SQSTM1/p62 and Hepatic Mallory Denk Body Formation in Alcohol-Associated Liver Disease

Autophagy is an evolutionarily conserved degradation process for removing protein aggregates or damaged organelles. Autophagy can be classified into three major types, chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy, according to how cargos that contain disposable materials are delivered to lysosomes.^, The detailed process of these three types of autophagy and the function of autophagy-related genes have been well described in previous reviews.^,, Studies on the role of microautophagy in liver biology and diseases are scarce likely because of the lack of specific tools to separate microautophagy from autophagy and CMA. CMA degrades a subset of proteins that consist of the KFERQ motif using the lysosome-associated membrane protein type 2A as the receptor. CMA declines in aged mouse livers, and loss of hepatic CMA in mice leads to increased hepatic oxidative stress, decreased ability of drug metabolism, and proteostasis, resulting in liver dysfunction. Future studies are needed to investigate the role and relevance of microautophagy and CMA in ALD. Macroautophagy (hereafter referred to as autophagy) initiates from the nucleation of phagophores (also called isolated membrane) and follows by the expansion of phagophores into double membrane autophagosome. Autophagosome and its engulfed cytoplasm materials and damaged organelles then fuse with lysosomes to form autolysosome, resulting in degradation of the engulfed contents. Accumulated evidence indicates that autophagy has emerged as a key player in liver physiology, and it helps maintain liver hemostasis, balance liver metabolism, and liver regeneration.^{,  ,  ,}  In line with this notion, deficiency of autophagy has been associated with multiple liver diseases, especially in ALD^{,  ,}  and HCC.^{,  ,}  Deletion of Atg7 or Atg5 using either albumin-Cre or adeno-associated virus encoding Cre-recombinase under the control of hepatocyte-specific thyroxine-binding globulin promoter (AAV-TBG-Cre) leads to severe liver injury, inflammation, and spontaneous liver tumors with persistent nuclear factor erythroid 2–related factor 2 (NRF2) activation.^{,  ,  ,  ,}  Interestingly, using an inducible Ubc-cre to delete whole-body Atg7 in mice only leads to mild liver pathologic changes with no persistent NRF2 activation. However, these differences are likely due to insufficient deletion of hepatic Atg7 by Ubc-cre in this inducible whole-body autophagy-related 7 knockout mice.

Prior studies suggest that autophagy has an essential role in protecting against acute alcohol-induced liver injury by the clearance of damaged mitochondria and hepatic LDs, whereas long-term alcohol consumption impairs autophagy and aggravates ALD.^{,,  ,}  Alcohol directly impairs autophagy activity by downregulating hepatic expression of transcriptional regulator EB via activation of mechanistic target of rapamycin complex 1 (mTORC1), which reduces lysosomal biogenesis and exacerbates alcohol-induced liver injury in mice. Overexpression of transcriptional regulator EB increases the expression of autophagy-related and lysosomal genes and protects against alcohol-induced steatosis and inflammation in mice. It has been reported that alcohol-fed mice had impaired or insufficient lipophagy, a selective autophagy for removing excess LDs, causing lipid accumulation and liver steatosis.^, Mechanistically, alcohol decreased hepatic RAB7 activity, resulting in decreased fusion of autophagosome with LDs. Alcohol also decreased levels of phosphorylated Src kinase and dynamin 2, causing decreased lysosomes and lysosome reformation and impaired LD breakdown.^, In addition, ethanol treatment elevated ubiquitin signals on LDs, which further recruited autophagy adaptor protein p62 to the LDs to promote lipophagy in AML12 cells. Moreover, enhanced lipophagy by quercetin ameliorated ethanol-induced liver steatosis. Although these findings on lipophagy in cultured cells are interesting, mice with genetic deletion of autophagy-related genes (ie, Atg5, Atg7, or Fip200 also called Rb1cc1) do not develop hepatic steatosis in response to physiologic starvation or partial hepatectomy or high-fat diet feeding.^{,  ,  ,}  Decreased steatosis in autophagy-deficient livers is partially due to the accumulation of nuclear receptor co-repressor 1, which inhibits liver X receptor α–mediated de novo lipogenesis and LD biogenesis. Notably autophagy-deficient livers have remarkably high levels of p62 that leads to NRF2 activation may also contribute to the resistance to starvation-induced hepatic steatosis. Therefore, the relevance of lipophagy in vivo needs to be further investigated.

