Tti2 Thesis Final Apr 8

Elucidating the Structure and Function of Protein Tti2

:: Selwyn Chui :: Dr. Christopher J Brandl Lab

April 8, 2015::

Department of Biochemistry

:: IntroductionTti2 is an essential yeast protein• Tti2 = Tel2 (TELO2 in humans) Interacting Protein 2

o 421 amino acids (~49 kDa)

o Predicted to be mainly α-helical + chaperone

o ASTRA (Assembly of Tel, Rvb and Atm-like kinase) complex

o Triple T (TTT) complex: Tti1/2 and Tel2 (Telomere maintenance protein 2)

Structure and Function of Tti2


:: IntroductionThe TTT complex and PIKKs:• TTT complex (along with co-chaperone Hsp90) helps with

folding/stabilization of PIKKsPIKKS (Phosphatidylinositol 3-kinase-related protein kinases) are involved

with several essential cellular processes: DDR, metabolism, transcription,

etc…



folding/stabilization of PIKKs PIKKS (Phosphatidylinositol 3-kinase-related protein kinases) are involved


etc…


folding/stabilization of PIKKs PIKKS (Phosphatidylinositol 3-kinase-related protein kinases) are involved


etc…


TTT complex helps these proteins function, but what role does Tti2 play?

:: IntroductionTti2 in other organisms• Tti2 studied in yeast, but conserved in fungi and mammals;

a human Tti2 homolog also exists


:: IntroductionTti2 in other organisms• Tti2 studied in yeast, but conserved in fungi and mammals;

a human Tti2 homolog also exists

• Implicated in human brain development


:: Goal

The ultimate goal is to elucidate Tti2 structure, function, and protein interactions.

To this end, we want to identify alleles for suppressor analysis.


:: Approach Need to characterize Tti2 – How?

• Genetics approach: Altering individual Tti2 residues – are they important for yeast growth in stress conditions? If yes, we can proceed with…

• Suppressor analysis: Finding functionally related proteins by selecting for suppressors of mutant phenotypes

Eg. Tra1 (PIKK) functionally linked to Tti2to Y

But how do we pick residues to investigate?


:: ApproachUnigenic evolution analysis highlights candidate key Tti2 residues



Some of these residues were also conserved betweenTti2 homologs…


L187: α-helix?

P228/P229: Random coil / turn?Try P228G/P229A


Some of these residues were also conserved betweenTti2 homologs…


(L187P tested… Resists azetidine-2-carboxylic acid)

L187: α-helix?Try L187R

P228/P229: Random coil / turn?Try P228G/P229A

:: Approach

Engineering mutant vectors:

1. Choose Tti2 alleles (L187R, P228G-P229A)

2. Site-directed mutagenic PCR of Tti2 gene

3. Digest & ligation into plasmid vector

4. Cloning in E. coli • Plasmid amplification


Tti2-mutant, LEU2, ampr plasmid

:: Approach

Plasmid Insertion into Yeast

5. Plasmid purification from E. coli

6. Plasmid insertion into (ΔTti2 ΔLEU2 ΔURA3)S. cerevisiae with URA3 Tti2WT plasmid

7. Plasmid Shuffling (ura+ yeast inviableon 5-fluoroorotic acid)


5-FOA,counterselectsura+ cells

Tti2-wt, URA3 plasmid

Tti2-mutant, LEU2, ampr plasmidGenome: ΔTti2 ΔLEU2 ΔURA3

:: Approach

Stress-Inducing Conditions

8. Observe yeast growth in response to stress Tti2 stabilizes Tra1 (PIKK)Crippled Tra1 grows slowly on ethanol / calcofluor white Thus mutant Tti2 may also grow slowly on those media

• Ethanol• Calcofluor white


:: Approach

Stress-Inducing Conditions

8. Observe yeast growth in response to stress Tti2 stabilizes Tra1 (PIKK)Crippled Tra1 grows slowly on ethanol / calcofluor white Thus mutant Tti2 may also grow slowly on those media

• Ethanol• Calcofluor white

L187P resists slow growth on azetidineL187R may also grow differently

• Azetidine-2-carboxylic acid

• Heat Shock (37oC) **


::ResultsTti2L187R and Tti2P228G-P229A: Ethanol

• Ethanol is a mild denaturant + growth inhibitor Suggests leu/pro’s not vital for Tti2 response to ethanol


::ResultsTti2L187R and Tti2P228G-P229A: Calcofluor White

• Calcofluor white (dye) binds the cell wall Suggests leu/pro not vital for cell wall integrity


::ResultsTti2L187R and Tti2P228G-P229A: Azetidine

• Azetidine-2-carboxylic acid toxic proline homologue(WT sensitive to azetidine, but L187P conferred resistance; L187R and prolines both same as WT)


::ResultsTti2L187R and Tti2P228G-P229A: Heat shock

• Slow growth by increasing heat shock response Suggests leu/pro not important for heat shock repsonse


::Results


Structural Interpretation of Results

Pro228/229 Ala, Gly (More rotational freedom) Prolines not functionally important? Perhaps a linker region, not active site

::Results




Leu187 (NP) Arg (+) But Leu187Pro had big effect?

Sidechain identity vs backbone structure Not active site + Alpha helix disruption?

::Results




Leu187 (NP) Arg (+) But Leu187Pro had big effect?

Sidechain identity vs backbone structure Not active site + Alpha helix disruption?

Without a non-WT growth phenotype, we can’t do suppressor analysis. Try a more severe allele?

::ResultsCombining proline and R1 region mutations:


Previously tested the (R1 region mutant) Tti2W161A-I162A-P164A allele: Seemed to have a slight effect (Data not published)

Will a combined allele give slower growth?





…So severe, it’s not viable!

No Tti2

Tti2-WTTti2 mutant

W161A-I162A-P164A-

P228G-P229A





…So severe, it’s not viable!

What next??

No Tti2

Tti2-WTTti2 mutant

W161A-I162A-P164A-

P228G-P229A

::Future DirectionsFurther test R1 region / proline residues

• Try lowering expression of Tti2 mutants for slow-growth phenotype

Continue investigating alleles

• Pick other hypomutable residues from unigenic evolution analysis

• Trying different combinations of alleles (Eg. L187R and the R1 region mutant)


::AcknowledgementsThanks to:

Dr. Christopher J Brandl, Julie Generaux, Kyle Hoffman and Matthew Berg.

Also to Western University for providing the opportunity and the facilities.

Questions?


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