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Related Concept Videos

The Unfolded Protein Response01:37

The Unfolded Protein Response

The ER is the hub of protein synthesis in a cell. It has robust systems to quality control protein folding and also for degradation of terminally misfolded proteins. Under normal conditions, a small proportion of misfolded proteins that cannot be salvaged need to be transported to the cytoplasm by the ER-associated degradation or ERAD pathways. However, if the ERAD cannot handle the misfolded proteins, the cell activates the unfolded protein response or UPR to adjust the protein folding...
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 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...
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...
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.
Regulation of the Unfolded Protein Response01:31

Regulation of the Unfolded Protein Response

Inositol-requiring kinase one or IRE1 is the most conserved eukaryotic unfolded protein response (UPR) receptor. It is a type I transmembrane protein kinase receptor with a distinctive site-specific RNase activity. As the binding mechanics of the misfolded proteins with the N-terminal domain of IRE-1 are unclear, three binding models — direct, indirect, and allosteric -- are proposed for receptor activation. Nevertheless, it is known that once a misfolded protein associates with IRE1, it...

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Related Experiment Video

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In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

E1-L2 activates both ubiquitin and FAT10.

Yu-Hsin Chiu1, Qinmiao Sun, Zhijian J Chen

  • 1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.

Molecular Cell
|September 25, 2007
PubMed
Summary

Researchers identified E1-L2, an enzyme activating ubiquitin and FAT10. This protein is crucial for FAT10 conjugation and embryonic development, highlighting its specialized role in ubiquitination pathways.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Ubiquitination is a key post-translational modification mediated by E1, E2, and E3 enzymes.
  • Ubiquitin-like proteins (UBLs) also regulate cellular processes through conjugation.
  • FAT10 is a UBL implicated in immune responses and cellular stress.

Purpose of the Study:

  • To identify and characterize a novel E1-like enzyme involved in ubiquitin and FAT10 activation.
  • To elucidate the enzymatic mechanism and cellular function of the identified E1-like protein, E1-L2.
  • To investigate the role of E1-L2 in FAT10 conjugation and embryonic development.

Main Methods:

  • Protein purification and in vitro enzymatic assays to assess E1-L2 activity.
  • Site-directed mutagenesis to identify key residues for E1-L2 and FAT10 interaction.

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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
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  • RNA interference (RNAi) to silence E1-L2 expression in cellular models.
  • Generation and analysis of E1-L2 knockout mice.

    • Main Results:

    • Identification of E1-L2, an E1-like enzyme that activates both ubiquitin and FAT10.
    • E1-L2 forms a thioester intermediate with FAT10, dependent on its active-site cysteine and FAT10's diglycine motif.
    • E1-L2 specifically conjugates FAT10 in response to TNF-alpha and IFN-gamma stimulation.
    • Silencing E1-L2 inhibits FAT10 conjugation, and its deletion in mice leads to embryonic lethality.

    Conclusions:

    • E1-L2 is a novel E1-enzyme with a specialized role in activating FAT10 for conjugation.
    • The enzymatic activity of E1-L2 is essential for FAT10 modification and cellular processes.
    • E1-L2 plays a critical, indispensable role in embryogenesis, underscoring its biological significance.