Ubiquitin Conjugation
Ubiquitin Conjugation - Related Videos
09:00
Detection of Protein Ubiquitination
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Cited by 96 •
2009
Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.
10:43
siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
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Cited by 4 •
2014
Here, we describe a methodology to perform a targeted siRNA “ubiquitome” screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia. This can be adapted to any biological pathway where a robust read out of reporter activity is available.
07:58
In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination
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2026
This protocol describes a method to ubiquitinate Streptococcus pneumoniae using mammalian cell lysate or pure ubiquitin enzyme complex, followed by treatment with purified VCP/p97-UFD1-NPLOC4 protein complex to assess its bacteriolytic activity, enabling the study of ubiquitin-dependent immune effectors.
04:00
Immunoaffinity Based Extraction of Ubiquitinylated Peptides: A Technique to Selectively Extract Ubiquitin Tagged Peptides from Purified Peptide Fractions
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2023
This video describes a method to extract and purify ubiquitinylated peptides containing remnant di-glycine peptides from a complex peptide mixture. The presented method may help in identifying original ubiquitination sites in the protein.
02:50
Conjugated Proteins
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2020
Simple proteins and protein complexes contain only amino acids. In contrast, many other proteins, called conjugated proteins, covalently bond with non-protein moieties. Nucleoproteins are protein complexes that contain nucleic acids, categorized as deoxyribonucleoproteins (DNPs) or ribonucleoproteins (RNPs) respectively. The nucleosome is a typical example of a DNP where nuclear DNA is associated with histone proteins. The major antigen for the Covid-19 virus SARS-CoV is an RNP that is critical...
FAQs
ATP supplies the energy required for E1 to activate ubiquitin before transfer through the enzyme cascade. This energy-dependent step prepares ubiquitin for handling by the downstream E2 enzyme. Because activation occurs before substrate recognition and attachment, it establishes the biochemical starting point for subsequent modification of a target protein.
E2 enzymes carry activated ubiquitin, whereas E3 ligases recognize particular substrate proteins and promote formation of the covalent isopeptide bond. This division separates ubiquitin transport from substrate selection. E3 recognition therefore provides an important source of specificity, helping determine which proteins receive the modification within a cellular context.
The number and arrangement of ubiquitin molecules can influence the outcome of modification. Repeated additions may create chains that direct a protein to the proteasome for degradation, while single ubiquitin molecules or distinct chain architectures can regulate DNA repair, intracellular trafficking, or signaling. Thus, ubiquitin attachment does not produce one universal cellular response.
Analysis should follow the cascade from ATP-dependent activation by E1, to carriage by E2, and then substrate recognition and bond formation promoted by E3. The resulting modification can be examined in terms of whether ubiquitin is added singly or repeatedly and what cellular consequence follows. This sequence links enzymatic steps with protein stability, location, and activity.
This pathway provides a framework for investigating how cells regulate protein stability, location, and activity. It is particularly relevant when examining protein quality maintenance, DNA repair, intracellular trafficking, or signaling, because different ubiquitin arrangements can support different outcomes. Studying these connections helps relate molecular modification events to broader cellular processes.
Researchers can examine whether altered ubiquitin conjugation affects protein quality control or the regulation of proteins involved in DNA repair, trafficking, and signaling. Comparing modification patterns with changes in protein stability, location, or activity can reveal how pathway disruption contributes to disease. The pathway therefore connects enzyme-cascade mechanisms with cellular dysfunction and disease research.