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1.  Gambogic Acid Induces Apoptosis in Imatinib-Resistant Chronic Myeloid Leukemia Cells via Inducing Proteasome Inhibition and Caspase-Dependent Bcr-Abl Downregulation 
Purpose
Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.
Experimental Design
CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts.
Results
Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid–induced Bcr-Abl downregulation and apoptotic cell death.
Conclusions
These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy.
doi:10.1158/1078-0432.CCR-13-1063
PMCID: PMC3938960  PMID: 24334603
2.  Anti-rheumatic agent auranofin induced apoptosis in chronic myeloid leukemia cells resistant to imatinib through both Bcr/Abl-dependent and -independent mechanisms 
Oncotarget  2014;5(19):9118-9132.
Resistance to Imatinib mesylate (IM) is an emerging problem for patients with chronic myelogenous leukemia (CML). T315I mutation in the Bcr-Abl is the predominant mechanism of the acquired resistance to IM and second generation tyrosine kinase inhibitors (TKI). Therefore it is urgent to search for new measures to overcome TKI-resistance. Auranofin (AF), clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer. In contrast to the reports that AF induces apoptosis by increasing intracellular reactive oxygen species (ROS) levels via inhibiting thioredoxin reductase, our recent study revealed that AF-induced apoptosis depends on inhibition of proteasomal deubiquitinases (UCHL5 and USP14). Here we report that (i) AF induces apoptosis in both Bcr-Abl wild-type cells and Bcr-Abl-T315I mutation cells and inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) AF inhibits Bcr-Abl through both downregulation of Bcr-Abl gene expression and Bcr-Abl cleavage mediated by proteasome inhibition-induced caspase activation; (iii) proteasome inhibition but not ROS is required for AF-induced caspase activation and apoptosis. These findings support that AF overcomes IM resistance through both Bcr/Abl-dependent and -independent mechanisms, providing great clinical significance for cancer treatment.
PMCID: PMC4253423  PMID: 25193854
Auranofin; proteasome; chronic myelogenous leukemia; imatinib resistance; Bcr-Abl
3.  The COP9 Signalosome Is Required for Autophagy, Proteasome-Mediated Proteolysis, and Cardiomyocyte Survival in Adult Mice 
Circulation. Heart failure  2013;6(5):10.1161/CIRCHEARTFAILURE.113.000338.
Background
The COP9 signalosome (CSN) is an evolutionarily conserved protein complex composed of 8 unique protein subunits (CSN1 through CSN8). We have recently discovered in perinatal mouse hearts that CSN regulates not only proteasome-mediated proteolysis but also macroautophagy. However, the physiological significance of CSN in a pots-mitotic organ of adult vertebrates has not been determined. We sought to study the physiological role of CSN8/CSN in adult mouse hearts.
Methods and Results
Csn8 was conditionally ablated in the cardiomyocytes of adult mice (CSN8CKO) using a temporally controlled Cre-LoxP system. Loss of CSN8 accumulated the neddylated forms of cullins and non-cullin proteins, increased ubiquitinated proteins, and stabilized a surrogate substrate of the proteasome in the heart. Autophagic flux was significantly decreased while autophagosomes were markedly increased in CSN8CKO hearts, indicative of impaired autophagosome removal. Furthermore, we observed increased oxidized proteins, massive necrotic cardiomyocytes, and morphological and functional changes characteristic of dilated cardiomyopathy in CSN8CKO mice.
Conclusions
CSN deneddylates substrates more than cullins and is indispensable to cardiomyocyte survival in not only perinatal hearts but also adult hearts. CSN8/CSN regulates both proteasome-mediated proteolysis and the autophagic-lysosomal pathway, critical to the removal of oxidized proteins in the heart.
doi:10.1161/CIRCHEARTFAILURE.113.000338
PMCID: PMC3835345  PMID: 23873473
COP9 signalosome; autophagy; proteasome; NEDD8; heart
4.  Ubiquilin-1 Protects Cells from Oxidative Stress and Ischemic Stroke Caused Tissue Injury in Mice 
The Journal of Neuroscience  2014;34(8):2813-2821.
