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Related Concept Videos

The Proteasome02:18

The Proteasome

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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.
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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.
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Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
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After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for...
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Tagging and Fusion Proteins

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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The Proteasome Structure01:17

The Proteasome Structure

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The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
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Mapping Cell Surface Proteolysis with Plasma Membrane-Targeted Subtiligase.

Aspasia A Amiridis1, Amy M Weeks2

  • 1Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 25, 2022
PubMed
Summary

This study introduces subtiligase-TM, a new method for analyzing cell surface proteins. It improves the identification of protein N termini using mass spectrometry, enhancing proteomic analysis.

Keywords:
Cell surfaceEnrichmentMass spectrometryN terminomicsPost-translational modificationProteolysisProteomics

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Area of Science:

  • Proteomics
  • Biochemistry
  • Cell Biology

Background:

  • N terminomics uses mass spectrometry (MS) to identify protein N termini and proteolytic cleavage sites.
  • Existing methods require cell lysis and offer limited coverage of cell surface proteins.

Purpose of the Study:

  • To develop a novel method for selective enrichment and analysis of cell surface N termini.
  • To improve the coverage and specificity of N terminomics for cell surface proteins.

Main Methods:

  • Application of subtiligase-TM, a plasma membrane-targeted peptide ligase.
  • Selective biotinylation of cell surface N termini.
  • Enrichment and analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Main Results:

  • Demonstrated successful selective biotinylation of cell surface N termini.
  • Achieved increased coverage and specificity for cell surface N-terminal peptides.
  • Confirmed compatibility with existing quantitative LC-MS/MS workflows.

Conclusions:

  • Subtiligase-TM enables efficient and specific profiling of cell surface N termini.
  • This method advances N terminomics by overcoming limitations of traditional workflows.
  • The technique is valuable for studying cell surface protein dynamics and functions.