In addition to lipophagy, mitophagy, a selective autophagy that specifically targets damaged mitochondria, plays a protective role in alcohol-induced liver injury by eliminating damaged mitochondria that otherwise can elevate intracellular reactive oxygen species and cause cell damage.^{,  ,  ,}  Alcohol consumption blunts mitophagy through reducing the mitochondrial fission protein dynamin-related protein 1 in mouse liver and augmented production of megamitochondria, a hallmark of ALD. Loss of hepatic dynamin-related protein 1increases metabolic stress through mitochondrial maladaptation likely due to impaired mitophagy because megamitochondria are difficult to remove by mitophagy because of their size. Both short-term and long-term ethanol exposure can induce parkin mitochondrial translocation and increase parkin-mediated mitophagy, which serves as another layer of adaptive protection against alcohol-induced liver injury.^,

Overall, current data seem to support a temporal role of autophagy in ALD and impaired autophagy, including dysfunction of mitophagy, lipophagy, and lysosome activity, in long-term alcohol exposure, resulting in aggravated ALD. Short-term alcohol exposure induces adaptive protective autophagy, whereas this adaptive autophagy process becomes impaired and lost in long-term alcohol exposure. Pharmacologic intervention of various types of autophagy may serve as new therapeutic avenues for ALD, such as balancing hepatic metabolism (mitophagy), increasing lipid degradation (lipophagy), and promoting clearance of hepatic protein aggregates.

One of the hallmarks of ALD is the accumulation of hepatic Mallory-Denk bodies (MDBs), which are hepatic inclusion bodes and protein aggregates. Sequestosome 1 (SQSTM1/p62, hereafter p62) is a main component of MDBs, which is also a substrate of autophagy that accumulates in autophagy-deficient mouse livers.^{,  ,}  Below we discuss the basic structure of p62, p62-mediated signaling pathways, and selective autophagy as well as their contributions to the pathogenesis of ALD.

p62 is a multidomain protein that interacts with various proteins via its different domains and acts as an intracellular signaling hub for many different pathways.^{,  ,}  The N-terminal Phox and Bem1 (PB1) domain leads to the formation of p62 homo-oligomerization. The PB1 domain of p62 also interacts with other PB1-containing proteins, such as interacting with atypical protein kinase Cζ (PKCζ) to form hetero-oligomers, and interacting with the neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor that has similar domain architecture with p62. Mitogen-activated protein kinase kinase kinase 3 (MEKK3) also contains a PB1 domain, which forms a heterodimer with the PB1 domain of p62 and binds to tumor necrosis receptor–associated factor 6 (TRAF6), a lysine 63 E3 ligase, to trigger NF-κB activation. The PB1 domain is followed by a ZZ-type zinc finger domain, and this ZZ domain of p62 is required for efficient starvation-induced autophagy in mouse embryonic fibroblasts. Moreover, the ZZ domain binds to the receptor interacting protein to regulate NF-κB activation. The TB domain next to the ZZ domain also activates NF-κB via interacting with TRAF6. Additionally, p62 interacts with the regulatory-associated protein of mTOR via the region between the ZZ and TB domains to activate mTORC1. p62 directly binds to LC3 through the LC3-interacting region (LIR) and thus acts as an autophagy receptor protein for selective autophagy.^, Followed by the LIR domain is a Kelch-like ECH-associated protein 1 (KEAP1) interacting region (KIR) that binds to KEAP1 and drives KEAP1 degradation by selective autophagy, resulting in NRF2 activation via the noncanonical KEAP1-NRF2 pathway.^{,,  ,}  p62- and KEAP1-positive aggregates have been observed in the autophagy-deficient mouse livers, causing the persistent activation of NRF2 in the liver.^,, The ubiquitin-associated (UBA) domain on C-terminal binds to ubiquitin-labeled proteins or damaged organelles and leads them into autophagosome for degradation.^,

