Browsing by Subject "Ubiquitination"

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Open Access
Activation of the ATF6 (Activating Transcription Factor 6) Signaling Pathway in Neurons Improves Outcome After Cardiac Arrest in Mice.
(Journal of the American Heart Association, 2021-06-11) Shen, Yuntian; Li, Ran; Yu, Shu; Zhao, Qiang; Wang, Zhuoran; Sheng, Huaxin; Yang, Wei
Background Ischemia/reperfusion injury impairs proteostasis, and triggers adaptive cellular responses, such as the unfolded protein response (UPR), which functions to restore endoplasmic reticulum homeostasis. After cardiac arrest (CA) and resuscitation, the UPR is activated in various organs including the brain. However, the role of the UPR in CA has remained largely unknown. Here we aimed to investigate effects of activation of the ATF6 (activating transcription factor 6) UPR branch in CA. Methods and Results Conditional and inducible sATF6-KI (short-form ATF6 knock-in) mice and a selective ATF6 pathway activator 147 were used. CA was induced in mice by KCl injection, followed by cardiopulmonary resuscitation. We first found that neurologic function was significantly improved, and neuronal damage was mitigated after the ATF6 pathway was activated in neurons of sATF6-KI mice subjected to CA/cardiopulmonary resuscitation. Further RNA sequencing analysis indicated that such beneficial effects were likely attributable to increased expression of pro-proteostatic genes regulated by ATF6. Especially, key components of the endoplasmic reticulum-associated degradation process, which clears potentially toxic unfolded/misfolded proteins in the endoplasmic reticulum, were upregulated in the sATF6-KI brain. Accordingly, the CA-induced increase in K48-linked polyubiquitin in the brain was higher in sATF6-KI mice relative to control mice. Finally, CA outcome, including the survival rate, was significantly improved in mice treated with compound 147. Conclusions This is the first experimental study to determine the role of the ATF6 UPR branch in CA outcome. Our data indicate that the ATF6 UPR branch is a prosurvival pathway and may be considered as a therapeutic target for CA.
Open Access
Aging Is Associated With Impaired Activation of Protein Homeostasis-Related Pathways After Cardiac Arrest in Mice.
(Journal of the American Heart Association, 2018-09) Shen, Yuntian; Yan, Baihui; Zhao, Qiang; Wang, Zhuoran; Wu, Jiangbo; Ren, Jiafa; Wang, Wei; Yu, Shu; Sheng, Huaxin; Crowley, Steven D; Ding, Fei; Paschen, Wulf; Yang, Wei
Background The mechanisms underlying worse outcome at advanced age after cardiac arrest ( CA ) and resuscitation are not well understood. Because protein homeostasis (proteostasis) is essential for cellular and organismal health, but is impaired after CA , we investigated the effects of age on proteostasis-related prosurvival pathways activated after CA . Methods and Results Young (2-3 months old) and aged (21-22 months old) male C57Bl/6 mice were subjected to CA and cardiopulmonary resuscitation ( CPR ). Functional outcome and organ damage were evaluated by assessing neurologic deficits, histological features, and creatinine level. CA / CPR -related changes in small ubiquitin-like modifier conjugation, ubiquitination, and the unfolded protein response were analyzed by measuring mRNA and protein levels in the brain, kidney, and spinal cord. Thiamet-G was used to increase O-linked β-N-acetylglucosamine modification. After CA / CPR , aged mice had trended lower survival rates, more severe tissue damage in the brain and kidney, and poorer recovery of neurologic function compared with young mice. Furthermore, small ubiquitin-like modifier conjugation, ubiquitination, unfolded protein response, and O-linked β-N-acetylglucosamine modification were activated after CA / CPR in young mice, but their activation was impaired in aged mice. Finally, pharmacologically increasing O-linked β-N-acetylglucosamine modification after CA improved outcome. Conclusions Results suggest that impaired activation of prosurvival pathways contributes to worse outcome after CA / CPR in aged mice because restoration of proteostasis is critical to the survival of cells stressed by ischemia. Therefore, a pharmacologic intervention that targets aging-related impairment of proteostasis-related pathways after CA / CPR may represent a promising therapeutic strategy.
Open Access
Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics.
