in Yeast

“…It is possible that in these latter tumors, the increase in Hsp60c inhibits the proteasome, making the cells more susceptible to apoptosis induced by chemotherapy. An excellent recent example for the use of a proteasome inhibitor is Bortezomib, which can restore Msh2 protein levels and its mismatch repair function in lowlevel variants and thereby reverse the resistance to cisplatin, a common chemotherapeutic (47). Fascinating future questions to be approached are whether Hsp60 inhibits proteasome activity in mammalian cells and in which disease processes it is involved.…”