Autophagic Regulation of p62 is Critical for Cancer Therapy

Cell, 2009

Allelic loss of the essential autophagy gene beclin1 occurs in human cancers and renders mice tumorprone suggesting that autophagy is a tumor-suppression mechanism. While tumor cells utilize autophagy to survive metabolic stress, autophagy also mitigates the resulting cellular damage that may limit tumorigenesis. In response to stress, autophagy-defective tumor cells preferentially accumulate p62/SQSTM1 (p62), endoplasmic reticulum (ER) chaperones, damaged mitochondria, reactive oxygen species (ROS), and genome damage. Moreover, suppressing ROS or p62 accumulation prevented damage resulting from autophagy defects indicating that failure to regulate p62 caused oxidative stress. Importantly, sustained p62 expression resulting from autophagy defects was sufficient to alter NF-κB regulation and gene expression and to promote tumorigenesis. Thus defective autophagy is a mechanism for p62 upregulation commonly observed in human tumors that contributes directly to tumorigenesis likely by perturbing the signal transduction adaptor function of p62 controlling pathways critical for oncogenesis.

BioMed Research International, 2014

The ubiquitin-proteasome system and autophagy were long viewed as independent, parallel degradation systems with no point of intersection. By now we know that these degradation pathways share certain substrates and regulatory molecules and show coordinated and compensatory function. Two ubiquitin-like protein conjugation pathways were discovered that are required for autophagosome biogenesis: the Atg12-Atg5-Atg16 and Atg8 systems. Autophagy has been considered to be essentially a nonselective process, but it turned out to be at least partially selective. Selective substrates of autophagy include damaged mitochondria, intracellular pathogens, and even a subset of cytosolic proteins with the help of ubiquitin-binding autophagic adaptors, such as p62/SQSTM1, NBR1, NDP52, and Optineurin. These proteins selectively recognize autophagic cargo and mediate its engulfment into autophagosomes by binding to the small ubiquitin-like modifiers that belong to the Atg8/LC3 family.

Cell Cycle, 2009

Autophagy is an evolutionary conserved cell survival process for degradation of long-lived proteins, damaged organelles and protein aggregates. The mammalian proteins p62 and NBR1 are selectively degraded by autophagy and can act as cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates. Despite differing in size and primary sequence, both proteins share a similar domain architecture containing an N-terminal PB1 domain, a LIR motif interacting with ATG8 family proteins, and a C-terminal UBA domain interacting with ubiquitin. The LIR motif is essential for their autophagic degradation, indicating that ATG8 family proteins are responsible for the docking of p62 and NBR1 to nucleating autophagosomes. p62 and NBR1 co-operate in the sequestration of misfolded and ubiquitinated proteins in p62 bodies and are both required for their degradation by autophagy. Here we discuss the role of p62 and NBR1 in degradation of ubiquitinated cargoes and the putative role of LIR as a general motif for docking of proteins to ATG8 family proteins.