Although p62 can interact with LC3, KEAP1, and ubiquitinated proteins, these interactions are considered relatively weak at the basal levels. However, these weak interactions can be enhanced by the oligomerization and phosphorylation of p62 on various sites. p62’s PB1 domain–mediated oligomerization increases the binding affinities between p62 and other proteins, including the UBA domain of p62 with ubiquitinated misfolded proteins and the LIR domain of p62 with LC3, thus promoting ubiquitinated protein aggregates relocated to autophagosomes for degradation. Casein kinase 2 directly phosphorylates Ser403 of p62 to increase the autophagic clearance of ubiquitinated proteins and protein aggregates. Additionally, polyubiquitinated mitochondria recruited TANK-binding kinase 1 (TBK1) through TBK1-binding adaptor proteins, including optineurin (OPTN). TBK1 is activated by autophosphorylation and phosphorylates p62 at S403, which further strengthens binding between the UBA domain and ubiquitin that drives the ubiquitinated mitochondria into autophagosome for degradation. mTORC1 phosphorylates p62 at Ser349 (humans) or Ser351 (mice) to enhance the binding affinity between the KIR domain of p62 and KEAP1, resulting in constant NRF2 activation through the noncanonical KEAP-NRF2 pathway. Both the tripartite motif (TRIM) 16 and TRIM21 proteins have E3 ligase activities, and they ubiquitylate p62 under oxidative and proteotoxic stress conditions.^, However, these two proteins play different roles during stress. TRIM16 acts as a scaffold protein to interact with p62, KEAP1, and ubiquitinated proteins, leading to the degradation of protein aggregates and stabilizing NRF2 activation against oxidative stress. In contrast to TRIM16, TRIM21 prevents p62 oligomerization and releases the p62 sequestrated KEAP1 by ubiquitylating PB1 domain of p62 at lysine (K)7 via lysine 63 linkage, leading to a decrease in p62-mediated autophagy activity and an increase in KEAP1-mediated NRF2 degradation to aggravate oxidative stress and liver carcinogenesis.^, In addition, cyclin-dependent kinase 1 phosphorylated p62 at Thr269 and Ser272 during mitosis, regulating cell cycle progression, cell proliferation, and tumorigenesis. Because of the unique protein structure of p62 and its capacity to interact with multiple proteins in various signaling pathways, it is not surprising that p62 has critical roles in regulating redox, proteostasis, cell death, cell survival, proliferation, and tumorigenesis.^,, In addition to phosphorylation and ubiquitination, p62 can also be acetylated by acetyltransferase TIP60 and deacetylated by deacetylase histone deacetylase 6. Acetylation at K420 and K435 increases the binding of p62 to ubiquitin and facilitates polyubiquitin chain–induced p62 phase separation by disrupting UBA dimerization. In cells under stress, increased p62 acetylation may thus promote selective degradation of ubiquitinated proteins by regulating the assembly of p62 bodies. The schematic domain structure and posttranslational modifications of p62 is illustrated in Figure 1 and its interacting partners in Table 1.^{,  ,  ,  ,  ,} 

Because of its unique multidomain structure and interactions with various proteins, p62 not only plays a role in selective autophagy but also contributes to the formation of stress granules (SGs) and ubiquitin-positive protein aggregates in the cytoplasm. p62 is a common component of many human disease–related cellular inclusion bodies, such as MDBs, IHBs, and α₁-antitrypsin aggregates in the liver^{,  ,}  as well as Lewy bodies, neurofibrillary tangles, and huntingtin aggregates in the brain.^, Notably, all these inclusion bodies are also positive for ubiquitin. Unlike soluble proteins that can be removed by ubiquitin proteasome system, insoluble protein aggregates can only be removed by autophagy.^, Therefore, the presence of hepatic protein aggregates may act as a sign for decreased autophagy activity.

