Possible Role of His-133 Phosphorylation on G-

Actin RegulationSumit Saha

INTRODUCTION

➢ 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

➢ 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

➢ 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

➢ 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

➢ 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

➢ 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

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

MATERIALS & METHODS

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

RESULTS

Figure 1: CD of Profilin-1 wild type Figure 2: Gel Electrophoresis of Profilin Protein

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

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

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

DISCUSSION

➢ 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

➢ 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

➢ 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

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)

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%

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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References

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