Requirement of dendritic Akt degradation by the ubiquitin-proteasome system for neuronal polarity

Asymmetric distributions of activities of the protein kinases Akt and glycogen synthase kinase 3beta (GSK-3beta) are critical for the formation of neuronal polarity. However, the mechanisms underlying polarized regulation of this pathway remain unclear. In this study, we report that the instability of Akt regulated by the ubiquitin-proteasome system (UPS) is required for neuron polarity. Preferential distribution in the axons was observed for Akt but not for its target GSK-3beta. A photoactivatable GFP fused to Akt revealed the preferential instability of Akt in dendrites. Akt but not p110 or GSK-3beta was ubiquitinated. Suppressing the UPS led to the symmetric distribution of Akt and the formation of multiple axons. These results indicate that local protein degradation mediated by the UPS is important in determining neuronal polarity.     

Activation of atypical protein kinase C zeta by caspase processing and degradation by the ubiquitin-proteasome system

Atypical protein kinase C zeta (PKCzeta) is known to transduce signals that influence cell proliferation and survival. Here we show that recombinant human caspases can process PKCzeta at three sites in the hinge region between the regulatory and catalytic domains. Caspase-3, -6, -7, and -8 chiefly cleaved human PKCzeta at EETD downward arrowG, and caspase-3 and -7 also cleaved PKCzeta at DGMD downward arrowG and DSED downward arrowL, respectively. Processing of PKCzeta expressed in transfected cells occurred chiefly at EETD downward arrowG and DGMD downward arrowG and produced carboxyl-terminal polypeptides that contained the catalytic domain. Epitope-tagged PKCzeta that lacked the regulatory domain was catalytically active following expression in HeLa cells. Induction of apoptosis in HeLa cells by tumor necrosis factor alpha plus cycloheximide evoked the conversion of full-length epitope-tagged PKCzeta to two catalytic domain polypeptides and increased PKCzeta activity. A caspase inhibitor, zVAD-fmk, prevented epitope-tagged PKCzeta processing and activation following the induction of apoptosis. Induction of apoptosis in rat parotid C5 cells produced catalytic domain polypeptides of endogenous PKCzeta and increased PKCzeta activity. Caspase inhibitors prevented the increase in PKCzeta activity and production of the catalytic domain polypeptides. Treatment with lactacystin, a selective inhibitor of the proteasome, caused polyubiquitin-PKCzeta conjugates to accumulate in cells transfected with the catalytic domain or full-length PKCzeta, or with a PKCzeta mutant that was resistant to caspase processing. We conclude that caspases process PKCzeta to carboxyl-terminal fragments that are catalytically active and that are degraded by the ubiquitin-proteasome pathway.     

Cigarette smoke suppresses the ubiquitin-dependent degradation of OLC1

The newly identified gene, overexpressed in lung cancer 1 (OLC1), is highly expressed as OLC1 protein in the tumor tissues of lung cancer patients with histories of cigarette smoking. However, the underlying mechanisms of how the gene is affected by cigarette smoke have been poorly characterized. In this study, we investigated how OLC1 is regulated in lung cancer cells by cigarette smoke condensate (CSC).Compared to the controls, CSC treatment increased OLC1 protein levels in a dose- and time-dependent manner without affecting OLC1 mRNA levels in lung cancer cells. Ubiquitination of OLC1 protein was blocked upon CSC treatment. Biochemical analysis revealed that the ubiquitin E3 ligase anaphase promoting complex (APC) and its activators cell-division cycle protein 20 (CDC20) and cadherin-1 (CDH1) are responsible for the degradation of OLC1. However, upon introducing CSC the binding of OLC1 to the proteins CDC20, CDH1, and APC2 was impaired. These results demonstrate that CSC regulates OLC1 expression in lung cancer cells by compromising its ubiquitination and subsequent degradation through the ubiquitin E3 ligase APC.     

Cigarette smoke and dopaminergic system

It has been reported that there is an ameliorative effect of cigarette smoking on certain neurological responses and neurodegenerative disorders. The purpose of this study was to examine the neurochemical and neurobehavioral response of cigarette smoke (CS) in the adult male guinea pig brain. Both acute and chronic CS exposure enhanced locomotor behavior and caused a decrease in midbrain dopamine (DA) levels and corresponding increase in 3,4-dihydroxyphenylacetic acid (DOPAC) levels. In addition, CS caused a significant increase in the protein levels of the dopamine D1 and D2 receptors. CS caused a significant increase in the binding capacity of the D1 receptor and a significant decrease in the binding capacity of D2. Furthermore, CS caused a significant increase in the binding capacity of the dopamine transporter (DAT). The mechanism by which cigarette smoke exposure increases locomotor activity remains to be elucidated but may include modulation of dopamine neuron activity that emerges after repeated direct smoke exposure.     

