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Related Concept Videos

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...
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...
The Proteasome02:18

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
The Proteasome01:13

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important among these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin...
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.
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...

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High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines
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Small-molecule control of protein degradation using split adaptors.

Joseph H Davis1, Tania A Baker, Robert T Sauer

  • 1Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

ACS Chemical Biology
|August 27, 2011
PubMed
Summary

We developed a novel split-adaptor system for targeted protein degradation in bacteria. This inducible system uses rapamycin to control the ClpXP protease, enabling precise control over protein levels for biological studies and biotechnology.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Microbial Genetics

Background:

  • Targeted intracellular degradation is crucial for understanding protein function and has biotechnological applications.
  • Current methods often rely on modifying endogenous proteases or de novo synthesis of components.
  • Adaptor proteins are key to directing substrates for proteolysis via degradation tags.

Purpose of the Study:

  • To engineer a novel, small-molecule-inducible split-adaptor system for targeted protein degradation in bacteria.
  • To demonstrate the system's efficacy and modularity for controlling protein levels.
  • To provide a new tool for bacterial research and biotechnology.

Main Methods:

  • Developed an engineered split-adaptor system controlled by rapamycin.
  • Utilized the ClpXP protease for substrate degradation.
  • Demonstrated degradation of tagged LacI repressor and FtsA proteins in E. coli.

Main Results:

  • The rapamycin-inducible system effectively degraded target proteins without modifying endogenous proteases.
  • Degradation was reversible upon rapamycin removal, as shown by FtsA depletion reversal.
  • The system exhibited robustness across various adaptor concentrations and required no new synthesis for initiation.

Conclusions:

  • The engineered split-adaptor system offers a modular and controllable method for bacterial protein degradation.
  • This system provides a versatile platform for future applications in synthetic biology and protein research.
  • The rapamycin-inducible control allows for precise manipulation of protein levels in bacteria.