Key players in Nitric Oxide Signaling
RegulationAlexander Ollerton, Sarah Young, William Montfort
The University of Arizona, BLAISER Program
Goals
Develop functional readout for Thrombospondin-1. (Calcium Assay) Obtain high purity and expression levels of Thrombospondin-1 in order
to measure the cytosolic calcium concentration increase via flow cytometry.
Discover what proximal protein is interacting with CD47 and Thrombospondin-1 to inhibit Nitric Oxide Signaling via BirA.
Key Players
50 kDa protein receptor for TSP-1
“Don’t eat me” signal to escape detection from immune system
Plays a role in NO signaling
Direct role in sGC inhibition not well characterized
CD47
GTP cGMP
α β
𝐹𝑒2+¿ ¿
Coiled-coil
H-NOX
PAS
Catalytic
150 kDa heterodimeric enzyme
NO binds to ferrous heme on beta strand
GTPcGMP physiological changes
Soluble Guanylyl Cyclase
TSP-1 450 kDa protein
Has calcium binding domain and C-terminal binding domain
Binds to N-terminus of CD47 and inhibits angiogenesis
E3CaG1 is a 63 kDa truncated TSP-1
Easier for experimental use
Thrombospondin-1 and E3CaG1NO N-
terminal ProcollagenThrombosondin repeats
EGF-like repeats
Calcium repeats
C-terminal
AngiogenesisCD47VEGFR2
Tumor Cell
Old Blood Vessel
New blood vessels
• Angiogenesis: New Blood Vessels forming from old blood vessels
• VEGFR2: protein receptor that may interact with CD47 to create signal for new blood vessel formation
Overview of Nitric Oxide Signaling
L-arginine+NADPH++2citrulline+ NO+
¿
GTP cGMP
𝐹𝑒2+¿ ¿
CD47X
·NO
TSP-1
• NO is a byproduct of nitric oxide synthase
• NO binds to sGC lowering cytosolic calcium concentrations
• TSP-1 binds to CD47 inhibiting sGC and NO signaling
Endothelial cellSmooth muscle cell
Results
Goal: Develop functional readout for E3CaG1
¿
GTP cGMP
𝐹𝑒2+¿ ¿
CD47X AT1
Angiotensin-II
TSP-1
Flow Cytometry
(A) (B) (C)
• (A) baseline measurement with 5µM fluo-3AM
• (B) angiotensin-II added and after 15 minutes measurements obtained • No increase in calcium concentrations, need further examination
• (C) Ionomycin was used for positive control
• All were measure with 488nm laser
¿
GTPcGMP𝐹𝑒2+¿ ¿
CD47
X
Angiotensin-II
Goal: Obtain high purity and expression levels of Thrombospondin-1
¿
GTP cGMP
𝐹𝑒2+¿ ¿
CD47X
TSP-1
Western blots and Coomassie gel(A)
1 2 3 4 5 6 7 8 9 10(B)
(left) E3CaG1 is eluting during 40mM imidazole wash step indication of weak binding to Ni column
(right) E3CaG1 eluted (lanes 3-5) and were combined and concentrated (lane 8).
Coomassie gel shows low purity or degradation of E3CaG1
Goal: Discover proximal protein interaction with CD47 via BirA
Xbioti
nbiotin
biotin
CD47 Bir
Abioti
n
Cloning Strategy
• Cloning of CD47-BirA was a two step process
• CD47 was cloned into pCMV-3tag 3A vector with restriction sites NotI/BamHI
• BirA was cloned into pCMV-3tag 3A vector with restriction sites BamHI/XhoI
• GGSG linker was added in between CD47-BirA DNA segments
PCR and Double Digest
500040003000
15001000700
50004000
15001000700
(A) (B)
(A) PCR product of NotI/ BamHI into CD47 (976 bp)
(B) Double digest of pCMV vector with restriction sites NotI/BamHI (4214 bp)
CD47
NotI BamHI
pEGFP-N3
NotI BamHI
pCMV-3tag 3A
3:1 Ligation Colony
• NotI-CD47-BamHI ligated into the pCMV vector with 3:1 ratio
• Transformed in DH5α E. coli cells
• Plates incubated for 13 hours with result of one clony
• Inoculated colony in culture and isolated DNA with concentrated of 10 ng/μL
pEGFP-N3
CD47
NotI
BamHI
NotI BamHI
pCMV-3tag 3A
Conclusions
• Vasoconstrictor angiotensin-II, which signals via calcium, did not elicit a response in preliminary experiments, suggesting receptor may be missing in these Jurkat cells
• E3CaG1 is expressing in Sf9 cells; however, expression levels and purification need optimization.
• Ligation and transformation led to a possible clone. Greater quantity of DNA is needed for sequencing and conformation of correct cloning.
Future Work
Jurkat T-cells will be treated with phorbol ester or T-cell receptor antibody to induce calcium signaling
Once E3CaG1 is prepared, its ability to induce calcium signaling will be examined by flow cytometry. This will allow for unraveling signaling mechanism.
Once cloning is complete and transfection optimized, proximity labeling by CD47-BirA will be used to isolate and identify co-receptors and signaling partners by mass spectrometry.
Acknowledgements
Thank you to the Montfort lab for allowing me to be a part of their research lab.
Thank you Sarah Young for teaching me new scientific techniques and for giving me great advice throughout this process.
Thank you to the BLAISER program for giving me this wonderful opportunity to research over the summer.
Funding: American Heart Association, NIH R01 GM117357
References
Kaur, S.et al. 2013, Sci. Rep. 3,1673 Kim, D.I et al. 2014, PNAS, 10.1073, E2453-E2461 Lawler,J., et al. 1992, Biochemistry., 31, 1173-1180 Ramanathan,S. et al. 2011, Biochemistry, 50, 7787-7799 Rogers, N.M. et al. 2012, AJP-renal ,303, F1117-1125 Roux,K. et al. 2012, JCB, 196, 801-810 Willingham, S.B., et al. 2012, PNAS, 109, 6662-6667
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