Autophagy regulates DNA repair through SQSTM1/p62

Macroautophagy/autophagy is primarily a degradative pathway that clears malfunctioning cellular components in response to various types of stress. Recent studies have indicated that autophagy also plays an important role in maintaining genome stability. Loss of autophagy is associated with increased damage to DNA, inappropriate amplification of genomic regions and abnormal chromosome number. In a recent paper by Wang et al. the authors uncover a mechanism through which autophagy regulates the ubiquitination of chromatin. In particular, the autophagy receptor and substrate SQSTM1/p62 inhibits the E3 ligase RNF168-dependent ubiquitination of histone in response to DNA double-strand breaks. Dysregulation of this process leads to a reduced ability to repair DNA and a corresponding increase in the sensitivity of cells to radiation-induced damage.     

Mitofusin 2-Deficiency Suppresses Cell Proliferation through Disturbance of Autophagy

Mitofusin2 (Mfn2), a mitochondrial outer membrane protein serving primarily as a mitochondrial fusion protein, has multiple functions in regulating cell biological processes. Defects of Mfn2 were found in diabetes, obesity, and neurodegenerative diseases. In the present study, we found that knockdown of Mfn2 by shRNA led to impaired autophagic degradation, inhibited mitochondrial oxygen consumption rate and cell glycolysis, reduced ATP production, and suppressed cell proliferation. Inhibition of autophagic degradation mimicked Mfn2-deficiency mediated cell proliferation suppression, while enhancement of autophagosome maturation restored the suppressed cell proliferation by Mfn2-deficiency. Thus, our findings revealed the role of Mfn2 in regulating cell proliferation and mitochondrial metabolism, and shed new light on understanding the mechanisms of Mfn2 deficiency related diseases.     

细胞自噬与肿瘤发生的关系

细胞自噬(autophagy)是将细胞内受损、变性或衰老的蛋白质以及细胞器运输到溶酶体进行消化降解的过程.正常生理情况下,细胞自噬利于细胞保持自稳状态;在发生应激时,细胞自噬防止有毒或致癌的损伤蛋白质和细胞器的累积,抑制细胞癌变;然而肿瘤一旦形成,细胞自噬为癌细胞提供更丰富的营养,促进肿瘤生长.因此,在肿瘤发生发展的过程中,细胞自噬的作用具有两面性.尽管大多数抑癌蛋白可以激活细胞自噬这一结论被广泛接受,但p53作为重要的抑癌蛋白,在细胞核和细胞浆不同的亚细胞定位中对细胞自噬有着截然相反的调控.对于细胞自噬和癌症发生之间关系亟待深入的研究,这将会有助于人类更好地认识并最终攻克癌症.本文将针对细胞自噬与肿瘤发生过程中主要的信号调节通路展开介绍.     

Autophagy Inhibitor LRPPRC Suppresses Mitophagy through Interaction with Mitophagy Initiator Parkin

Autophagy plays an important role in tumorigenesis. Mitochondrion-associated protein LRPPRC interacts with MAP1S that interacts with LC3 and bridges autophagy components with microtubules and mitochondria to affect autophagy flux. Dysfunction of LRPPRC and MAP1S is associated with poor survival of ovarian cancer patients. Furthermore, elevated levels of LRPPRC predict shorter overall survival in patients with prostate adenocarcinomas or gastric cancer. To understand the role of LRPPRC in tumor development, previously we reported that LRPPRC forms a ternary complex with Beclin 1 and Bcl-2 to inhibit autophagy. Here we further show that LRPPRC maintains the stability of Parkin that mono-ubiquitinates Bcl-2 to increase Bcl-2 stability to inhibit autophagy. Under mitophagy stress, Parkin translocates to mitochondria to cause rupture of outer mitochondrial membrane and bind with exposed LRPPRC. Consequently, LRPPRC and Parkin help mitochondria being engulfed in autophagosomes to be degraded. In cells under long-term mitophagy stress, both LRPPRC and Parkin become depleted coincident with disappearance of mitochondria and final autophagy inactivation due to depletion of ATG5-ATG12 conjugates. LRPPRC functions as a checkpoint protein that prevents mitochondria from autophagy degradation and impact tumorigenesis.     