Regulation of ubiquitin-proteasome system mediated degradation by cytosolic stress

ER-associated, ubiquitin-proteasome system (UPS)-mediated degradation of the wild-type (WT) gap junction protein connexin32 (Cx32) is inhibited by mild forms of cytosolic stress at a step before its dislocation into the cytosol. We show that the same conditions (a 30-min, 42 degrees C heat shock or oxidative stress induced by arsenite) also reduce the endoplasmic reticulum (ER)-associated turnover of disease-causing mutants of Cx32 and the cystic fibrosis transmembrane conductance regulator (CFTR), as well as that of WT CFTR and unassembled Ig light chain. Stress-stabilized WT Cx32 and CFTR, but not the mutant/unassembled proteins examined, could traverse the secretory pathway. Heat shock also slowed the otherwise rapid UPS-mediated turnover of the cytosolic proteins myoD and GFPu, but not the degradation of an ubiquitination-independent construct (GFP-ODC) closely related to the latter. Analysis of mutant Cx32 from cells exposed to proteasome inhibitors and/or cytosolic stress indicated that stress reduces degradation at the level of substrate polyubiquitination. These findings reveal a new link between the cytosolic stress-induced heat shock response, ER-associated degradation, and polyubiquitination. Stress-denatured proteins may titer a limiting component of the ubiquitination machinery away from pre-existing UPS substrates, thereby sparing the latter from degradation.     

Diversity of degradation signals in the ubiquitin-proteasome system

The ubiquitin-proteasome system degrades an enormous variety of proteins that contain specific degradation signals, or 'degrons'. Besides the degradation of regulatory proteins, almost every protein suffers from sporadic biosynthetic errors or misfolding. Such aberrant proteins can be recognized and rapidly degraded by cells. Structural and functional data on a handful of degrons allow several generalizations regarding their mechanism of action. We focus on different strategies of degron recognition by the ubiquitin system, and contrast regulatory degrons that are subject to signalling-dependent modification with those that are controlled by protein folding or assembly, as frequently occurs during protein quality control.     

Inhibition of the ubiquitin-proteasome system induces stress granule formation

The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2alpha by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2alpha phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21(cip1) mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.     

Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system

We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2(VHL) substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2(VHL) or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC(50) = 1.7 μM) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system.     

Cigarette smoke extract induces endothelial cell injury via JNK pathway

Cigarette smoking is the most crucial factor responsible for chronic obstructive pulmonary disease (COPD). The precise mechanisms of the development of the disease have, however, not been fully understood. Recently, impairment of pulmonary endothelial cells has been increasingly recognized as a critical pathophysiological process in COPD. To verify this hypothesis, we examined how cigarette smoke extract (CSE) damages human umbilical vein endothelial cells (HUVECs). CSE activated c-Jun N-terminal kinase (JNK), and treatment of HUVECs with SP600125, a specific inhibitor of the JNK pathway, significantly suppressed endothelial cell damage by CSE. In contrast, inhibition of the extracellular-regulated kinase or the p38 pathway did not affect the cytotoxicity of CSE. Furthermore, anti-oxidants superoxide dismutase and catalase reduced CSE-induced JNK phosphorylation and endothelial cell injury. These results indicate that CSE damages vascular endothelial cells through the JNK pathway activated, at least partially, by oxidative stress.     

Phosphorelay-regulated degradation of the yeast Ssk1p response regulator by the ubiquitin-proteasome system

