The chaperonin-related protein Tcm62p ensures mitochondrial gene expression under heat stress

Tcm62p, distantly related to chaperonins, is required for the assembly of succinate dehydrogenase in mitochondria of Saccharomyces cerevisiae and was proposed to exert chaperone activity. We demonstrate here crucial functions of Tcm62p under heat stress. It ensures mitochondrial gene expression at elevated temperatures and prevents heat-aggregation of the ribosomal subunit Var1p. Similar to chaperonins, Tcm62p forms a high molecular mass protein complex of approximately 850 kDa in the mitochondrial matrix space. These results suggest a more general chaperone function of Tcm62p in mitochondria.     

The mitochondrial small heat-shock protein protects NADH:ubiquinone oxidoreductase of the electron transport chain during heat stress in plants

Functional inactivation of the mitochondrial small heat-shock protein (lmw Hsp) in submitochondrial vesicles using protein-specific antibodies indicated that this protein protects NADH:ubiquinone oxidoreductase (complex I), and consequently electron transport from complex I to cytochrome c:O₂ oxidoreductase (complex IV). Lmw Hsp function completely accounted for heat acclimation of complex I electron transport in pre-heat-stressed plants. Addition of purified lmw Hsp to submitochondrial vesicles lacking this Hsp increased complex I electron transport rates 100% in submitochondrial vesicles assayed at high temperatures. These results indicate that production of the mitochondrial lmw Hsp is an important adaptation to heat stress in plants.     

p62 provides dual cytoprotection against oxidative stress in the retinal pigment epithelium

As a signaling hub, p62/sequestosome plays important roles in cell signaling and degradation of misfolded proteins. p62 has been implicated as an adaptor protein to mediate autophagic clearance of insoluble protein aggregates in age-related diseases, including age-related macular degeneration (AMD), which is characterized by dysfunction of the retinal pigment epithelium (RPE). Our previous studies have shown that cigarette smoke (CS) induces oxidative stress and inhibits the proteasome pathway in cultured human RPE cells, suggesting that p62-mediated autophagy may become the major route to remove impaired proteins under such circumstances. In the present studies, we found that all p62 mRNA variants are abundantly expressed and upregulated by CS induced stress in cultured human RPE cells, yet isoform1 is the major translated form. We also show that p62 silencing exacerbated the CS induced accumulation of damaged proteins, both by suppressing autophagy and by inhibiting the Nrf2 antioxidant response, which in turn, increased protein oxidation. These effects of CS and p62 reduction were further confirmed in mice exposed to CS. We found that over-expression of p62 isoform1, but not its S403A mutant, which lacks affinity for ubiquitinated proteins, reduced misfolded proteins, yet simultaneously promoted an Nrf2-mediated antioxidant response. Thus, p62 provides dual, reciprocal enhancing protection to RPE cells from environmental stress induced protein misfolding and aggregation, by facilitating autophagy and the Nrf2 mediated antioxidant response, which might be a potential therapeutic target against AMD.     

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.     

Drp1-dependent mitochondrial fission regulates p62-mediated autophagy in LPS-induced activated microglial cells

Microglial activation is known to be an important event during innate immunity, but microglial inflammation is also thought to play a role in the etiology of neurodegenerative diseases. Recently, it was reported that autophagy could influence inflammation and activation of microglia. However, little is known about the regulation of autophagy during microglial activation. In this study, we demonstrated that mitochondrial fission-induced ROS can promote autophagy in microglia. Following LPS-induced autophagy, GFP-LC3 puncta were increased, and this was suppressed by inhibiting mitochondrial fission and mitochondrial ROS. Interestingly, inhibition of mitochondrial fission and mitochondrial ROS also resulted in decreased p62 expression, but Beclin1 and LC3B were unaffected. Taken together, these results indicate that ROS induction due to increased LPS-stimulated mitochondrial fission triggers p62 mediated autophagy in microglial cells. Our findings provide the first important clues towards understanding the correlation between mitochondrial ROS and autophagy.

Abbreviations: Drp1; Dynamin related protein 1, LPS; Lipopolysaccharide, ROS; Reactive Oxygen Species, GFP; Green Fluorescent Protein, CNS; Central Nervous System, AD; Alzheimer’s Disease, PD; Parkinson’s Disease, ALIS; Aggresome-like induced structures, iNOS; inducible nitric oxide synthase, Cox-2; Cyclooxygenase-2, MAPK; Mitogen-activated protein kinase; SODs; Superoxide dismutase, GPXs; Glutathione Peroxidase, Prxs; Peroxiredoxins     

p62蛋白的分子功能及其在疾病中的研究进展

螯合体1(SQSTM1/p62)是一种选择性自噬接头蛋白,在清除待降解蛋白、维持细胞内蛋白质稳态中发挥重要的调控作用。p62蛋白具有多个功能结构域,介导与多种蛋白质发生相互作用进而精确调节特定的信号通路,从而将p62蛋白与氧化防御系统、炎症反应和营养感知等重要生命过程联系起来。研究表明p62的突变或者表达异常与多种疾病的发生发展过程密切相关,包括神经退行性疾病、肿瘤、感染性疾病、遗传性疾病以及慢性疾病等。本文综述了p62蛋白的结构特征、分子功能,并系统介绍其在蛋白质稳态和信号通路调控中的多种功能,总结了p62在疾病发生发展中的复杂性与多面性,以期为p62蛋白的功能与相关疾病研究提供参考。     