(Journal of proteome research, 2012-02) Yang, W; Thompson, JW; Wang, Z; Wang, L; Sheng, H; Foster, MW; Moseley, MA; Paschen, W
Transient cerebral ischemia dramatically activates small ubiquitin-like modifier (SUMO2/3) conjugation. In cells exposed to 6 h of transient oxygen/glucose deprivation (OGD), a model of ischemia, SUMOylation increases profoundly between 0 and 30 min following re-oxygenation. To elucidate the effect of transient OGD on SUMO conjugation of target proteins, we exposed neuroblastoma B35 cells expressing HA-SUMO3 to transient OGD and used stable isotope labeling with amino acids in cell culture (SILAC) to quantify OGD-induced changes in levels of specific SUMOylated proteins. Lysates from control and OGD-treated cells were mixed equally, and HA-tagged proteins were immunoprecipitated and analyzed by 1D-SDS-PAGE-LC-MS/MS. We identified 188 putative SUMO3-conjugated proteins, including numerous transcription factors and coregulators, and PIAS2 and PIAS4 SUMO ligases, of which 22 were increased or decreased more than ±2-fold. In addition to SUMO3, the levels of protein-conjugated SUMO1 and SUMO2, as well as ubiquitin, were all increased. Importantly, protein ubiquitination induced by OGD was completely blocked by gene silencing of SUMO2/3. Collectively, these results suggest several mechanisms for OGD-modulated SUMOylation, point to a number of signaling pathways that may be targets of SUMO-based signaling and recovery from ischemic stress, and demonstrate a tightly controlled crosstalk between the SUMO and ubiquitin conjugation pathways.
Open Access
Characterization of the ubiquitin-modified proteome regulated by transient forebrain ischemia.
(Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2014-03) Iwabuchi, Masahiro; Sheng, Huaxin; Thompson, J Will; Wang, Liangli; Dubois, Laura G; Gooden, David; Moseley, Marthur; Paschen, Wulf; Yang, Wei
Ubiquitylation is a posttranslational protein modification that modulates various cellular processes of key significance, including protein degradation and DNA damage repair. In animals subjected to transient cerebral ischemia, ubiquitin-conjugated proteins accumulate in Triton-insoluble aggregates. Although this process is widely considered to modulate the fate of postischemic neurons, few attempts have been made to characterize the ubiquitin-modified proteome in these aggregates. We performed proteomics analyses to identify ubiquitylated proteins in postischemic aggregates. Mice were subjected to 10 minutes of forebrain ischemia and 4 hours of reperfusion. The hippocampi were dissected, aggregates were isolated, and trypsin-digested after spiking with GG-BSA as internal standard. K-ɛ-GG-containing peptides were immunoprecipitated and analyzed by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. We identified 1,664 peptides to 520 proteins containing at least one K-ɛ-GG. Sixty-six proteins were highly ubiquitylated, with 10 or more K-ɛ-GG peptides. Based on selection criteria of greater than fivefold increase and P<0.001, 763 peptides to 272 proteins were highly enriched in postischemic aggregates. These included proteins involved in important neuronal functions and signaling pathways that are impaired after ischemia. Results of this study could serve as an important platform to uncover the mechanisms linking insoluble ubiquitin aggregates to the functions of postischemic neurons.
Open Access
O-linked β-N-acetylglucosamine modification of proteins is activated in post-ischemic brains of young but not aged mice: Implications for impaired functional recovery from ischemic stress.
(Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2016-02) Liu, Shuai; Sheng, Huaxin; Yu, Zhui; Paschen, Wulf; Yang, Wei
To evaluate the effect of age on the response of brains to an ischemic challenge, we subjected young and aged mice to transient forebrain ischemia, and analyzed the heat shock response and unfolded protein response, ubiquitin conjugation and SUMO conjugation, and O-linked β-N-acetylglucosamine modification of proteins (O-GlcNAcylation). The most prominent age-related difference was an inability of aged mice to activate O-GlcNAcylation. Considering many reports on the protective role of O-GlcNAcylation in various stress conditions including myocardial ischemia, this pathway could be a promising target for therapeutic intervention to improve functional recovery of aged patients following brain ischemia.
Open Access
Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway.
(Science (New York, N.Y.), 2019-05) Lee, Yu-Ru; Chen, Ming; Lee, Jonathan D; Zhang, Jinfang; Lin, Shu-Yu; Fu, Tian-Min; Chen, Hao; Ishikawa, Tomoki; Chiang, Shang-Yin; Katon, Jesse; Zhang, Yang; Shulga, Yulia V; Bester, Assaf C; Fung, Jacqueline; Monteleone, Emanuele; Wan, Lixin; Shen, Chen; Hsu, Chih-Hung; Papa, Antonella; Clohessy, John G; Teruya-Feldstein, Julie; Jain, Suresh; Wu, Hao; Matesic, Lydia; Chen, Ruey-Hwa; Wei, Wenyi; Pandolfi, Pier Paolo
Activation of tumor suppressors for the treatment of human cancer has been a long sought, yet elusive, strategy. PTEN is a critical tumor suppressive phosphatase that is active in its dimer configuration at the plasma membrane. Polyubiquitination by the ubiquitin E3 ligase WWP1 (WW domain-containing ubiquitin E3 ligase 1) suppressed the dimerization, membrane recruitment, and function of PTEN. Either genetic ablation or pharmacological inhibition of WWP1 triggered PTEN reactivation and unleashed tumor suppressive activity. WWP1 appears to be a direct MYC (MYC proto-oncogene) target gene and was critical for MYC-driven tumorigenesis. We identified indole-3-carbinol, a compound found in cruciferous vegetables, as a natural and potent WWP1 inhibitor. Thus, our findings unravel a potential therapeutic strategy for cancer prevention and treatment through PTEN reactivation.