Autophagy, 2010

Abbreviations: ALFY, autophagy-linked FYVE protein; BEACH, beige and chediak-higashi; GFP, enhanced green fluorescent protein; LC3, light chain 3; PB1 domain, Phox and Bem1 domain; siRNA, small interfering RNA; UBA domain; ubiquitin-associated domain accumulation of ubiquitinated protein aggregates and neurodegeneration both in mice and Drosophila. 11-14 Moreover, an increase in intracellular, ubiquitinated protein aggregates is observed upon aging. 13,15 The Ub-binding adaptor protein p62/SQSTM1 accumulates in Ub-positive inclusions in neurodegenerative diseases and proteinopathies of the liver, 16-18 as well as in the brains of mice with the lysosomal storage diseases multiple sulfatase deficiency or mucopolysacharidosis type IIIA. 19 Moreover, ubiquitin-and p62-positive protein bodies (named p62 bodies or ALIS; aggresome-like inducible structures) form in response to cellular stress, such as amino acid starvation, oxidative stress, accumulation of defective ribosomal products, or inhibition of autophagy. 5,6-8 Both p62 and its Drosophila homologue Ref(2)P has been found to be required for the formation of ubiquitinated protein aggregates, both in cell culture and in vivo. 5,6,20,21 The 440-amino acid p62 protein homopolymerizes and interacts with atypical protein kinase Cs, MEK5 and NBR1 via its N-terminal PB1 domain. 22-24 Moreover, it has a C-terminal UBA (Ub-associated) domain that binds both Accumulation of ubiquitinated proteins in cytoplasmic and/or nuclear inclusions is a hallmark of several diseases associated with premature cell death. sQsTM1/p62 is known to bind ubiquitinated substrates and aid their aggregation and degradation by macroautophagy. We show here that p62 is required to recruit the large phosphoinositide-binding protein ALFY to cytoplasmic p62 bodies generated upon amino acid starvation or puromycin-treatment. ALFY, as well as p62, is required for formation and autophagic degradation of cytoplasmic ubiquitin-positive inclusions. Moreover, both p62 and ALFY localize to nuclear promyleocytic leukemia (PML) bodies. The Drosophila p62 homologue Ref(2) P accumulates in ubiquitinated inclusions in the brain of flies carrying mutations in the ALFY homologue Blue cheese, demonstrating that ALFY is required for autophagic degradation of p62-associated ubiquitinated proteins in vivo. We conclude that p62 and ALFY interact to organize misfolded, ubiquitinated proteins into protein bodies that become degraded by autophagy.

Journal of Biological Chemistry, 2007

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related ␥-aminobutyrate receptor-associated protein and ␥-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62-and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62-and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 m diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.

Autophagy, 2010

Atg8 interacts with Atg11, which brings Atg19-associated vacuolar enzyme aminopeptidase I to autophagosomes for packaging. 11-14 Recent studies revealed that mitophagy, a specific form of autophagy that mediates the degradation of mitochondria, is also bridged through an interaction between a mitochondrial membrane-associated protein Atg32 and Atg8. 15,16 In mammalian cells, p62, as well as NBR1 (next to BRCA1 gene 1 protein) and Alfy (autophagy-linked FYVE protein), 17,18 serve as crucial adaptors between LC3-decorated autophagosomes and ubiquitin-conjugated protein aggregates. 19,20 p62 binds to LC3 via an LC3-interacting region (LIR) and ubiquitin chains through a UBA (ubiquitin-associated) domain. 21 Ubiquitin conjugates accumulate in autophagy knockout mice and lead to cytotoxicity in multiple tissues and cells. 5-7 Concomitant deletion of p62 rescued the cytotoxic phenotype of liver cells in autophagy mutant mice. 9 Moreover, p62 accumulation is frequently observed in human tumors due to impairment of the autophagy pathway, which leads to the oxidative stress and inflammation responses that correlate with tumorigenesis. 22 However, how p62 links autophagy, oxidative stress and ubiquitin aggregates together is still unclear. Here we report that an oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) interacts with p62 and LC3. Upon puromycin treatment, the majority of Keap1 localizes to cellular puncta that are also positive for p62, ubiquitin conjugates, and LC3. Furthermore,

Frontiers in Cell and Developmental Biology, 2022

The key tumor suppressor protein p53, additionally known as p53, represents an attractive target for the development and management of anti-cancer therapies. p53 has been implicated as a tumor suppressor protein that has multiple aspects of biological function comprising energy metabolism, cell cycle arrest, apoptosis, growth and differentiation, senescence, oxidative stress, angiogenesis, and cancer biology. Autophagy, a cellular self-defense system, is an evolutionarily conserved catabolic process involved in various physiological processes that maintain cellular homeostasis. Numerous studies have found that p53 modulates autophagy, although the relationship between p53 and autophagy is relatively complex and not well understood. Recently, several experimental studies have been reported that p53 can act both an inhibitor and an activator of autophagy which depend on its cellular localization as well as its mode of action. Emerging evidences have been suggested that the dual role o...

Methods in Enzymology, 2009

Autophagy, 2008

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