The Adaptor Protein p62 Is Involved in RANKL-induced Autophagy and Osteoclastogenesis

Previous studies have implicated autophagy in osteoclast differentiation. The aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis. Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation. The data showed that several key autophagy-related markers including p62 were significantly altered during RANKL-induced osteoclast differentiation. In addition, the expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by western blot and immunofluorescence analyses, respectively. Importantly, the knockdown of p62 obviously attenuated RANKL-induced expression of autophagy- and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, accumulation of LC3, as well as formation of F-actin ring. Our study indicates that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclastogenesis-related diseases.     

Proliferation of Toxoplasma gondii Suppresses Host Cell Autophagy

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.     

Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1

The scaffolding adaptor protein p62/SQSTM1 (p62) has been shown to be an autophagy receptor that acts as a link between the ubiquitination and autophagy machineries. However, the roles of autophagy and p62 in human keratinocytes are not well understood. In this study, we show that keratinocyte autophagy negatively regulates p62 expression, which is essential for the prevention of excessive inflammation and the induction of cathelicidin in human keratinocytes. Stimulation of TLR2/6 or TLR4 in primary human keratinocytes robustly activated autophagy pathways and up-regulated p62 expression through induction of NADPH oxidases 2 and 4 and the generation of reactive oxygen species. MyD88 and TNFR-associated factor 6, key signaling molecules that mediate TLR activation, played an essential role in the induction of autophagy and p62 expression. Additionally, blockade of autophagy significantly increased the generation of inflammatory cytokines and expression of p62 in primary human keratinocytes. Notably, silencing hp62 through RNA interference resulted in a significant decrease in NF-κB activation, inflammatory cytokine production, cathelicidin expression, and cell proliferation (as well as cyclin D1 expression) in keratinocytes. Epidermal expression of p62 was further found to be significantly higher in psoriatic skin than in skin affected by atopic dermatitis or from healthy controls. Collectively, our data provide new insights into the roles of autophagy and p62 in controlling cutaneous inflammation.     

细胞自噬在个体发育和肿瘤发生中的作用

细胞自噬是一种广泛存在于真核生物细胞中的代谢过程,参与调控胞内物质合成、降解和重新利用之间的代谢平衡。自噬体与溶酶体的有效融合能有效地保障胞内多余物质的降解及其再利用,是真核细胞所特有的一种自我保护机制。细胞自噬近年来受到广泛的关注,其不仅能充当胞内一个合格的质检员,有效地降解胞内受损的蛋白质成分和细胞器,进而阻止细胞损伤和凋亡,也与肿瘤发生、衰老、神经退行性疾病、人体自身免疫性疾病、肥胖症和糖尿病等多种疾病的发生及发展密切相关。本文旨在对细胞自噬过程和其调控机制进行介绍,并侧重对自噬在生长发育和肿瘤发生中的作用进行综述,为预防与治疗多种人类重大疾病提供理论依据。     

Autophagy substrate SQSTM1/p62 regulates chromatin ubiquitination during the DNA damage response

The importance of autophagy in the DNA damage repair process is clear; however, the detailed molecular mechanism is still largely unknown. Here we found that DNA damage-induced histone H2A ubiquitination is suppressed in autophagy-deficient cells in a SQSTM1/p62 dependent manner. SQSTM1 binds and inhibits E3 ligase RNF168s activity, which is essential for H2A ubiquitination. As a result, several important factors for DNA repair cannot be recruited to the sites of DNA double-strand breaks (DSBs) in autophagy-deficient cells, leading to diminished DNA repair and increased sensitivity of cells to radiation.     

Dusquetide modulates innate immune response through binding to p62

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Signal integration and diversification through the p62 scaffold protein

Signal specificity of multifunctional enzymes is achieved through protein-protein interactions involving specific domains on scaffold proteins. p62 (also known as sequestosome 1) is such a scaffold protein that possesses PB1 and UBA domains, and the TRAF6 binding sequence. Proteins recruited to these domains enable p62 to integrate kinase-activated and ubiquitin-mediated signaling pathways. The biological function of p62 has been studied in diverse systems and processes such as osteoclastogenesis, inflammation, differentiation, neurotrophin biology and obesity. The availability of mice in which p62 has been genetically inactivated is providing new insight into the mechanism and function of p62 at a whole-organism level.     

p62/SQSTM1: A Missing Link between Protein Aggregates and the Autophagy Machinery