Open Access
SPOP Promotes Nanog Destruction to Suppress Stem Cell Traits and Prostate Cancer Progression.
(Developmental cell, 2019-02) Zhang, Jinfang; Chen, Ming; Zhu, Yasheng; Dai, Xiangpeng; Dang, Fabin; Ren, Junming; Ren, Shancheng; Shulga, Yulia V; Beca, Francisco; Gan, Wenjian; Wu, Fei; Lin, Yu-Min; Zhou, Xiaobo; DeCaprio, James A; Beck, Andrew H; Lu, Kun Ping; Huang, Jiaoti; Zhao, Cheryl; Sun, Yinghao; Gao, Xu; Pandolfi, Pier Paolo; Wei, Wenyi
Frequent SPOP mutation defines the molecular feature underlying one of seven sub-types of human prostate cancer (PrCa). However, it remains largely elusive how SPOP functions as a tumor suppressor in PrCa. Here, we report that SPOP suppresses stem cell traits of both embryonic stem cells and PrCa cells through promoting Nanog poly-ubiquitination and subsequent degradation. Mechanistically, Nanog, but not other pluripotency-determining factors including Oct4, Sox2, and Klf4, specifically interacts with SPOP via a conservative degron motif. Importantly, cancer-derived mutations in SPOP or at the Nanog-degron (S68Y) disrupt SPOP-mediated destruction of Nanog, leading to elevated cancer stem cell traits and PrCa progression. Notably, we identify the Pin1 oncoprotein as an upstream Nanog regulator that impairs its recognition by SPOP and thereby stabilizes Nanog. Thus, Pin1 inhibitors promote SPOP-mediated destruction of Nanog, which provides the molecular insight and rationale to use Pin1 inhibitor(s) for targeted therapies of PrCa patients with wild-type SPOP.
Open Access
Structural basis of JAZ repression of MYC transcription factors in jasmonate signalling.
(Nature, 2015-09) Zhang, Feng; Yao, Jian; Ke, Jiyuan; Zhang, Li; Lam, Vinh Q; Xin, Xiu-Fang; Zhou, X Edward; Chen, Jian; Brunzelle, Joseph; Griffin, Patrick R; Zhou, Mingguo; Xu, H Eric; Melcher, Karsten; He, Sheng Yang
The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development. Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase. The hormone-dependent formation of the COI1-JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.
Open Access
The Skp2 Pathway: A Critical Target for Cancer Therapy.
(Seminars in cancer biology, 2020-12) Cai, Zhen; Moten, Asad; Peng, Danni; Hsu, Che-Chia; Pan, Bo-Syong; Manne, Rajeshkumar; Li, Hong-Yu; Lin, Hui-Kuan
Strictly regulated protein degradation by ubiquitin-proteasome system (UPS) is essential for various cellular processes whose dysregulation is linked to serious diseases including cancer. Skp2, a well characterized component of Skp2-SCF E3 ligase complex, is able to conjugate both K48-linked ubiquitin chains and K63-linked ubiquitin chains on its diverse substrates, inducing proteasome mediated proteolysis or modulating the function of tagged substrates respectively. Overexpression of Skp2 is observed in various human cancers associated with poor survival and adverse therapeutic outcomes, which in turn suggests that Skp2 engages in tumorigenic activity. To that end, the oncogenic properties of Skp2 are demonstrated by various genetic mouse models, highlighting the potential of Skp2 as a target for tackling cancer. In this article, we will describe the downstream substrates of Skp2 as well as upstream regulators for Skp2-SCF complex activity. We will further summarize the comprehensive oncogenic functions of Skp2 while describing diverse strategies and therapeutic platforms currently available for developing Skp2 inhibitors.
Open Access
The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1.
(J Cell Biol, 2012-04-30) Huang, Nai-Jia; Zhang, Liguo; Tang, Wanli; Chen, Chen; Yang, Chih-Sheng; Kornbluth, Sally
Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/C(Cdh1)) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/C(Cdh1)-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid-mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.
Open Access
Ubiquitylation of p53 by the APC/C inhibitor Trim39.
(Proc Natl Acad Sci U S A, 2012-12-18) Zhang, Liguo; Huang, Nai-Jia; Chen, Chen; Tang, Wanli; Kornbluth, Sally
Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.

Browsing by Subject "Ubiquitination"

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