MDBs, which are membrane-less cytoplasmic protein aggregates, are found in three-fourths of patients with AH and in nearly all patients with alcoholic cirrhosis. MDBs were first discovered in a patient with AH by professor Frank B. Mallory in 1911. MDBs have been found in ballooned hepatocytes and are associated with ALD. The existence of MDBs in hepatocytes is a hallmark of ALD, although whether and how MDBs would contribute to ALD remains elusive.^, Along with that, there are multiple factors that contribute to MDB formation. Mice exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine and high-fat diet show increased MDB formation and develop severe liver injury and inflammation.^, Sex also plays a crucial role in MDB formation, with one study showing that male mice formed significantly more MDBs than female mice, which is likely due to higher levels of estradiol and lower levels of oxidative stress in female mice. In addition, aging mice are more susceptible to MDB formation compared with young mice due to increased oxidative stress and decreased autophagy and proteasome activities. Animal models are essential to investigate the pathogenesis and mechanisms of ALD. Unfortunately, current ALD animal models do not faithfully phenocopy the full spectrum of human ALD because these animal models fail to recapitulate the characteristics of severe human ALD, such as fibrosis, ductular reaction, and accumulation of MDBs and hepatic progenitor cells. One potential approach is to combine a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet and alcohol feeding in mice, which increases cholestasis and hepatic progenitor cells, although MDBs were not investigated in this model.

The major components of MDBs are keratin 8/18, ubiquitinated proteins, chaperone proteins, misfolded proteins, transglutaminase-2, and p62. The indispensable step of MDB formation is stress-induced up-regulation of K8, which is cross-linked by transglutaminase-2.^, The increased ubiquitinated K8 and ubiquitinated misfolded proteins overwhelm proteasome and other protein quality control machineries, resulting in the accumulation of MDBs. The UBA domain of p62 directly binds to ubiquitinated K8 and ubiquitinated misfolded proteins, which further sequesters them into aggresome or MDBs via the PB1 domain of p62. p62 appears to play a critical role in MDB formation because p62 knockout mice manifest defective MDB maturation and fail to form large MDBs, although this do not affect modifications of keratin. Some studies indicated that p62 reduces cytotoxicity by sequestering soluble misfolded protein into insoluble and less toxic aggresomes or sequestosome-like aggregates, such as MDBs. In addition to regulating MDB formation, as a substrate of selective autophagy, p62 also mediates MDB degradation by autophagy. Therefore, the balance between the formation and degradation mediated by p62 may determine the levels of MDBs in ALD.

Although the reticular MDBs have multiple components, the globular intracytoplasmic hyaline bodies only consist of p62 and ubiquitin or only p62. A previous study showed that if the expression of p62 prevails by itself, the p62-containing aggregates IHBs will form; however, if abnormal keratins and p62 are present at the same time, the MDBs will form. Both MDBs and IHBs have been detected in HCC, in which MDBs occur in approximately 20% to 30% of HCCs, whereas IHBs are seen in approximately 20% of the cases.^,, In addition, HCCs with IHB show worse prognosis than HCCs without IHBs, but the function of IHBs is still largely unclear. IHBs are also eliminated by p62-mediated autophagy, and increased p62-containing protein aggregates is regarded as a marker of impaired autophagy.^, It remains unclear whether MDBs and IHBs would also contain KEAP1 and regulate noncanonic NRF2 activation. Future studies are needed to further investigate whether and how p62-positive IHBs or MDBs contribute to the liver injury and tumorigenesis in autophagy-deficient livers and ALD.

