自噬与p62/SQSTM1在肝缺血再灌注中的作用

自噬是溶酶体依赖性的降解系统,p62作为重要的蛋白载体,可促进受损蛋白经蛋白酶体和自噬体清除。p62在线粒体自噬等选择性自噬中发挥重要作用。p62与Keap1结合并通过自噬清除Keap1,这导致游离的Nrf2水平增加,而Nrf2又可促进p62表达。因此自噬、p62、Nrf2三者间存在密切的联系,相互调控。同时,Nrf2还可促进谷胱甘肽(Glutathione,GSH)和硫氧还蛋白(Thioredoxin,TXN)等多种分子的表达,而在肝缺血再灌注损伤过程中,肝细胞内这些分子可能参与细胞死亡的过程。因此,自噬与p62在肝缺血再灌注中的具体机制仍需进一步研究明确。     

多功能蛋白p62调节细胞自噬及相关疾病的研究进展

Sequestosome 1 （p62/SQSTM1）是一种由应激诱导产生的细胞内蛋白并作用于选择性自噬的多功能蛋白.p62包含多种蛋白作用域,可结合聚泛素化蛋白并将其进一步降解,在信号转导过程中发挥重要作用.研究表明,p62参与多种疾病的病理过程,如神经退行性疾病、糖尿病、肥胖甚至是癌症.因而从p62的结构和功能这两方面阐述其在相关疾病中的作用很有必要.     

p62/SQSTM1 synergizes with autophagy for tumor growth in vivo

Autophagy is crucial for cellular homeostasis and plays important roles in tumorigenesis. FIP200 (FAK family-interacting protein of 200 kDa) is an essential autophagy gene required for autophagy induction, functioning in the ULK1–ATG13–FIP200 complex. Our previous studies showed that conditional knockout of FIP200 significantly suppressed mammary tumorigenesis, which was accompanied by accumulation of p62 in tumor cells. However, it is not clear whether FIP200 is also required for maintaining tumor growth and how the increased p62 level affects the growth in autophagy-deficient FIP200-null tumors in vivo. Here, we describe a new system to delete FIP200 in transformed mouse embryonic fibroblasts as well as mammary tumor cells following their transplantation and show that ablation of FIP200 significantly reduced growth of established tumors in vivo. Using similar strategies, we further showed that either p62 knockdown or p62 deficiency in established FIP200-null tumors dramatically impaired tumor growth. The stimulation of tumor growth by p62 accumulation in FIP200-null tumors is associated with the up-regulated activation of the NF-κB pathway by p62. Last, we showed that overexpression of the autophagy master regulator TFEB^S142A increased the growth of established tumors, which correlated with the increased autophagy of the tumor cells. Together, our studies demonstrate that p62 and autophagy synergize to promote tumor growth, suggesting that inhibition of both pathways could be more effective than targeting either alone for cancer therapy.     

Autoantibodies to IGF-II mRNA binding protein p62 and overexpression of p62 in human hepatocellular carcinoma

Studies of autoantibodies in systemic rheumatic diseases have provided abundant evidence suggesting that autoimmune responses are antigen-driven and that autoantibodies often can be viewed as reporters of the immune system revealing the identity of antigens which might be playing roles in the pathophysiology of the disease process. Recent data from our laboratory suggest a similar mechanistic process may be involved in humoral immune responses in certain cancers such as hepatocellular carcinoma (HCC). HCC is unique in that one can follow a cohort of patients with chronic liver disease who will likely progress to develop malignancy over a period of 10 or more years. It has been observed that during transition from chronic liver disease to HCC, novel autoantibodies can appear which are not detected prior to pre-malignant conditions. The hypothesis is that these novel antibody responses may be stimulated by cellular proteins which are involved in carcinogenesis. By immunoscreening an expression library to isolate cDNA clones of autoantigens, a RNA-binding autoantigen p62 has been identified in HCC recently and autoantibodies to p62 were found in 21% of a cohort of HCC patients. p62 is a cytoplasmic protein which binds to mRNA encoding insulin-like growth factor II (IGF-II), a growth factor which is known to be overexpressed in HCC and is tumorigenic in transgenic animals. The expression of p62 is developmentally regulated, and expressed in fetal, but not in adult liver. Our recent observations showing that p62 was aberrantly expressed in 30% of unselected HCC suggest that it could play a role in HCC and other tumors by upregulating expression of growth factor IGF-II in the milieu of other oncogenic factors.     

