Functional consequences of NLS mutations in human MLH1 Alex Dukes Dr. Andrew Buermeyer Department of...
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Transcript of Functional consequences of NLS mutations in human MLH1 Alex Dukes Dr. Andrew Buermeyer Department of...
Functional consequences Functional consequences of NLS mutations in of NLS mutations in
human MLH1human MLH1Alex DukesAlex Dukes
Dr. Andrew BuermeyerDr. Andrew BuermeyerDepartment of Environmental & Department of Environmental &
Molecular ToxicologyMolecular ToxicologyOregon State UniversityOregon State University
HHMI Program, Summer 2007HHMI Program, Summer 2007
The Importance of The Importance of Mismatch Repair (MMR)Mismatch Repair (MMR)
Cellular mechanism for preventing DNA mutations, resulting from:Cellular mechanism for preventing DNA mutations, resulting from: Replication errorsReplication errors Recombination pathwaysRecombination pathways Exogenous DNA damaging agentsExogenous DNA damaging agents
Necessary to trigger apoptosis in event of extensive DNA damageNecessary to trigger apoptosis in event of extensive DNA damage
MMR and Colon CancerMMR and Colon Cancer
Colorectal cancers Colorectal cancers account for 10% of all account for 10% of all new cancers new cancers
Lynch Syndrome, a Lynch Syndrome, a type of hereditary type of hereditary colon cancer (HNPCC)colon cancer (HNPCC)
Caused by genetic Caused by genetic defects in MMR genesdefects in MMR genes
©2005 American Cancer Society, National Cancer Institute
A Component of MMR: A Component of MMR: MutLMutLαα
Heterodimer composed of MLH1 Heterodimer composed of MLH1 and PMS2 proteinsand PMS2 proteins
In the absence of MMR, cells In the absence of MMR, cells display:display: Increased mutation rate Increased mutation rate Decreased apoptotic response to Decreased apoptotic response to
genotoxins genotoxins Mutations in MLH1 account for 30-Mutations in MLH1 account for 30-
40% of Lynch Syndrome cases40% of Lynch Syndrome cases
Two Mutations of Two Mutations of InterestInterest
Missense mutations both identified in cancer Missense mutations both identified in cancer patientspatients
Nuclear localization sequence (NLS): Nuclear localization sequence (NLS): Acts as a cellular “password” to allow protein into Acts as a cellular “password” to allow protein into
nucleus via the nuclear porenucleus via the nuclear pore amino acids 470 – 474 in MLH1amino acids 470 – 474 in MLH1
Image courtesy of : www.scripps.edu/.../proj/NPC/NucleusWhite.gif
Image courtesy of: Wu, X., et al, Dimerization of MLH1 and PMS2 Limits Nuclear Localization of MutL. Molecular and Cellular Biology, 2003. 23(9): p. 3320–3328
Mutation #1Mutation #1 Amino acid 474Amino acid 474 Replaces arginine (R) with Replaces arginine (R) with
glutamine (Q)glutamine (Q) Charged Charged Charged Charged
R474QR474Q
Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif
Mutation #2Mutation #2
Amino acid 472Amino acid 472Replaces arginine (R) with isoleucine Replaces arginine (R) with isoleucine (I)(I)Charged Charged Non-polar (more Non-polar (more deleterious?)deleterious?)
R472IR472I
Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif
Project ObjectivesProject Objectives Examine two NLS mutations in MLH1 and Examine two NLS mutations in MLH1 and
identify the phenotypic consequences of identify the phenotypic consequences of eacheach
Experimental MethodsExperimental Methods Transient transfectionTransient transfection Stable transfectionStable transfection
The Transient The Transient TransfectionTransfection
Plasmids encoding mutated MLH1 and PMS2 Plasmids encoding mutated MLH1 and PMS2 are introduced into MutLare introduced into MutLαα- deficient cells- deficient cells
Cells assessed to determine protein Cells assessed to determine protein expression and localization patternexpression and localization pattern
Is the mutant MutLIs the mutant MutLαα heterodimer stable? heterodimer stable?Does the mutation interfere with normal localization patterns?Does the mutation interfere with normal localization patterns?