RelA-independent (p)ppGpp accumulation and heat shock protein induction after salt stress in Bacillus subtilis

Abstract Sodium chloride treatment triggered the accumulation of (p)ppGpp in the Bacillus subtilis relA ⁺ strain IS58 as well as in its relaxed counterpart IS56 . Besides this relA -independent (p)ppGpp induction the GTP and ATP pools decreased dramatically.
In previous papers we found a direct correlation between (p)ppGpp accumulation and stress protein induction. In B. subtilis relA the (p)ppGpp accumulation was accompanied by the induction of general stress proteins whose synthesis rates were also enhanced by heat stress, amino acid limitation or oxygen starvation. Specific heat shock proteins were not induced by salt stress.
We suggest that these general stress proteins are induced under non-growing conditions in general.     

Assurance of mitochondrial integrity and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cascade

Sqstm1/p62 functions in the non-canonical activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, its physiological relevance is not certain. Here, we show that p62(-/-) mice exhibited an accelerated presentation of ageing phenotypes, and tissues from these mice created a pro-oxidative environment owing to compromised mitochondrial electron transport. Accordingly, mitochondrial function rapidly declined with age in p62(-/-) mice. In addition, p62 enhanced basal Nrf2 activity, conferring a higher steady-state expression of NAD(P)H dehydrogenase, quinone 1 (Nqo1) to maintain mitochondrial membrane potential and, thereby, restrict excess oxidant generation. Together, the p62-Nrf2-Nqo1 cascade functions to assure mammalian longevity by stabilizing mitochondrial integrity.     

SQSTM1/p62 (sequestosome 1) senses cellular ubiquitin stress through E2-mediated ubiquitination

The alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. We recently reported that the macroautophagy/autophagy receptor SQSTM1/p62, functions as a novel Ub sensor to activate autophagy upon Ub⁺ stress (upregulation of the Ub level). First, SQSTM1 was found to undergo extensive ubiquitination and activate autophagy under Ub⁺ stress induced by prolonged Bortezomib (BTZ) treatment, Ub overexpression or by heat shock. Mechanistically, Ubiquitination of SQSTM1 disrupts its dimerization of the UBA domain, switching it from an auto-inhibitory conformation to recognize poly-ubiquitinated cargoes, promoting autophagic flux. Interestingly, Ub⁺ stress-responsive SQSTM1 ubiquitination is mediated by Ub conjugating enzymes, UBE2D2/3, in a unique E2-dependent manner. Our work has thus revealed a novel mechanism for how SQSTM1 senses cellular Ub stress conditions and regulates selective autophagy in response to diverse intrinsic or extrinsic challenges.     

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.     

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.     

核孔蛋白p62促进非受体酪氨酸激酶c-Abl进入细胞核

非受体酪氨酸激酶c-Abl广泛表达于各组织细胞中,其序列高度保守,它的亚细胞定位与其功能密切相关。c-Abl借助其C端的3个核定位信号（NLS）和1个核输出信号（NES）完成细胞核一细胞质问的穿梭过程。关于c-Abl核-质穿梭的详细机制还不清楚。通过酵母双杂交系统,以人类Ib型c-Abl作为诱饵蛋白进行HeLa细胞eDNA文库的筛选,获得了可能在c-Abl核-质穿梭过程中具有调控怍用核孔蛋白p62。核孔复合物（NPC）是大分子物质进行核-质运输的惟一通道,p62是NPC的重要组成部分,它位于中央通道内侧,在许多物质的核-质穿梭过程中具有调节作用。免疫共沉淀和体外结合实验证实,c-Abl和p62之间具有相互作用,而且这种相互作用是通过c-Abl的SH3结构域与p62的P299位点之间的结合实现的;p62可被c-Abl部分磷酸化此外,在293和DKO细胞株中共转染c-Abl和p62,发现核内的c-Abl分布增多。以上结果表明,p62具有促进e-Abl进入细咆核的作用。     

The lncRNA SNHG16 affects prognosis in hepatocellular carcinoma by regulating p62 expression

Long noncoding RNAs (lncRNAs) regulate tumor development and progression by promoting proliferation, invasion, and metastasis. The oncogenic role of lncRNA SNHG16 in hepatocellular carcinoma (HCC) has not been revealed. LncRNA SNHG16 is upregulated in HCC and correlates with poorer prognosis. Patients with high SNHG16 expression showed lower rates of overall and disease-free survival than patients with low SNHG16 expression. Multivariate Cox regression revealed that SNHG16 expression was an independent predictor of poor overall and disease-free survival. In vitro, SNHG16 promoted HCC cell proliferation, migration, and invasion while inhibiting apoptosis; in vivo, it accelerated tumor development. Altering SNHG16 expression altered levels of miR-17-5p, which in turn modified expression of p62, which has been shown to regulate the mTOR and NF-κB pathways. Indeed, altering SNHG16 expression in HCC cells activated mTOR and NF-κB signaling. These results reveal a potential mechanism for the oncogenic role of SNHG16 in HCC. SNHG16 may therefore be a promising diagnostic marker as well as therapeutic target in HCC.     

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression: functional analysis of p62 and autophagy in hypoxic neuronal cells

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.     

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.     

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.