酒精诱导嗜铬细胞瘤细胞自噬及其与P62作用的关系

目的 采用不同浓度酒精作用于大鼠嗜铬细胞瘤（PC12）细胞,观察细胞自噬发生及其与P62变化的关系。 方法 用四甲基偶氮唑盐比色法（MTT）观察酒精对PC12细胞生存率的影响;间接免疫荧光法检测细胞自噬标志性蛋白LC3和P62的变化;高内涵活细胞成像系统检测细胞LC3荧光强度;透射电镜检测细胞自噬的超微结构;Western blotting方法检测P62蛋白量的表达。 结果 50～800mmol/L浓度酒精对PC12细胞的增殖有显著的抑制作用,呈浓度依赖性。酒精致PC12细胞自噬标志性蛋白LC3在细胞核周围密度增高,并与P62形成点状聚集共定位,其中在200mmol/L浓度酒精作用2h,PC12细胞LC3自噬荧光强度最高;透射电镜也观察到酒精作用的PC12细胞质中自噬体和自噬溶酶体。Western blotting结果显示,不同浓度酒精处理PC12细胞2h,P62蛋白表达量显著增加 (P<0.01);用200mmol/L浓度酒精处理PC12细胞,P62蛋白表达在2h达到最高值。 结论 酒精诱导PC12细胞的自噬作用,P62蛋白参与自噬调控过程。     

Chaperone-mediated autophagy degrades mutant p53

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells.     

The effect of RNAi silencing of p62 using an osmotic polysorbitol transporter on autophagy and tumorigenesis in lungs of K-ras mice

Treating cancer patients by conventional chemotherapy to achieve prolonged survival still remains complicated. Autophagy is a topic of considerable interest in recent times, as it may contribute greatly to tumor suppression. Recent studies indicate that autophagy-deficient cells accumulate high levels of p62, an ubiquitin-binding scaffold protein, involved greatly in tumorigenesis. Here, we synthesized an osmotically active polysorbitol-mediated transporter (PSMT) to downregulate p62 using an RNAi strategy and described the mechanism of how p62 silencing using PSMT/siRNA p62 system activates autophagy and contributes to tumor suppression in the lungs of K-ras^LA1 mice. Downregulation of p62 by PSMT/siRNA p62 activated autophagy confirmed by the formation of autophagosomes and swelling of Golgi apparatus with a decreasing level of GM130, a cis-Golgi matrix protein. Activation of osmotic PSMT-mediated autophagy remarkably reduced the size and number of tumors by suppressing proliferative cell nuclear antigen, cluster of differentiation 31, and vascular endothelial growth factor levels. Furthermore, an increase in apoptosis was observed in the lungs of PSMT/siRNA p62-delivered K-ras^LA1 mice.     

Exonucleolytic degradation of RNA by p53 protein in cytoplasm

p53 in cytoplasm displays an intrinsic 3'-->5' exonuclease activity. To understand the significance of p53 exonuclease activity in cytoplasm, cytoplasmic extracts of various cell lines were examined for exonuclease activity with different single-stranded RNA (ssRNA) substrates. Using an in vitro RNA degradation assay, we observed in cytoplasmic extracts of LCC2 cells, expressing high levels of endogenous wtp53, an efficient 3'-->5' exonuclease activity with RNA substrates, removing the 3'-terminal nucleotides. Interestingly, RNA containing AU-rich sequences (ARE) is the permissive substrate for exonucleolytic degradation. Evidence that exonuclease function with RNA detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: (1) this activity closely parallels with status and levels of endogenous cytoplasmic p53; (2) the endogenous exonuclease exerts identical RNA substrate specificity and excision profile characteristic for purified baculovirus-or bacterially-expressed wtp53s; (3) the exonuclease activity with ARE RNA is competed out by the presence of ss or double-stranded DNA substrate utilized by p53 protein in cytoplasm; (4) immunoprecipitation by specific anti-p53 antibodies markedly reduced the exonuclease activity with both RNA and DNA substrates; and (5) transfection of the wtp53, but not exonuclease-deficient mutant p53-R175H, into p53-null H1299 or HCT116 cells induced high levels of exonuclease activity with ARE RNA substrate in cytoplasm with characteristic excision profile. The efficient ARE RNA degradation correlates with the efficient binding of p53 to ARE RNA in cytoplasm. The possible role of p53 exonuclease activity in ARE-mRNA destabilization in cytoplasm, which may be important for expression of proteins that control cell growth and/or apoptosis is discussed.     