In eukaryotic cells short-lived proteins are degraded in a specific process by the ubiquitin-proteasome system (UPS), whereas long-lived proteins and damaged organelles are degraded by macroautophagy (hereafter referred to as autophagy). A growing body of evidence now suggests that autophagy is important for clearance of protein aggregates that form in cells as a consequence of ageing, oxidative stress, alterations that elevate the amounts of certain aggregation-prone proteins or expression of aggregating mutant variants of specific proteins. Autophagy is generally considered to be a non-specific, bulk degradation process. However, a recent study suggests that p62/SQSTM1 may link the recognition of polyubiquitinated protein aggregates to the autophagy machinery.1 This protein is able to polymerize via its N-terminal PB1 domain and to recognize polyubiquitin via its C-terminal UBA domain. It can also recruit the autophagosomal protein LC3 and co-localizes with many types of polyubiquitinated protein aggregates.1 Here we discuss possible implications of these findings and raise some questions for further investigation.     

Autophagy and p62/sequestosome 1 generate neo-antimicrobial peptides (cryptides) from cytosolic proteins

In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.Key words: autophagy, tuberculosis, ribosome, ubiquitin, antimicrobial peptidesAutophagy is an evolutionarily conserved cytoplasm-homeostatic process with a multitude of functions supporting, for the most part, cellular viability. During autophagy, cytoplasmic targets ranging from protein aggregates to whole organelles such as mitochondria and intracellular microbes are sequestered into a double-membrane bound organelle called the autophagosome. Autophagosomes mature into autolysosomes through fusion with lysosomes or their transport intermediates, bringing about acidification and acquisition of hydrolases leading to the digestion of the captured substrates. It is generally assumed that autophagy produces terminal degradative products such as free amino acids that are then used by the cell or the body as nutrients at times of starvation. Recently, we have discovered that autophagy generates, by proteolysis of captured cytosolic proteins, a mixture of peptides conferring potential cryptic biological functions, termed “cryptides.” Some of the cryptides with thus far assigned biological functions are the neo-antimicrobial peptides liberated from innocuous cytoplasmic proteins such as the ribosomal protein precursor FAU and ubiquitin.Our study was motivated by the search for factors or ingredients that make autophagic organelles particularly mycobactericidal, as Mycobacterium tuberculosis can survive the environment of the conventional phagolysosome. This was shown in the 1970s by the classical work of Armstrong and D''Arcy Hart at the same time when these authors established the more broadly appreciated and well-ingrained reputation of the tubercle bacillus as inhibiting the conventional phagosome-lysosome fusion. The approach to identifying such hypothetical ingredients was to first examine the steps of the autophagic pathway that are necessary for the mycobactericidal nature of macrophages induced for autophagy by, for example, starvation. We have found that not only are all stages of autophagy (initiation, elongation/closure and maturation) required for full mycobactericidal potency, but that p62, the first autophagic adapter characterized by the Johansen group, and also known as sequestosome 1, is absolutely required for autophagic elimination of M. tuberculosis. Sequestosome 1/p62 recognizes ubiquitinated protein aggregates and possibly ubiquitinated depolarized mitochondria and other targets, and delivers them to nascent autophagosomes; p62 also binds to the mammalian Atg8 paralog LC3 via its LC3-interaction region (LIR), thus conveniently bridging the targets with forming phagophores.At first blush, it may seem that mycobacteria follow the same fate demonstrated for several other bacteria, whereby p62 or another autophagic adapter, NDP52, capture cytosolic microbes and deliver them to autophagosomes. For example, the fraction of Salmonellae that are no longer retained within phagosomes and are free in the cytosol, or Shigella and Listeria that actively escape into the cytosol, are associated with ubiquitinated material or become otherwise recognized by p62 or NDP52, and end up being sequestered into autophagosomes. However, we found no evidence for p62 acting directly to transfer intraphagosomal mycobacteria into autophagic vacuoles. Instead, we observed p62-positive organelles as periodically fusing with mycobacterial phagosomes. At the same time, we found by imaging and biochemical means that proteins recruited by p62 from the cytosol into conventional autophagic organelles are subsequently transferred to model (latex bead phagosomes formed upon feeding 1 µm beads to macrophages) or mycobacterial phagosomes, as they gradually acquire autolysosomal characteristics. Next, we established that p62-captured cytosolic proteins (ribosomal protein rpS30 precursor FAU and ubiquitin) are proteolytically degraded into smaller peptides, and that specific peptides from these complex mixtures show antimycobacterial activity. Thus, the emerging model posits that autophagy captures cytosolic proteins and converts them into neo-antimycobacterial peptides that can then kill M. tuberculosis upon delivery to mycobacteria-containing phagosomes, which in turn gradually acquire autolysosomal properties (Fig. 1).Open in a separate windowFigure 1Elimination of M. tuberculosis by autophagy and p62. Mycobacteria are phagocytosed by macrophages and at least for some time reside within phagosomes. Upon induction of autophagy, p62, as a bifunctional agent interacting with autophagic substrates and with LC3, recruits into autophagosomes pre-antimicrobicidal cytosolic substrates. Autophagosome maturation including acquisition of lysosomal hydrolases leads to the proteolytic cleavage of p62 substrates and their conversion into peptides (cryptides) that can act as antimicrobial peptides.In contrast to the direct mechanism of capturing bacteria employed in some instances described above, in the case of M. tuberculosis, an organism that resides within the phagosomes, the adapter molecule p62 exerts its anti-microbial action through an indirect, but rather sophisticated mechanism. By sequestering into autophagosomes the initially harmless cytosolic components and by proteolytically processing them within maturing autophagosomes, p62 and autophagy liberate antimicrobial peptides from the otherwise innocuous substrates. This amounts to a resourceful utilization by the cell of otherwise spent or to-be-discarded cytoplasmic proteins and gives them an after-function upon completion of their “day jobs” that they performed as whole proteins.Our studies have uncovered a previously unappreciated function for autophagy in generating neo-antimicrobial peptides, and perhaps also opened the prospect for other biological functions potentially engendered by the products of autophagic proteolysis. Given that autophagy has the capacity to capture en masse and subject to digestion large sections of the cytoplasm, most cellular proteins are undergoing, or can undergo, processing into peptides or peptide intermediates within autophagic organelles. We postulate that the antimicrobial peptide production revealed in our studies thus far is only one manifestation of a spectrum of potential biological functions of cryptides generated by autophagy.     

