Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, R, M||WB, FC, ICC||M||Purified||Monoclonal Antibody|
|Presentation||Purified mouse monoclonal IgMκ in buffer containing PBS with 0.05% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at 2-8°C from date of receipt.|
|Material Size||100 µg|
Anti-p62 (Sequestosome-1) Antibody, clone 11C9.2 SDS
|Reference overview||Pub Med ID|
|Suppression of experimental arthritis through AMP-activated protein kinase activation and autophagy modulation.|
Yan, H; Zhou, HF; Hu, Y; Pham, CT
Journal of rheumatic diseases and treatment 1 5 2015
Autophagy plays a central role in various disease processes. However, its contribution to inflammatory arthritides such as rheumatoid arthritis (RA) is unclear. We observed that autophagy is engaged in the K/BxN serum transfer model of RA but autophagic flux is severely impaired. Metformin is an anti-diabetic drug that has been shown to stimulate autophagy. Induction of autophagic flux, through metformin-mediated AMP-activated protein kinase (AMPK) activation and interruption of mammalian target of rapamycin (mTOR) signaling mitigated the inflammation in experimental arthritis. Further investigation into the effects of metformin suggest that the drug directly activates AMPK and dose-dependently suppressed the release of TNF-α, IL-6, and MCP-1 by macrophages while enhancing the release of IL-10 in vitro. In vivo, metformin treatment significantly suppressed clinical arthritis and inflammatory cytokine production. Mechanistic studies suggest that metformin exerts its anti-inflammatory effects by correcting the impaired autophagic flux observed in the K/BxN arthritis model and suppressing NF-κB-mediated signaling through selective degradation of IκB kinase (IKK). These findings establish a central role for autophagy in inflammatory arthritis and argue that autophagy modulators such as metformin may represent potential therapeutic agents for the treatment of RA.
|Fumagillin prodrug nanotherapy suppresses macrophage inflammatory response via endothelial nitric oxide.|
Zhou, HF; Yan, H; Hu, Y; Springer, LE; Yang, X; Wickline, SA; Pan, D; Lanza, GM; Pham, CT
ACS nano 8 7305-17 2014
Antiangiogenesis has been extensively explored for the treatment of a variety of cancers and certain inflammatory processes. Fumagillin, a mycotoxin produced by Aspergillus fumigatus that binds methionine aminopeptidase 2 (MetAP-2), is a potent antiangiogenic agent. Native fumagillin, however, is poorly soluble and extremely unstable. We have developed a lipase-labile fumagillin prodrug (Fum-PD) that eliminated the photoinstability of the compound. Using αvβ3-integrin-targeted perfluorocarbon nanocarriers to deliver Fum-PD specifically to angiogenic vessels, we effectively suppressed clinical disease in an experimental model of rheumatoid arthritis (RA). The exact mechanism by which Fum-PD-loaded targeted nanoparticles suppressed inflammation in experimental RA, however, remained unexplained. We herein present evidence that Fum-PD nanotherapy indirectly suppresses inflammation in experimental RA through the local production of endothelial nitric oxide (NO). Fum-PD-induced NO activates AMP-activated protein kinase (AMPK), which subsequently modulates macrophage inflammatory response. In vivo, NO-induced AMPK activation inhibits mammalian target of rapamycin (mTOR) activity and enhances autophagic flux, as evidenced by p62 depletion and increased autolysosome formation. Autophagy in turn mediates the degradation of IkappaB kinase (IKK), suppressing the NF-κB p65 signaling pathway and inflammatory cytokine release. Inhibition of NO production by N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, reverses the suppression of NF-κB-mediated inflammatory response induced by Fum-PD nanotherapy. These unexpected results uncover an activity of Fum-PD nanotherapy that may be further explored in the treatment of angiogenesis-dependent diseases.
|Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling.|
Valapala, M; Wilson, C; Hose, S; Bhutto, IA; Grebe, R; Dong, A; Greenbaum, S; Gu, L; Sengupta, S; Cano, M; Hackett, S; Xu, G; Lutty, GA; Dong, L; Sergeev, Y; Handa, JT; Campochiaro, P; Wawrousek, E; Zigler, JS; Sinha, D
Autophagy 10 480-96 2014
In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.
|Human stefin B role in cell's response to misfolded proteins and autophagy.|
Polajnar, M; Zavašnik-Bergant, T; Škerget, K; Vizovišek, M; Vidmar, R; Fonović, M; Kopitar-Jerala, N; Petrovič, U; Navarro, S; Ventura, S; Žerovnik, E
PloS one 9 e102500 2014
Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.
|AMPK involvement in endoplasmic reticulum stress and autophagy modulation after fatty liver graft preservation: a role for melatonin and trimetazidine cocktail.|
Zaouali, Mohamed Amine, et al.
J. Pineal Res., (2013) 2013
Ischemia/reperfusion injury (IRI) associated with liver transplantation plays an important role in the induction of graft injury. Prolonged cold storage remains a risk factor for liver graft outcome, especially when steatosis is present. Steatotic livers exhibit exacerbated endoplasmic reticulum (ER) stress that occurs in response to cold IRI. In addition, a defective liver autophagy correlates well with liver damage. Here, we evaluated the combined effect of melatonin and trimetazidine as additives to IGL-1 solution in the modulation of ER stress and autophagy in steatotic liver grafts through activation of AMPK. Steatotic livers were preserved for 24 hr (4°C) in UW or IGL-1 solutions with or without MEL + TMZ and subjected to 2-hr reperfusion (37°C). We assessed hepatic injury (ALT and AST) and function (bile production). We evaluated ER stress (GRP78, PERK, and CHOP) and autophagy (beclin-1, ATG7, LC3B, and P62). Steatotic livers preserved in IGL-1 + MEL + TMZ showed lower injury and better function as compared to those preserved in IGL-1 alone. IGL-1 + MEL + TMZ induced a significant decrease in GRP78, pPERK, and CHOP activation after reperfusion. This was consistent with a major activation of autophagic parameters (beclin-1, ATG7, and LC3B) and AMPK phosphorylation. The inhibition of AMPK induced an increase in ER stress and a significant reduction in autophagy. These data confirm the close relationship between AMPK activation and ER stress and autophagy after cold IRI. The addition of melatonin and TMZ to IGL-1 solution improved steatotic liver graft preservation through AMPK activation, which reduces ER stress and increases autophagy.
|Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy.|
Papadakis, M; Hadley, G; Xilouri, M; Hoyte, LC; Nagel, S; McMenamin, MM; Tsaknakis, G; Watt, SM; Drakesmith, CW; Chen, R; Wood, MJ; Zhao, Z; Kessler, B; Vekrellis, K; Buchan, AM
Nature medicine 19 351-7 2013
Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia and the tolerance conferred by ischemic preconditioning (IPC), would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo, suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.