Nuclear Cytoplasmic
MutLα present
MutLα absent
The Transient The Transient TransfectionTransfection
Lipid-based transfection
The Stable TransfectionThe Stable Transfection
Plasmid encoding mutated MLH1 is Plasmid encoding mutated MLH1 is introduced into MLH1- deficient cellsintroduced into MLH1- deficient cells
Cells exhibiting reduced MMR Cells exhibiting reduced MMR function identified by:function identified by: Increased mutation levels Increased mutation levels Loss of apoptotic responseLoss of apoptotic response
Does the mutation result in reduced MMR function? Does the mutation result in reduced MMR function?
This assay also can be used to confirm the This assay also can be used to confirm the heterodimer stability and localization pattern heterodimer stability and localization pattern
found in the transient transfection.found in the transient transfection.
The Stable TransfectionThe Stable Transfection
Selection with cytotoxic G418
Positive ControlNegative Control R472I R474Q
Electroporation
Plating from the Stable Plating from the Stable TransfectionTransfection
Freeze cell lines
Ouabain AssayMutation frequency Survival following
DNA damage response
6-TG Assay
Pass 1:10
Pass 1:10
Interpretation of possible Interpretation of possible outcomesoutcomes
LocalizationLocalization MMR MMR PhenotypePhenotype
Interpretation of ResultsInterpretation of Results
CytoplasmicCytoplasmic DeficientDeficientMutation interferes with Mutation interferes with localization causing MMR localization causing MMR deficiencydeficiency
Hypothesis: Both mutations interfere with Hypothesis: Both mutations interfere with localization and cause MMR deficiency.localization and cause MMR deficiency.
CytoplasmicCytoplasmic ProficientProficient Low levels of MutLLow levels of MutLαα enter enter nucleus and are sufficient for nucleus and are sufficient for MMR phenotypeMMR phenotype
NuclearNuclear DeficientDeficient
NuclearNuclear ProficientProficient
Mutation interferes with Mutation interferes with enzymatic function or other enzymatic function or other protein activity, not localizationprotein activity, not localizationMutation does not interfere Mutation does not interfere with normal localizationwith normal localization
Transient Transfection Transient Transfection ResultsResults
PMS2
Only
R474Q
R47
2I
1 2 3 4W
ild T
ype
MSH6
PMS2
MLH1
Both R474Q and R472I mutants stabilize PMS2
Stable Transfection Stable Transfection Results: Results:
Protein Expression LevelsProtein Expression Levels
MLH1 accumulates similar to wild type
MLH1 stabilizes PMS2 similar to wild
type
Stable Transfection Stable Transfection Results: Results:
Cellular LocalizationCellular Localization
Mutation #2: R472IMutation #1: R474Q
Both mutants exhibit nuclear localization
Stable Transfection Stable Transfection Results: Results: Mutation FrequencyMutation Frequency
MMR Proficient
(LOW mutant frequency)
MMR Deficient
(HIGH mutant frequency)
Stable Transfection Stable Transfection Results: Results: Cytotoxic ResponseCytotoxic Response
MMR Proficient
(LOW survival)
MMR Deficient (HIGH
survival)
Summary of ResultsSummary of Results Localization?Localization? NUCLEAR
PROFICIENT Mismatch repair phenotype?Mismatch repair phenotype?
Possible ExplanationsPossible Explanations
A redundant NLS sequence allowed the protein into the A redundant NLS sequence allowed the protein into the nucleus (Another in MLH1? In PMS2?) nucleus (Another in MLH1? In PMS2?)
1
2
The mutation in MLH1 did not critically interfere with the NLSThe mutation in MLH1 did not critically interfere with the NLS
Colon Cancer Colon Cancer ImplicationsImplications
Our functional assays show no Our functional assays show no reduced MMR activityreduced MMR activity
Other lab groups obtained similar Other lab groups obtained similar results for R474Qresults for R474Q
Clinical data is not conclusive in Clinical data is not conclusive in linking mutations to Lynch Syndromelinking mutations to Lynch Syndrome
CONCLUSIONSCONCLUSIONS1.1. The mutations are not pathogenic.The mutations are not pathogenic.2.2. The mutations are moderately The mutations are moderately
pathogenic and our assays are not pathogenic and our assays are not sensitive enough.sensitive enough.
AcknowledgementsAcknowledgements
Howard Hughes Medical InstituteHoward Hughes Medical Institute Dr. Andrew Buermeyer, mentorDr. Andrew Buermeyer, mentor Dr. Kevin Ahern, program coordinatorDr. Kevin Ahern, program coordinator
Generously funded by grants Generously funded by grants from the HHMI Programfrom the HHMI Program