In Saccharomyces cerevisiae, a phosphorelay signal transduction pathway composed of Sln1p, Ypd1p, and Ssk1p, which are homologous to bacterial two-component signal transducers, is involved in the osmosensing mechanism. In response to high osmolarity, the phosphorelay system is inactivated and Ssk1p remains unphosphorylated. Unphosphorylated Ssk1p binds to and activates the Ssk2p mitogen-activated protein (MAP) kinase kinase kinase, which in turn activates the downstream components of the high-osmolarity glycerol response (HOG) MAP kinase cascade. Here, we report a novel inactivation mechanism for Ssk1p involving degradation by the ubiquitin-proteasome system. Degradation is regulated by the phosphotransfer from Ypd1p to Ssk1p, insofar as unphosphorylated Ssk1p is degraded more rapidly than phosphorylated Ssk1p. Ubc7p/Qri8p, an endoplasmic reticulum-associated ubiquitin-conjugating enzyme, is involved in the phosphorelay-regulated degradation of Ssk1p. In ubc7Delta cells in which the degradation is hampered, the dephosphorylation and/or inactivation process of the Hog1p MAP kinase is delayed compared with wild-type cells after the hyperosmotic treatment. Our results indicate that unphosphorylated Ssk1p is selectively degraded by the Ubc7p-dependent ubiquitin-proteasome system and that this mechanism downregulates the HOG pathway after the completion of the osmotic adaptation.     

Cigarette smoke condensate induces MMP-12 gene expression in airway-like epithelia

Cigarette smoke (CS)-induced emphysema is attributable to matrix metalloproteinase-12 (MMP-12) in mice, however, a relationship between CS and MMP-12 is absent in human emphysema. Here, we show that cigarette smoke condensate (CSC) induces MMP-12 gene expression in airway-like epithelia through a hydrogen peroxide (H(2)O(2))-dependent pathway involving NADPH oxidase, AP-1, and TNF-alpha. Cigarette smoke condensate-induced H(2)O(2) production and MMP-12 gene expression were inhibited by apocynin, a specific inhibitor of NADPH oxidases, while 3-aminobenzamide, an inhibitor of AP-1, attenuated CSC-induced MMP-12 gene expression. Messenger RNAs encoding phagocytic NADPH oxidase components and a homologue of p67phox, p51 (NOXA1), were detected, while mRNA of dual oxidase (Duox)1 was unchanged by CSC. Enbrel, an inhibitor of TNF-alpha function, reduced CSC-induced H(2)O(2) production and MMP-12 expression. These findings provide novel evidence of a direct relationship between CS exposure and MMP-12 in human airway epithelia and suggest several targets for modulation of this potentially pathogenic pathway.     

Cigarette smoke induces anaphase bridges and genomic imbalances in normal cells

Exposure to cigarette smoke has long been linked to carcinogenesis, but the emphasis has been placed on mutational changes in the DNA sequence caused by the carcinogens in smoke. Here, we report an additional role for cigarette smoke exposure in contributing to chromosomal aberrations in cells. We have found that cigarette smoke condensate (CSC) induces anaphase bridges in cultured human cells, which in a short time lead to genomic imbalances. The frequency of the induced bridges within the entire population decreases with time, and this decrease is not dependent upon the p53-mediated apoptotic pathway. Additionally, we show that CSC induces DNA double stranded breaks (DSBs) in cultured cells and purified DNA. The reactive oxygen species (ROS) scavenger, 2' deoxyguanosine 5'-monophosphate (dGMP) prevents CSC-induced DSBs, anaphase bridge formation and genomic imbalances. Therefore, we propose that CSC induces bridges and genomic imbalances via DNA DSBs. Furthermore, since the amount of CSC added to the cultures was substantially less than that extracted from a single cigarette, our results show that even low levels of cigarette smoke can cause irreversible changes in the chromosomal constitution of cultured cells.     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts

Cigarette smoke is a mixture of chemicals having direct and/or indirect toxic effects on different lung cells. We investigated the effect of cigarette smoke on human lung fibroblasts (HFL-1) oxidation and apoptosis. Cells were exposed to various concentrations (1, 5, and 10%) of cigarette smoke extract (CSE) for 3 h, and oxidative stress and apoptosis were assessed by fluorescence-activated cell sorting and confocal laser fluorescence microscopy. Both oxidative stress and apoptosis exhibited a dose-response relationship with CSE concentrations. Lung fibroblasts also showed marked DNA fragmentation at the Comet assay after exposure to 10% CSE. Coincubation of HLF-1 cells with N-acetylcysteine (1 mM) during CSE exposure significantly reduced oxidative stress, apoptosis, and DNA fragmentation, whereas preincubation (3 h) with the glutathione-depleting agent buthionine sulfoximine (125 microM) produced a significant increase of oxidative stress. Cigarette smoke is a potent source of oxidative stress, DNA damage, and apoptosis for HFL-1 cells, and we speculate that this could contribute to the development of pulmonary emphysema in the lungs of smokers.