脂质诱导的p62dok高表达增加肝葡萄糖异生

目的:探讨p62^dok表达与肝细胞葡萄糖异生间的相互关系。方法:利用小鼠高脂饮食模型,观察p62^dok表达与肝组织胰岛素信号转导和葡萄糖异生调节蛋白表达间的关系;利用原代培养的小鼠肝细胞,采用基因沉默和过表达等方法,研究p62^dok表达量对胰岛素信号通路和葡萄糖异生的影响。采用蛋白免疫印迹法检测蛋白和磷酸化蛋白表达。结果:在高脂饮食处理的小鼠肝组织和游离脂肪酸处理的原代培养小鼠肝细胞中, p62^dok的表达量显著增加、蛋白激酶B(Akt)和叉头框蛋白O1(FoxO1)磷酸化程度降低,葡萄糖异生调节蛋白葡萄糖-6-磷酸酶(G6Pase)和磷酸烯醇丙酮酸羧激酶(PEPCK)的含量增加。沉默p62^dok可增加游离脂肪酸处理的原代培养小鼠肝细胞中Akt和FoxO1磷酸化,降低G6Pase和PEPCK的含量。结论:高脂可上调肝细胞 p62^dok表达,并通过抑制胰岛素信号转导而调节葡萄糖异生。     

Sequestosome‐1 (p62) expression reveals chaperone‐assisted selective autophagy in immune‐mediated necrotizing myopathies

Diffuse myofiber necrosis in the context of inflammatory myopathy is the hallmark of immune‐mediated necrotizing myopathy (IMNM). We have previously shown that skeletal muscle fibers of IMNM patients may display nonrimmed vacuoles and sarcoplasmic irregularities. The dysfunctional chaperone activity has been linked to the defective assembly of skeletal muscle proteins and their degradation via lysosomes, autophagy and the proteasomal machinery. This study was undertaken to highlight a chaperone‐assisted selective autophagy (CASA) pathway, functionally involved in protein homeostasis, cell stress and the immune response in skeletal muscle of IMNM patients. Skeletal muscle biopsies from 54 IMNM patients were analyzed by immunostaining, as well as by qPCR. Eight biopsies of sIBM patients served as pathological controls, and eight biopsies of nondisease control subjects were included. Alteration of autophagy was detectable in all IMNM biopsy samples highlighted via a diffuse sarcoplasmic staining pattern by p62 and LC3 independent of vacuoles. This pattern was at variance with the coarse focal staining pattern mostly confined to rimmed vacuoles in sIBM. Colocalization of p62 with the chaperone proteins HSP70 and αB‐crystalline points to the specific targeting of misfolded proteins to the CASA machinery. Bcl2‐associated athanogene 3 (BAG3) positivity of these fibers emphasizes the selectivity of autophagy processes and these fibers also express MHC class I sarcolemma. Expression of genes involved in autophagy and endoplasmic reticulum (ER) stress pathways studied here is significantly upregulated in IMNM. We highlight that vacuoles without sarcolemmal features may arise in IMNM muscle biopsies, and they must not be confounded with sIBM‐specific vacuoles. Further, we show the activation of selective autophagy and emphasize the role of chaperones in this context. CASA occurs in IMNM muscle, and specific molecular pathways of autophagy differ from the ones in sIBM, with p62 as a unique identifier of this process.     

Molecular cloning of a truncated p62Dok1 isoform,p22Dokdel

The p21Ras GTPase activating protein‐associated 62‐kDa protein, p62Dok1, is an early substrate of various tyrosine phosphorylation pathways. Its recent cloning in human myeloid cells and in murine pre‐B cells revealed an N‐terminal pleckstrin‐homology domain and a tyrosine‐ and proline‐rich C‐terminal tail in its sequence. Here, we characterized a new 1261‐bp cDNA identical to that of p62Dok1, but with a central 185‐bp deletion (bp 456–640). This induced a frameshift leading to a premature stop codon. The deduced protein, designated p22Dok^del, corresponded to a truncated p62Dok1 isoform of 177 amino acids that can be expressed both in vitro and in vivo with an apparent molecular mass of 22 kDa. This newly identified molecule was composed of the N‐terminal PH domain of p62Dok1 followed by a new 25‐amino acid C‐terminal sequence containing a typical class II proline‐rich motif, suggesting a specific role for p22Dok^del in signal transduction pathways.     

