Stability Analysis of Cystic Fibrosis Transmembrane Conductance Regulator via Tryptic Digestion

Chris Holmes, Biophysical Society John Riordan (PI) & Timothy Jensen (Mentor) , CF Center

Symptoms of Cystic Fibrosis (CF)

http://smithbiologyp3.wikispaces.com/Cystic+Fibrosishttp://upload.wikimedia.org/wikipedia/commons/4/4e/ClubbingCF.JPG

http://1.bp.blogspot.com/-VoDtcyKhUew/T0IT9WdRpvI/AAAAAAAAAE4/utY1XRNhuFE/s640/Caylee's+1st+Vest+Treatment+010edit2.jpg

Background Methods Results Conclusions

Cystic Fibrosis: The Resultant from the Disruption of the Cystic Fibrosis Transmembrane

Conductance Regulator (CFTR)

http://4.bp.blogspot.com/-NypSdLKYx_0/T38RrbomnwI/AAAAAAAAABw/wZpOLyNcXz0/s1600/cf-1.jpg

Background Methods Results Conclusions

Acquiring a Complete CFTR Crystal Structure is Presently Unsuccessful

Molinski, S.; Eckford, P. D. W.; Pasyk, S.; Ahmadi, S.; Chin, S.; Bear, C. E. Functional rescue of F508del-CFTR using small molecule correctors. Frontiers in Pharmacology 2012, 3.

Background Methods Results Conclusions

X

Limited Tryptic Digestion and Sonication of CFTR

Western Blot of Digested CFTR

Repeat with Mutant CFTR and Correctors

Procedure for Indirectly Determining the Structural Stability of CFTR

Background Methods Results Conclusions

Expectations and Objective from the Tryptic Digestion of CFTR

Investigate the structural stability of CFTR

Wild-type CFTR (natural) and ΔF508 CFTR (mutated) Adenosine triphosphate (ATP) and a non-hydrolysable ATP-

analogue (AMP-PNP)

VX-809 (Lumacaftor) was used because of its success with CF

Currently in phase three of clinical trials

http://upload.wikimedia.org/wikipedia/commons/d/d3/Lumacaftor_skeletal.svghttp://upload.wikimedia.org/wikipedia/

commons/0/07/ATP_structure.svghttp://patentimages.storage.googleapis.com/WO2003076333A2/imgf000010_0002.png

Background Methods Results Conclusions

Using Tryptic Digestion: Cleavage Locations at Lysine Residues

Background Methods Results Conclusions

Henderson, M. J.; Singh, O. V.; Zeitlin, P. L. Applications of proteomic technologies for understanding the premature proteolysis of CFTR. Expert Rev Proteomics 2010, 7, 473–486.

Trypsin cleaves the protein on the carboxyl side of lysine or arginine residues

The more digestion that occurs, the more exposed those lysine and arginine residues are.

0μL

1μL

3μL

9μL

Using Sonication to Permit the Passage of Treatments and Trypsin into the CFTR Cells

Background Methods Results Conclusions

= CFTR Membrane

= Treatment (VX-809, ATP, AMP-PNP)

= Trypsin

Using Western Blot Band Strength as an Indicator of Structural Stability

Background Methods Results Conclusions

The More Intense the Band

The More Intact the CFTR

The Higher the Stability

IR Detection Using Primary and Secondary Antibodies

Membrane

Antigen

1° Antibody

2° Antibody

Fluorophore

Background Methods Results Conclusions

1° Antibodies = 660 & 7692° Antibodies = IgG1 & IgG2b

Limited Tryptic Digestion as an Indicator of CFTR Stability

Cui, L.; Aleksandrov, L.; Chang, X.; Hou, Y.; He, L.; Hegedus, T.; Gentzsch, M.; Aleksandrov, A.; Balch, W. E.; Riordan, J. R. Domain Interdependence in the Biosynthetic Assembly of CFTR. J. Mol. Biol. 2007, 365, 981-994

Background Methods Results Conclusions

NBD 2

Western Blotting of Wild-Type CFTR after Tryptic DigestionNBD 1

AMP-PNP Wild-Type ATP VX-809

0µL 1µL 3µL 9µL

Background Methods Results Conclusions

~170 kDa

Data Quantification of Wild-Type CFTR Membranes is Significant

Background Methods Results Conclusions

0 1 3 90

10

20

30

40

50

60

70

80

90

100

110

NBD 1AMP-PNP

VX-809

WT

ATP

Trypsin Concentration (µL)

