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Student Resources: Student Projects: Kwabena Bonsu, Beremis Perez
Biochemical Characterization of the Waf-1 p53 “Bindomers”   view PDF

Slide 1

INTRODUCTION

What is P53 gene?

Human P53 gene is a tumor suppressor gene and its resulting protein (p53 protein) is found in human cells.

Why is it the focus of so much research?

Previous research has shown that in about 70% of all human cancers, P53 gene is mutated.

Due to the mutation (deletion) to the P53 gene, the resulting p53 protein is sometimes abnormal or dysfunctional, contributing to the formation of cancerous cells


Slide 2

What does normal p53 protein do?

  • The function of the p53 protein is to prevent cells with DNA damage from going through the cell cycle.
  • p53 protein binds in sequence specific manner to sites (p53-Response Elements) in certain genes (p53-Target Genes) such as WAF-1, BAX, MDM2 etc as a transcription factor.
  • Resulting regulatory protein checks the cell cycle and directly initiates DNA-damage repair or cell destruction (Apoptosis) based on the degree of DNA damage.


Slide 3

What determines p53’s affinity to the Target genes?

  • P53 binding sites (REs) in Target genes follow a general consensus sequence and are found to be very loose

    General consensus sequence:
    5’(Pu-Pu-Pu-C A/T T/A G-Py-Py-Py)3’
Hypothesis:
The degree of phosphorylation of the p53 protein modulates it affinity for the different p53-REs.


Slide 4

Research design:
To investigate patterns of phosphorylation as a possible factor behind p53 proteins' Affinity to p53-REs in target genes,

  • The p53 protein would be made to bind to several of its target genes beginning with Waf-1.
  • The p53 proteins that bind to the target genes will be isolated and collected in an elution using a DNA-Affinity Chromatography System.
  • P53 proteins that bound to different target genes will then be studied for specific patterns of phosphorylation using the Western Blot Technique.


Slide 5

RESULTS

Western blot of DLD Cell extracts
(photo)
Lane 1: molecular markers. Lane 2: empty space. Lane 3 DLD cells nuclear extract, induced (D+). Lane 4: empty space. Lane 5: DLD cells nuclear extract, uninduced (D-).

Waf 1 bindomers affinity chromatography
(photo)
WAF 1 affinity chromatography of the DLD cells nuclear extract. Lane one: flow through showing p53 protein bands. Lane 2: elution fraction from induced DLD cells. Lanes 3: elution fraction from DLD uninduced cells.


Slide 6

Western Blot

  • The collected elutions will be run on a polyacrylamide gel and the proteins be transferred to a nitrocellulose paper for probing.
  • In order to be able to see the results, series of specific phosphorylation antibodies will be used against p53

Discussion and Conclusion

  • By using antibodies against phosphorylated forms of p53 on a nitrocellulose paper which will be developed using Kodak Image Station 2000, we will be able to identify the phosphorylation status of the p53 molecules that bind to the p53 binding sites located in the target genes.

Slide 7

Mixture is poured through a magnetic column and microbeads-biotin-Waf 1-p53 complex is attracted to walls of column. P53 is isolated from complex by series of wash with KCl solution ranging from 0.1M to1.0M

(figure)

Slide 8

P53 is expected to bind to its Waf 1 binding site (probe), and the µMacs Streptavidin micro beads bind to the biotin attached to probe to form a microbead-biotin-waf1-p53 complex

(figure)


Slide 9

Abstract

P53 is a tumor suppressor protein, which functions abnormally in most human cancer. Further investigation into the mechanism behind p53 function is very useful in developing more effective anticancer preventive measures. While in normal human cells p53 is broken down by the ubiquitin protease system as soon as it is made, in damaged cells, p53 is stabilized, and activated by mechanism yet unknown. The activated p53 induces cell cycle arrest, thus allowing for cell damage repair before entry into the S phase or by destroying the damaged cell completely. P53 does this by regulating the transcription of many known target genes such as BAX, MDM2, WAF-1 etc. For p53 to regulate gene expression, p53 binds in a sequence specific manner to a p53- RESPONSE ELEMENT (p53-RE) found in the target gene, initiating its transcription. The p53-REs of target genes follow the general consensus sequence: PuPuPuC (A/T) (T/A) GPyPyPy. We suggest a p53 control mechanism based on the phosphorylation pattern of the p53 molecule. The differential phosphorylation of the various p53 molecules allows them to distinguish between different p53-REs. As of now, we have done a polyacrylamide gel electrophoresis to confirm the activation of p53 after Doxycycline was added to the cell culture. We made a nuclear extract to isolate the nuclei where p53 is found. Then we did a Polyacrylamide Gel Electrophoresis immunodetection to separate and detect p53 from other proteins respectively. After seeing p53 in the nuclear extract, and knowing that it is functional according to results from the western blot and growth curve respectively, we are set with the preliminary part of the experiment and will use the nuclear extract for the DNA Affinity step.


Slide 10

Biochemical Characterization of the Waf-1 p53 “Bindomers”

Presenter: Kwabena Bonsu (718-681-4752)
Co-research assistant: Beremis Perez
Research advisors: Dr. Patricia Molina, Dr. Sarah Salm, Dr. Lauren Goodwyn
LS-AMP, Borough of Manhattan Community College