During the past few years, the presence of a number of non–membrane-bound body or organelles inside the cell, such as SGs, germ granules, Cajal body, and nucleolus, has drawn a lot of research attention and expanded our current knowledge on their cellular organization and functions. SGs can rapidly form in cells during stress as a mechanism to combat the potentially negative effect on cell health elicited by the stress, and dysregulation of SG is implicated in many diseases.^, These non–membrane-bound organelles, which are mainly composed of proteins and RNAs, have liquidlike properties. They can fuse and flow like a liquid droplet, and molecules can undergo rapid exchange within the droplet as well as with their surrounding cytoplasm because of the lack of a physical barrier of lipid layers (which is commonly observed in membrane-bound organelles, such as mitochondria). Some of these non–membrane-bound organelles can mature into a gel-like state or solidify into amyloidlike aggregates. The formation of non–membrane-bound organelles is driven by the liquid-liquid phase separation from surrounding cytoplasm, leading to a condensed phase with participating molecules.^, The liquid-liquid phase separation is mediated by multivalent interactions from proteins and RNAs,^{,,  ,}  among which the RNA-binding proteins with intrinsically disordered regions are overrepresented.

As a multivalent protein, p62 has emerged to undergo liquid–liquid-phase separation. p62 oligomerizes via its PB1 domain and thus clusters UBA domains from each p62 in the oligomers, creating a multivalent hub that is accessible to multiple partners.^,, Overexpression of NBR1 blocks selective degradation of p62 and promotes the accumulation of phosphorylated p62 in liquidlike bodies. p62 droplets formed in vivo also show liquidlike properties, such as high sphericity, the ability to undergo fusion, and recovery after photobleaching. Liquidlike properties of the condensates are crucial in the initiation of aggrephagy.^,, p62 acts as an RNA-binding protein, and p62 binds with vault RNAs, which are approximately 88- to 100-nt long noncoding RNAs, via the ZZ domain and impairs selective autophagy for ubiquitinated proteins. Subsequent interactome studies show that p62 is enriched with recombinant signal binding proteins. Among these, seven of the 46 candidate p62 interactors are known as SG components^, In this way, p62 might also be involved in SG formation. SGs play a protective role in cells during the initial insult from the environment. However, an increasing body of evidence has suggested that prolonged stress can cause the vitrification of SGs and turn into solid aggregates, which eventually develop into cytosolic inclusion bodies. Those inclusion bodies are also characterized with ubiquitinated proteins and are abundant with p62^{,  ,}  (Figure 2). Whether and how alcohol would affect SGs in hepatocytes are not clear, but it is likely that alcohol consumption may increase hepatic SG formation in early ALD, which may further progress to MDBs in patients with severe AH. Future studies are needed to further dissect the role of p62 in regulating intracellular SG formation and homeostasis in the pathogenesis of ALD.

As we mentioned above, the formation of MDB and IHB inclusions caused by the aggregation of misfolded proteins is a hallmark of ALD. Many studies point out that soluble protein aggregates, such as a dimer, oligomer, or fibril, can be cytotoxic in the disease state due to increased proteotoxicity, whereas the formation of insoluble protein aggregates or inclusion is protective due to decreased proteotoxicity.^, There are two major protein degradation pathways in eukaryotic cells: the ubiquitin-proteasome system (UPS) and autophagy.^,, Misfolded proteins typically refold with the help of chaperons or get degraded by UPS. However, when chaperons and UPS fail to take care of misfolded proteins, these misfolded proteins tend to accumulate into protein aggregates and are removed by a selective form of autophagy called aggrephagy.^, Ubiquitination of misfolded proteins serves as a key signal for aggrephagy, where the ubiquitinated aggregates can be recognized by various autophagy receptors for degradation. There are various autophagy cargo receptor proteins that have been reported in aggrephagy, and all these cargo receptors contain LIR domain or putative LC3-interacting regions to interact with LC3 and drive misfolded proteins or aggregates for aggrephagy. These autophagy cargo receptor proteins include p62, NBR1, OPTN, nuclear dot protein 52, Toll-interacting protein, and tax1 binding protein 1 (a homology to nuclear dot protein 52).^{,,  ,}  The eukaryotic chaperonin TCP-1 ring complex subunit chaperonin-containing TCP-1 subunit 2 acts as a chaperone and an aggrephagy receptor, which regulates aggrephagy by promoting autophagosome incorporation and clearance of protein aggregates via interacting with LC3 and ubiquitinated proteins.