3. ➢ Human profilin (PFN1) is an actin-binding
protein with involvement in
restructuring the actin cytoskeleton and
has shown to be a possible area of study
to decrease breast cancer metastasis
➢ Profilin-1 is constructed around an
antiparallel beta-pleated sheet which is
between two alpha-helix segments
➢ The His-133 residue is located in an area
of high solvent exposure and in between
alpha-helix 1 and 4
➢ In the poly-L-proline binding site there is
not only the His-133, but also Tyr-6, Tyr-
139, Trp-3, and Trp-31
Background
Figure 1: Ribbon Diagram of Profilin-
1
4. ➢ It is expected that given a
phosphorylation of the His-133,
there be dislocations of the other
four residues
➢ His-133 is located on alpha-helix 4
near the C-terminus and is semi-
buried in a hydrophobic groove; this
groove functions as the polyproline
ligand binding site
Background
Figure 1: Ribbon Diagram of Profilin-
1
5. ➢ Profilin has been proposed to
regulate several mechanisms of
actin.
➢ The 1:1 complex that profilin
creates when bound to actin, shows
to change the conformation and
structure of the actin protein
sequestering actin from other
monomers
➢ This allows increased actin
availability in the cytosol
Background
Figure 2: Ribbon diagram of Profilin-
1 and Actin
6. ➢ Actin has shown to have a significant role in cancer metastasis
○ G-actin dynamically localizes to the leading edge of metastatic
cancer cells.
○ This localization augments cell movement due to an increase in G-
actin concentrations
○ Actin has shown to play a significant position in cytokinesis during
the last phases of cell division
○ During cytokinesis, a contractile ring is formed by actin and myosin
allowing cleavage of the original single cell into two identical
offspring
○ It has been shown in the yeast Schizosaccharomyces pombe that in
this contractile ring is an active site for actin polymerization. This
polymerization can only occur, however, through the use of profilin-
1
Background
7. ➢ A large percentage of cancer patients are in stage IV in which the
cancer is metastatic due to the high density of blood and lymph
vessels in that area allowing the cancer to spread
➢ Chemotherapy has shown to only minimally destroys cancer cells
(only 2.1% effective toward a five year survival)
➢ Alternative approaches such as inhibition of uniquely modified
proteins such as human profilin with phosphorylated amino acids
could lead to a more efficient treatment option
➢ A protein based approach, such as phosphorylated profilin protein
might result in a broad -based treatment of breast cancer
metastasis, since this approach would inhibit the tumor locomotion
and division.
Significance
8. ➢ G-actin was seen to have a significant relationship with the
cell motility of cancer cells
➢ It is seen that G-actin dynamically localizes itself to the
leading edge of growing neuroblastomas and thus causes
great cell movement of the carcinoma [6]
➢ Profilin’s interaction with actin was also seen in the
contractile ring of actin and myosin since actin
polymerization during cytokinesis of cell division [11]
➢ Phosphorylated profilin has been shown under in vivo
conditions to have increased affinity to poly-L-proline
sequences in vitro and in vivo. [12]
Literature Review
9. Research Problem: This project uniquely investigates the
conformational change of the profilin-1 protein by
phosphorylation of His-133. Conformational change of the
profilin-1 was applied to find possible changes in actin-
profilin interactions.
Research Hypothesis: It was predicted that there be a
significant change in the secondary and tertiary structure
of the profilin-1 with addition of the phosphohistidine
Problem & Hypothesis
11. Effect of Potassium Phosphoramidate on Secondary Structure of
Profilin: All CD spectra were collected on a Jasco 720
spectropolarimeter at room temperature in either 1 mm or 5mm path
length cuvettes. CD was done with four 1mL samples of increasing
concentrations of phosphoramidate from wavelengths of 190 to 230 nm.
Effect of Potassium Phosphoramidate on Tertiary Structure of
Profilin: Data were collected on the same four 1mL samples of
increasing concentration of potassium phosphoramidate using a Photon
Technology International (PTI) spectrofluorometer model QM-4/2005.
The slit-widths were set as 2 nm and the excitation wavelengths were set
at 290 nm and 280 nm in context to tryptophan and tyrosine, respectively.
The emission spectrum was recorded from 300 to 400 nm in 0.5
increments.
