A Complete Guide of Ubiquitination

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Introduction of Ubiquitination

Proteins are frequently modified post-translationally through a process known as protein ubiquitylation, which involves one or more ubiquitin molecules (a polypeptide made up of 76 amino acids called ubiquitin) and a number of specialized enzymes. Protein ubiquitylation is the process of categorizing proteins, choosing target protein molecules, and specifically altering target proteins.

 

Ubiquitination is an important post-translational modification, and the ubiquitin-proteasome system mediates 80%-85% of eukaryotic protein degradation. In addition to participating in protein degradation, ubiquitination modification is also involved in the regulation of almost all life activities such as cell cycle, proliferation, apoptosis, differentiation, transcription regulation, gene expression, transcription regulation, signal transmission, damage repair, inflammation and immunity. Ubiquitination is closely related to the pathogenesis of tumors, cardiovascular diseases and other diseases. Therefore, as a major achievement of biochemical research in recent years, it has become a new target for research and development of new drugs.

 

The process of protein ubiquitination and degradation

 

Ubiquitinated protein degradation needs to be achieved through ubiquitinated proteasome, which exists in the cytoplasm and nucleus and is a protein complex composed of ten to twenty subunits. The 26s proteasome commonly exists in biological cells, and its composition is divided into two parts, one part is the 20s proteasome, which is the core component, and the other part is two 19s proteasomes, which play a regulatory role. Most proteins in the cell are degraded by the 26s proteasome. The ubiquitination and degradation of target proteins is accomplished through several sequential processes:

 

  1. When ATP supplies energy, ubiquitin activating enzyme E1 activates ubiquitin molecules;
  2. Ubiquitin activating enzyme E1 transfers activated ubiquitin molecules to ubiquitin conjugating enzyme E2;
  3. Ubiquitin ligase E3 connects the ubiquitin bound to E2 to the target protein;
  4. The 26s proteasome specifically recognizes the ubiquitin-labeled substrate protein and degrades it.

 

How to detect if a protein is ubiquitinated

  1. Western blot and immunoprecipitations

A specific protein and its associated proteins are isolated by co-immunoprecipitation. The separated protein was subjected to SDS electrophoresis and western blot analysis. This method can clarify which lysine residues of specific proteins are ubiquitinated.

 

  1. Western blot and strip

All ubiquitinated protein bands were detected by western blot, and the membrane was stripped after taking pictures. Then react with antibodies to specific proteins and antibodies to specific ubiquitination sites, develop color and take pictures. By comparing the positive bands, it can be preliminarily judged that ubiquitination has occurred at a specific site of a specific protein.

 

  1. In vitro ubiquitin-binding assay

 

Transfect the gene of the protein we want to study (ubiquitinated protein) into 293 cells, express it in large quantities and then purify it. Or directly extract endogenous protein. In vitro incubation with K63- or K48-linked ubiquitin chain peptides followed by SDS electrophoresis and detection with ubiquitinated antibodies. This method can identify which lysine residues of the protein of interest are prone to ubiquitination.

 

  1. In vitro ubiquitination assay

 

The target gene to be studied was transfected into 293 cells to express it in large quantities. After 24h, the target protein was extracted and separated. In the in vitro reaction buffer, combine the protein A we want to study (the one that is ubiquitinated) with UBE1, UbeH13-Uev 1 a heterodimer complex, HA-ubiquitin and the protein B we want to study (the one that causes protein A ubiquitination), and incubated together. The incubated products were subjected to IP and WB analysis. This method can clarify which protein is the E3 ligase that causes the ubiquitination of a protein.

 

 

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