Related Experiment Video

Updated: Jul 27, 2026

Detection of Protein Ubiquitination
09:00

Detection of Protein Ubiquitination

Published on: August 19, 2009

Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization

Z Lai1, K V Ferry, M A Diamond

  • 1Cancer Research, DuPont Pharmaceuticals Company, Glenolden, Pennsylvania 19036, USA.

The Journal of Biological Chemistry
|June 9, 2001
PubMed

Insights

The MDM2 protein targets p53 for degradation via ubiquitination. This study simplifies the MDM2-p53 ubiquitination process, revealing p53 is modified by multiple single ubiquitin tags, not chains.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cellular Regulation

Background:

  • MDM2 is a key E3 ubiquitin ligase regulating p53 stability and function.
  • Ubiquitin conjugation targets proteins for proteasomal degradation.
  • The RING finger domain of MDM2 is essential for its ligase activity.

Purpose of the Study:

  • To simplify and enzymatically analyze the MDM2-mediated p53 ubiquitination cascade.
  • To characterize the ubiquitination pattern of p53 by MDM2.
  • To elucidate the kinetic mechanism of MDM2-mediated p53 ubiquitination.

Main Methods:

  • Purification of three recombinant proteins: ubiquitin-conjugated E2, MDM2, and p53.
  • Enzymatic analysis of the simplified ligase reaction.
  • Kinetic analysis of the ubiquitination transfer reaction.

Main Results:

  • A simplified three-component system recapitulates p53 ubiquitination.
  • p53 is modified with multiple mono-ubiquitin moieties, not poly-ubiquitin chains.
  • Kinetic analysis suggests a modified Ping Pong or Rapid Equilibrium Random Bi Bi mechanism.

Conclusions:

  • The MDM2-p53 ubiquitination cascade can be studied using a minimal set of purified components.
  • MDM2 primarily attaches multiple single ubiquitin molecules to p53.
  • The ubiquitination reaction involves complex enzyme-substrate interactions and conformational changes.

More Related Videos

Purification of Ubiquitinated p53 Proteins from Mammalian Cells10:55

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model09:00

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

Published on: April 17, 2026

Related Concept Videos

Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
DNA Damage can Stall the Cell Cycle02:36

DNA Damage can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
Regulated Protein Degradation02:58

Regulated Protein Degradation

It is vital to regulate the activity of enzymatic as well as non-enzymatic proteins inside the cell. This can be achieved either through creating a balance between their rate of synthesis and degradation or regulating the intrinsic activity of the protein. Both these regulation mechanisms play an essential role in the normal functioning of cells.
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
Master Transcription Regulators02:23

Master Transcription Regulators

Master transcription regulators are regulatory proteins that are predominantly responsible for regulating the expression of multiple genes. Often these genes work in concert to drive a  complex process. Activation of a master transcription regulator can lead to a cascade of transcriptional activation necessary for that outcome. These regulators can directly bind to the regulatory sequences of the various genes involved, or they can indirectly regulate transcription by binding to regulatory...
Abnormal Proliferation02:23

Abnormal Proliferation

Under normal conditions, most adult cells remain in a non-proliferative state unless stimulated by internal or external factors to replace lost cells. Abnormal cell proliferation is a condition in which the cell's growth exceeds and is uncoordinated with normal cells. In such situations, cell division persists in the same excessive manner even after cessation of the stimuli, leading to persistent tumors. The tumor arises from the damaged cells that replicate to pass the damage to the daughter...
Interactions Between Signaling Pathways01:19

Interactions Between Signaling Pathways

Signaling cascades usually lack linearity. Multiple pathways interact and regulate one another, allowing cells to integrate and respond to diverse environmental stimuli.
Convergence and divergence, and cross-talk between signaling pathways
Two distinct signaling pathways can converge on a single functional unit, which may either be a single protein or a complex of proteins. The response is either functionally distinct or synergistic between the two pathways but different from the response...