Materials & Methods
13. Figure 1: CD of Profilin-1 wild type Figure 2: Gel Electrophoresis
of Profilin Protein
14. Figure 3: CD of PFN1 with
Increasing Concentration of
Potassium Phosphoramidate (1ul
PFN1/10uL Solution)
Figure 4: CD of PFN1 and
Phosphorylated His-133 PFN1
15. Figure 5: Fluorescence of PFN1
with Increasing Concentration of
Potassium Phosphoramidate at
290nm excitation wavelength
Figure 6: Fluorescence of PFN1
and Phosphorylated His-133 PFN1
at 290nm excitation wavelength
16. Figure 7: Fluorescence of PFN1
with Increasing Concentration of
Potassium Phosphoramidate at
280nm excitation wavelength
Figure 8: Fluorescence of PFN1
and Phosphorylated His-133
PFN1 at 280nm excitation
wavelength
18. ➢ This projects finds that with the addition of the
phosphohistidines, profilin-1 undergoes a change
in secondary structure and specifically in the poly-
L-proline binding site.
➢ These results show potential to regulating G-actin
as G-actin must bind to profilin-1 to perform
processes in locomotion and cytokinesis
Discovery
19. ➢ In response to this research, interactions between profilin-1
with the phosphohistidines and actin should be viewed to
see changes from normal interactions
➢ The effect of the phosphohistidines should be monitored in
vivo to see if the interaction indeed inhibits cancer growth
and movement.
➢ If inhibition of profilin-actin interactions were to occur, it
would harm both cancer and somatic cells. Further research
should focus on isolating the protein action to only cancer
cells.
Further Research & Limitations
20. ➢ The profilin migrates to the 15 kD band, the expected
molecular weight
➢ The CD curve displays characteristic relative minimas at
208nm 222nm, and characteristic absolute minimum at
215nm, confirming the secondary structure of profilin.
➢ There is a consistent trend of a growing minimum as the
concentration of the potassium phosphoramidate
increases. Since the minima of the profilin-1 depict the
alpha helices and beta pleaded sheets, it is conclusive
that the phosphoramidate influenced the secondary
structure of the protein
Analytical Results
21. Analytical Results
➢ The greatest change in secondary structure was seen in the sample
with a 1:1 (100uL:100uL) volumetric ratio between profilin and
potassium phosphoramidate. The greatest percent change in
structure was 19.05%
➢ This difference gives insight to a change in the secondary structure
of the protein due to the combination of effects from
phosphohistidines 133 and 119
➢ Alpha-helix 4 residues (on which His-133 is located) interact
directly with beta-sheet of profilin by side chain-side chain
contacts [8]. Thus, there are possible structural perturbations of
the Arg 135 (alpha-helix 4) and Phe 83 (beta-strand 5)
22. Analytical Results
➢ The left maxima of the profilin do not show change reflecting
stability of the profilin with increasing concentrations of the
phosphoramidate
➢ The excitation wavelength of 290 nm and 280nm show change
in the tryptophan and tyrosine residues, respectively
➢ There is a constant trend in which the fluorescence signal
measures a greater maximum with addition of the
phosphohistidines
➢ Isolation of the greatest and most consistent maxima, as seen
in the sample with a 1:1 volumetric ratio between profilin and
phosphoramidate, shows a percent change of 14.6%
23. Analytical Results
➢ This change significantly indicates that the aromatic amino
acid tryptophan might have moved a slight distance away
from the phosphohistidine
➢ The poly-L-proline binding site remains hydrophobic and
intact as suggested by fluorescence maximum at 344 nm
➢ This residue movement may also shed light on more changes
in the poly-L-proline site since the chemical shift of each
residue is influenced by the local environment
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Retrieved November 12, 2013
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References
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Intermediate of Human Platelet Profilin. In Albert Einstein College of Medicine. Retrieved November 12, 2013
[9] Mitchison, T. J., & Cramer, L. P. (1996, February 9). Actin-Based Cell Motility and Cell Locomotion. In Cell.
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[10] Pantaloni, D., & Carlier, M. (1993, December 3). How profilin promotes actin filament assembly in the
presence of thymosin β4. In Cell. Retrieved November 12, 2013
[11] Pelham, R. J., & Chang, F. (2002, June 27). Actin dynamics in the contractile ring during cytokinesis in fission
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