miR-100 Induces Epithelial-Mesenchymal Transition but Suppresses Tumorigenesis,Migration and Invasion

Whether epithelial-mesenchymal transition (EMT) is always linked to increased tumorigenicity is controversial. Through microRNA (miRNA) expression profiling of mammary epithelial cells overexpressing Twist, Snail or ZEB1, we identified miR-100 as a novel EMT inducer. Surprisingly, miR-100 inhibits the tumorigenicity, motility and invasiveness of mammary tumor cells, and is commonly downregulated in human breast cancer due to hypermethylation of its host gene MIR100HG. The EMT-inducing and tumor-suppressing effects of miR-100 are mediated by distinct targets. While miR-100 downregulates E-cadherin by targeting SMARCA5, a regulator of CDH1 promoter methylation, this miRNA suppresses tumorigenesis, cell movement and invasion in vitro and in vivo through direct targeting of HOXA1, a gene that is both oncogenic and pro-invasive, leading to repression of multiple HOXA1 downstream targets involved in oncogenesis and invasiveness. These findings provide a proof-of-principle that EMT and tumorigenicity are not always associated and that certain EMT inducers can inhibit tumorigenesis, migration and invasion.     

Small Ribosomal Protein Subunit S7 Suppresses Ovarian Tumorigenesis through Regulation of the PI3K/AKT and MAPK Pathways

Small ribosomal protein subunit S7 (RPS7) has been reported to be associated with various malignancies, but the role of RPS7 in ovarian cancer remains unclear. In this study, we found that silencing of RPS7 by a specific shRNA promoted ovarian cancer cell proliferation, accelerated cell cycle progression, and slightly reduced cell apoptosis and response to cisplatin treatment. Knockdown of RPS7 resulted in increased expression of P85α, P110α, and AKT2. Although the basal levels of ERK1/2, MEK1/2, and P38 were inconsistently altered in ovarian cancer cells, the phosphorylated forms of MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204), JNK1/2 (Thr183/Tyr185), and P38 (Thr180/Tyr182) were consistently reduced after RPS7 was silenced. Both the in vitro anchorage-independent colony formation and in vivo animal tumor formation capability of cells were enhanced after RPS7 was depleted. We also showed that silencing of RPS7 enhanced ovarian cancer cell migration and invasion. In sum, our results suggest that RPS7 suppresses ovarian tumorigenesis and metastasis through PI3K/AKT and MAPK signal pathways. Thus, RPS7 may be used as a potential marker for diagnosis and treatment of ovarian cancer.