Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1

Bjørkøy, Geir; Lamark, Trond; Pankiv, Serhiy; Øvervatn, Aud; Brech, Andreas; Johansen, Terje

doi:10.1016/s0076-6879(08)03612-4

Cited by 998 publications

(793 citation statements)

References 28 publications

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“…This feature mainly occurs because this organelle is essential in the final step of the autophagic process, wherein autophagosomes fuse with lysosomes and subsequent protein digestion occurs [41]. The autophagic substrate, p62, also known as sequestosome 1 (SQSTM1), binds ubiquitinated proteins for subsequent degradation in lysosomes, where it is also degraded [42]. Our results show that there is no p62 degradation, indicating an impairment in the autophagic flux and consequent accumulation of unprocessed autophagosomes or autolysosomes [43].…”

Section: Discussionmentioning

confidence: 79%

Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer

Rodilla

Korrodi‐Gregório

Hernando

et al. 2017

Biochemical Pharmacology

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Current pharmacological treatments for lung cancer show very poor clinical outcomes, therefore, the development of novel anticancer agents with different mechanisms of action is urgently needed. Cancer cells have a reversed pH gradient compared to normal cells, which favors cancer progression by promoting proliferation, metabolic adaptation and evasion of apoptosis. In this regard, the use of ionophores to modulate intracellular pH appears as a promising new therapeutic strategy. Indeed, there is a growing body of evidence supporting ionophores as a novel antitumor drugs. Despite this, little is known about the implications of pH deregulation and homeostasis imbalance triggered by ionophores at the cellular level. In this work, we deeply analyze for the first time the anticancer effects of tambjamine analogues, a group of highly effective anion selective ionophores, at the cellular and molecular level. First, their effects on cell viability were determined in several lung cancer cell lines and patient-derived cancer stem cells, demonstrating their potent cytotoxic effects. Then, we have characterized the induced lysosomal deacidification, as well as, the massive cytoplasmic vacuolization observed after treatment with these compounds, which is consistent with mitochondrial swelling. Finally, the activation of several proteins involved in stress response, autophagy and apoptosis was also detected, although necrosis was the main mechanism of cell death induced.Altogether, these evidences suggest that tambjamine analogues provoke an imbalance in cellular ion homeostasis that triggers mitochondrial dysfunction and lysosomal deacidification leading to a potent cytotoxic effect through necrosis in lung cancer cell lines and cancer stem cells.

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Section: Discussionmentioning

confidence: 79%

Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer

Rodilla

Korrodi‐Gregório

Hernando

et al. 2017

Biochemical Pharmacology

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“…1D, E and 2C), indicating an inhibition in autophagic flux. 17 Baf treatment at this dose also led to a slight increase in the number of apoptotic chondrocytes (0 § 0% versus 1.04 § 0. Fig.…”

Section: Pharmacological Inhibitors Of Lysosomal Activity Stimulatedmentioning

confidence: 84%

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

et al. 2015

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These authors contributed equally to this publication.Keywords: autophagy, bafilomycin, bone, chondrocyte, lysosome, MTOR, MTORC1Abbreviations: 3-MA, 3-methyladenine; ACAN, aggrecan; ACP5/TRAP, acid phosphatase 5, tartrate-resistant; ACTB, actin, b; ALPL, alkaline phosphatase, liver/bone/kindey; ATG, autophagy related; ATG5cKO, ATG5 conditional knockout in chondrocytes; BC, avidin-biotin complex; Baf, bafilomycin A 1 ; bGP, b-glycerophosphate; BrdU, bromodeoxyuridine; C5.18 cells, RCJ 3.1C5.18 rat mesenchymal cell line; COL2A1, collagen, type II, a 1; COL10A1, collagen, type X, a 1; CQ, chloroquine; DAPI, 4 0 , 6-diamidino-2-phenylindole, dihydrochloride; Dox, doxycycline; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; FBS, fetal bovine serum; GAG, glycosaminoglycan; IGF1, insulin-like growth factor 1 (somatomedin C); LAMP2, lysosomalassociated membrane protein 2; MEFs, mouse embryonic fibroblasts; MAP1LC3A, microtubule-associated protein 1 light chain 3 a;MTOR, mechanistic target of rapamycin (serine/threonine kinase); MTORC, MTOR complex; p-, phosphorylated-; PFA, paraformaldehyde; RPS6, ribosomal protein S6; RPS6KB1/p70(S6K)-a/PS6K/S6K1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; RPTOR, regulatory associated protein of MTOR, complex 1; SGK1, serum/glucocorticoid regulated kinase 1; TSC, tuberous sclerosis complex; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; v-ATPase, vacuolar-type H C -ATPase.Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H C -ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A 1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

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“…28,29 This led us to speculate that autophagymediated degradation of 14-3-3η was occurring during inflammation. We investigated the presence of autophagy in the mucosa of colitic mice by analyzing the conversion of the autophagosomal marker LC3-I to LC3-II 31,32 and the presence of ATG5, a protein that is essential for autophagosome formation. 30 As shown in Figure 4b, the amount of LC3-II and ATG5 augmented in mucosal samples obtained from DSS-treated mice.…”

Section: Resultsmentioning

confidence: 99%

“…30 As shown in Figure 4b, the amount of LC3-II and ATG5 augmented in mucosal samples obtained from DSS-treated mice. Moreover, immunofluorescence assays revealed that LC3-II and the autophagosomal marker p62 31 were enriched in vesicles present in IECs (white arrow) and in cells located at the lamina propria (red star; control mice). The number of both cell types was increased in the mucosa of colitic mice (Figures 4c and d).…”

Section: Resultsmentioning

confidence: 99%

14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells

et al. 2016

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Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1

Cited by 998 publications

References 28 publications

Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer

Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells

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