LC3, a mammalian homologue of yeast ATG8, is a soluble protein that is distributed ubiquitously in mammalian tissues and cultured cells. LC3 has been studied most extensively and is frequently used as an autophagy marker in mammals. Newly translated LC3 (proLC3) is immediately processed at the C-terminus in order to form LC3-I. During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes.
Mechanisms of Autophagy
The ubiquitin-conjugating protein p62/SQSTM1 is thought to be a scaffold protein that interacts with a variety of molecules involved in toll-like receptor (TLR) signaling, such as TRAF6, ERK, and aPKC. Recent evidence suggests that p62 binds directly to the autophagosome marker LC3 and is then selectively degraded by autophagy.
p62 contains multiple phosphorylation sites. Sequential phosphorylation of these sites regulates biological defense mechanisms such as selective autophagy and the Keap1-Nrf2 system. Therefore, the failure of the p62 pathway is associated with various diseases such as cancer and many neurodegenerative diseases.
To learn more about the mechanisms of p62, please refer to our whitepaper written by Drs. Masaaki Komatsu and Yoshinobu Ichimura, Niigata University. They are leading researchers in Autophagy, especially p62 and Keap1-Nrf2 interaction. Dr. Komatsu was recognized as one of the most highly cited researchers in Autophagy in 2014 and 2015 by Clarivate Analytics.
How do you detect p62 or phospho-p62 using antibodies? Western blotting? Immunohistochemistry? Quantification of biomarkers is essential for medical and diagnostic application. Selective autophagy and Keap1-Nrf2 are already being discussed for their use as a diagnostic as well as other medical applications.
MBL International has developed new sandwich ELISA kits that can semi-quantify total p62 and its phosphorylation level at Ser403 and Ser349. Phospho-p62 ELISA kits are the first kits to quantify these markers by ELISA in the world research market. It is a chance to get a new discovery in the Autophagy area!
|CY-7055: CycLex® Total p62 ELISA Kit|
|CY-7057: CycLex® Phospho-p62 Ser403 ELISA Kit|
|CY-7056: CycLex® Phospho-p62 Ser349 ELISA Kit|
These kits can help you to prepare cell lysate easily including cell lysis buffers. Phosphatase inhibitor solution is also included in the phosho-p62 kits. They can be used for human and mouse samples.
Total p62 and phospho-p62 levels in HeLa cells
The process of autophagy was first recognized in the late 1950s and it was thought of as bulk “junk” removal. Then, dedicated scientists did a little more digging and found there is an amazing methodology to the “junk” removal. It was discovered that there is rhyme and reason for how a cell decides if and when its components should undergo degradation. Autophagy is rather specific and aids in cell survival by making sure the cell has essential components during times of flux. Autophagy can be selective (i.e. mitophagy) or non-selective (i.e. starvation-induced). Using this method, the cell can have non-essential components recycled or send them to onto autophagolysosomes to be degraded. It is more efficient for a cell to be able to recycle as much as it can, rather than to wait for new proteins to come into the picture. Autophagy is also crucial for clearing out “junk” such as misfolded or aggregated proteins. Research in autophagy has helped provide a better understanding for how a cell is able to survive even under poor conditions.
What is mitophagy? It is when damaged mitochondria are removed from the cell by autophagy. The damaged mitochondria end up in lysosomes for their final disposal. This whole process is to maintain and assure proper cellular function. The importance of this biological process is that it has been implicated in disease states such as cancer and Parkinson’s disease.
What does Autophagy Flux even mean? It’s not in the Urban dictionary. Dear reader, you have come to the right place to learn a little more about autophagy flux and how our Autophagy Watch kit can help you.