Norm

aliz

ed

Valu

e o

f In

itia

l Ban

d

(%)

0 1 3 90

10

20

30

40

50

60

70

80

90

100

110NBD 2

AMP-PNP

VX-809

WT

ATP

Trypsin Concentration (µL)

Norm

alize

d V

alu

e o

f In

itia

l B

and

(%)

*

*

*

*

NBD 2

Western Blotting of ΔF508 CFTR after Tryptic Digestion

NBD 1

AMP-PNP ΔF508 ATP VX-809

0µL1µL3µL9µL

Background Methods Results Conclusions

Data Quantification of ΔF508 CFTR Membranes is NOT Significant

Background Methods Results Conclusions

0 1 3 90

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8NBD 1

AMP-PNP

VX-809

ΔF

ATP

Trypsin Concentration (µL)

Norm

aliz

ed

Valu

e o

f In

itia

l Ban

d

(%)

0 1 3 90

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8NBD 2

AMP-PNP

VX-809

ΔF

ATP

Trypsin Concentration (µL)

Norm

alize

d V

alu

e o

f In

itia

l B

and

(%)

At least not yet!<1%

100100

Adjusting the Quantified Band from the Full-Length CFTR to Approximately Half-Length

CFTR for a More Definitive Reading

170 kDa

Background Methods Results Conclusions

AMP-PNP ΔF508 ATP VX-809

0µL 1µL 3µL 9µLMWM 0µL1µL 3µL 9µLMWM 0µL1µL 3µL 9µLMWM 0µL1µL 3µL 9µLMWM

~72 kDa

The Difference Between 170 kDa and 72 kDa in CFTR Membranes

Cui, L.; Aleksandrov, L.; Chang, X.; Hou, Y.; He, L.; Hegedus, T.; Gentzsch, M.; Aleksandrov, A.; Balch, W. E.; Riordan, J. R. Domain Interdependence in the Biosynthetic Assembly of CFTR. J. Mol. Biol. 2007, 365, 981-994

Background Methods Results Conclusions

0 1 3 90

2

4

6

8

10

12

14

16

18

NBD 1AMP-PNP

VX-809

ΔF

ATP

Trypsin Concentration (µL)

Norm

aliz

ed

Valu

e o

f In

itia

l Ban

d

(%)

Data Quantification of ΔF508 CFTR Membranes IS Significant

Background Methods Results Conclusions

>1%

0 1 3 90

2

4

6

8

10

12

14

16

18

NBD 2AMP-PNP

VX-809

ΔF

ATP

Trypsin Concentration (µL)

Norm

aliz

ed

Valu

e o

f In

itia

l Ban

d

(%)

* *

*

100 100

Implications of the Stabilizing Effects of ATP and AMP-PNP

Background Methods Results Conclusions

Bound ATP and AMP-PNP

Favorable Interactions and Conformational Adjustments

Lower Susceptibility to Proteolysis (Tryptic Digestion) & Increased Stability

Future Directions for the Stability Analysis of CFTR

Background Methods Results Conclusions

Since VX-809 was not particularly effective Investigate the effects of VX-661 and/or VX-770

Since AMP-PNP and ATP were effective Investigate other ATP analogues or treatments that would

extort the stabilizing effects of the ATP binding pocket

Since the ΔF508 mutation only represents one class of CFTR mutations Investigate the stabilities of other mutations(e.g. G551D)

Since the experiment was small scale and did not account for other mechanisms Investigate the treatments in vivo

Acknowledgments

Special thanks to the University of Mount Union, the Department of Chemistry and Biochemistry, UNC, the Biophysical Society, and the

Department of Biochemistry and Biophysics for providing me with this opportunity

University of North Carolina

In the Laboratory:

Dr. John Riordan

Dr. Tim Jensen

Dr. Luba Aleksandrov

Mr. Mohamed Dumbuya

In the Summer Program:

Dr. Mike Jarstfer

Dr. Barry Lentz

Lisa Phillippie

Ellen Mackall

Dr. Jaime Campbell-Fox

Patrick McCarter

Lior Vered

University of Mount Union

Mentoring:

Dr. Robert Woodward

Dr. Keith Miller

Student Assistants:

Amanda Dragan

Questions?

http://www.waid-observatory.com/arp194-2012-08-10-HLA-1092.html