TBK1 inhibitors enhance transfection efficiency by suppressing p62/SQSTM1 phosphorylation

DNA transfection is an essential technique in the life sciences. Non-viral transfection reagents are widely used for transfection in basic science. However, low transfection efficiency is a problem in some cell types. This low efficiency can be primarily attributed to the intracellular degradation of transfected DNA by p62-dependent selective autophagy, specifically by p62 phosphorylated at the S403 residue (p62-S403-P). To achieve efficient DNA transfection, we focused on a phosphorylation process that generates p62-S403-P and investigated whether inhibition of this process affects transfection efficiency. One of the kinases that phosphorylate p62 is TBK1. The TBK1 gene depletion in murine embryonic fibroblast cells by genome editing caused a significant reduction or loss of p62-S405-P (equivalent to human S403-P) and enhanced transfection efficiency, suggesting that TBK1 is a major kinase that phosphorylates p62 at S403. Therefore, TBK1 is a viable target for drug treatment to increase transfection efficiency. Transfection efficiency was enhanced when cells were treated with one of the following TBK1 inhibitors BX795, MRT67307, or amlexanox. This effect was synergistically improved when the two inhibitors were used in combination. Our results indicate that TBK1 inhibitors enhanced transfection efficiency by suppressing p62 phosphorylation.     

Increased degradation in rat liver induced by antilipolytic agents: a model for studying autophagy and protein degradation in liver?

A dramatic increase in the plasma glucagon/insulin ratio can be induced by treating fasted rats with antilipolytic drugs (e.g., with 3,5-dimethylpyrazole, 12 mg/kg body wt). These hormone changes are the physiologically appropriate response to a rapid decrease in free fatty acids and glucose plasma levels. Under this experimental condition, many vacuolated lysosomes can be observed at the electron microscopic level as early as 30 min and autophagic vacuoles are detectable in the liver cells 1 hr after the administration of the drug. By 1 hr and 45 min, vacuoles often contain recognizable peroxisomes. At the biochemical level, liver proteolysis in vitro is increased significantly. Very interestingly, changes in peroxisomal (but not mitochondrial or reticulum or cytosolic) enzyme activities are detected that are preventable by the administration of glutamine (i.e., of an inhibitor of proteolysis in vivo) but not by an isocaloric amount of glycine or alanine. It is concluded that the administration of antilipolytic agents to fasted animals may provide a convenient (i.e., an inexpensive, highly reproducible and timable) physiologic model to study hormone-induced autophagy in liver cells.     

p62 protein is expressed in pancreatic beta cells

p62 is a cellular protein that plays an adapter role in signal transduction pathways involved in such diverse biological functions as proliferation, differentiation, reaction to oxidative stress and immune response. Furthermore, p62 has recently been detected as a component of intracytoplasmic protein aggregates (inclusion bodies), which are hallmarks of a variety of chronic degenerative disorders, such as Parkinson's disease and Alzheimer's disease, but also of steatohepatitis. Here we report that p62 and insulin are co-expressed in a diffuse fashion in beta cells in normal human pancreas as well as in primary chronic pancreatitis and in normal pancreas from mouse and swine. In contrast, p62 protein is absent from, or only focally and very weakly expressed in, insulinomas, glucagonomas or non-functioning pancreatic neuroendocrine tumours or carcinomas that express insulin or other pancreatic as well as extrapancreatic hormones. Although the biological function of p62 in beta cells is unknown, the co-expression of p62 and insulin in non-neoplastic beta cells suggests that, in the beta cell, p62 may play a role in specific insulin-related signalling. Since p62 may also be involved in pro-apototic signal transduction, the loss of p62 expression in neuroendocrine neoplasms of the pancreas may render the tumour cells less sensitive to pro-apototic signals. Further research is necessary to elucidate the role of p62 in beta cell-specific signal transduction.