p62/SQSTM1 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

p62/SQSTM1 (ubiquitin-binding protein p62/Sequestrosome-1) is an intracellular protein (theoretical molecular weight 47.7 kDa) which localizes to the cytoplasm, nucleus, autophagosomes and lysosomes in all tissues (1). Structurally, p62/SQSTM1 consists of multiple domains (e.g., Phox1 and Bem1p-PB1, zinc finger-ZZ, TRAF6 binding domain-TB, Keap1-interacting region-KIR, LC3 interacting region-LIR and ubiquitin-associated domain-UBA) for interaction with various protein targets. Additional domains include nuclear export (NES) and nuclear localization signals (NLS1 and NLS2). p62/SQSTM1 is a multifunctional scaffold protein which forms oligomers with itself or with other proteins through interactions with PB1 domains.

Abnormal function of p62/SQSTM1 is associated with a range of disease states such as neurodegeneration, cancer, and metabolic disorders (2). Mutations in the p62/SQSTM1 sequence have been linked to Paget's disease of the bone, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration. In Parkinson's disease, p62/SQSTM1 has been linked to microglia activation and subsequent neuroinflammation (3). Functionally, p62/SQSTM1 is involved in a broad range of cellular processes such as amino acid sensing by interaction with mTORC1, oxidative stress response through interaction with Keap1, and targeting cargo for autophagy by interacting with ubiquitin labeled proteins (1).

To induce selective autophagy, p62/SQSTM1 forms long oligomers or helical filaments which interact with LC3 and ubiquitin labeled proteins and lead to the initiation of the autophagosome formation (2). p62/SQSTM1 is not only a selective autophagy receptor but also an autophagy substrate, as its engulfed by the autophagosome and degraded by the autophagolysosome. Monitoring LC3 levels is the standard for assessing autophagic flux, however monitoring p62/SQSTM1 levels by Western blot in the presence and absence of autophagy inhibitors (e.g., Chloroquine) is also a common practice (4). Besides its activity as a selective autophagy receptor, p62/SQSTM1 also plays a role as an adaptor in signaling cascades leading to NFkB activation downstream of TNF-R, IL-1 beta R, TrkA and p75NTR. Briefly, for NFkB signaling downstream of the TNF-R activation, p62/SQSTM1 engages RIP1 kinase and PKC iota/lambda through the ZZ and PB1 domains, respectively (5).

References

1.Katsuragi, Y., Ichimura, Y., & Komatsu, M. (2015). P62/SQSTM1 functions as a signaling hub and an autophagy adaptor. FEBS Journal. https://doi.org/10.1111/febs.13540

2. Sanchez-Martin, P., & Komatsu, M. (2018). p62/SQSTM1 - Steering the cell through health and disease. Journal of Cell Science. https://doi.org/10.1242/jcs.222836

3. Yao, L., Zhu, Z., Wu, J., Zhang, Y., Zhang, H., Sun, X., ... Lu, G. (2019). MicroRNA-124 regulates the expression of p62/p38 and promotes autophagy in the inflammatory pathogenesis of Parkinson's disease. The FASEB Journal. https://doi.org/10.1096/fj.201900363r

4 Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H., Arozena, A. A., ... Zughaier, S. M. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. https://doi.org/10.1080/15548627.2015.1100356

5. Bitto, A., Lerner, C. A., Nacarelli, T., Crowe, E., Torres, C., & Sell, C. (2014). p62/SQSTM1 at the interface of aging, autophagy, and disease. Age. https://doi.org/10.1007/s11357-014-9626-3

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-16446

• p62/SQSTM1 Antibodies
• p62/SQSTM1 ELISA Kit
• p62/SQSTM1 Lysate
• p62/SQSTM1 Proteins