Ubiquilin-1 (Ubqln1 or Ubqln), a ubiquitin-like protein, mediates degradation of misfolded proteins and has been implicated in a number of pathological and physiological conditions. To better understand its function in vivo, we recently generated transgenic (Tg) mice that globally overexpress mouse Ubqln in a variety of tissues and ubqln conditional knock-out mice. The Tg mice were viable and did not show any developmental or behavioral abnormalities compared with their wild-type (WT) littermates. When subjected to oxidative stress or ischemia/reperfusion, however, ubqln Tg mice but not the WT littermates showed increased tolerance to these insults. Following ischemic stroke, ubqln Tg mice recovered motor function more rapidly than did the WT mice. In contrast, KO of ubqln exacerbated neuronal damage after stroke. In addition, KO of ubqln also caused accumulation of ubiquitinated proteins. When ubqln KO mice were crossed with a ubiquitin-proteasome system function reporter mouse, the accumulation of a proteasome surrogate substrate was observed. These results suggest that Ubqln protects mice from oxidative stress and ischemic stroke-caused neuronal injury through facilitating removal of damaged proteins. Thus, enhanced removal of unwanted proteins is a potential therapeutic strategy for treating stroke-caused neuronal injury.
doi:10.1523/JNEUROSCI.3541-13.2014
PMCID: PMC3953589  PMID: 24553923
5.  Protein Kinase G Positively Regulates Proteasome-Mediated Degradation of Misfolded Proteins 
Circulation  2013;128(4):365-376.
Background
Proteasome functional insufficiency is implicated in a large subset of cardiovascular diseases and may play an important role in their pathogenesis. The regulation of proteasome function is poorly understood, hindering the development of effective strategies to improve proteasome function.
Methods and Results
Protein kinase G (PKG) was manipulated genetically and pharmacologically in cultured cardiomyocytes. Activation of PKG increased proteasome peptidase activities, facilitated proteasome-mediated degradation of surrogate (GFPu) and bona fide misfolded proteins (CryABR120G), and attenuated CryABR120G overexpression-induced accumulation of ubiquitinated proteins and cellular injury. PKG inhibition elicited the opposite responses. Differences in the abundance of the key 26S proteasome subunits Rpt6 and □5 between PKG manipulated and the control groups were not statistically significant, but the isoelectric points of were shifted by PKG activation. In transgenic mice expressing a surrogate substrate (GFPdgn), PKG activation by sildenafil increased myocardial proteasome activities and significantly decreased myocardial GFPdgn protein levels. Sildenafil treatment significantly increased myocardial PKG activity and significantly reduced myocardial accumulation of CryABR120G, ubiquitin conjugates, and aberrant protein aggregates in mice with CryABR120G-based desmin-related cardiomyopathy. No discernible effect on bona fide native substrates of the ubiquitin-proteasome system was observed from PKG manipulation in vitro or in vivo.
Conclusions
PKG positively regulates proteasome activities and proteasome-mediated degradation of misfolded proteins likely through posttranslational modifications to proteasome subunits. This may be a new mechanism underlying the benefit of PKG stimulation in treating cardiac diseases. Stimulation of PKG by measures such as sildenafil administration is potentially a new therapeutic strategy to treat cardiac proteinopathies.
doi:10.1161/CIRCULATIONAHA.113.001971
PMCID: PMC3761383  PMID: 23770744
Protein kinase G; proteasome; misfolded proteins; desmin-related cardiomyopathy
6.  Emission of Metals from Pelletized and Uncompressed Biomass Fuels Combustion in Rural Household Stoves in China 
Scientific Reports  2014;4:5611.
Effort of reducing CO2 emissions in developing countries may require an increasing utilization of biomass fuels. Biomass pellets seem well-suited for residential biomass markets. However, there is limited quantitative information on pollutant emissions from biomass pellets burning, especially those measured in real applications. In this study, biomass pellets and raw biomass fuels were burned in a pellet burner and a conventional stove respectively, in rural households, and metal emissions were determined. Results showed that the emission factors (EFs) ranged 3.20–5.57 (Pb), 5.20–7.58 (Cu), 0.11–0.23 (Cd), 12.67–39.00 (As), 0.59–1.31 mg/kg (Ni) for pellets, and 0.73–1.34 (Pb), 0.92–4.48 (Cu), 0.08–0.14 (Cd), 7.29–13.22 (As), 0.28–0.62 (Ni) mg/kg for raw biomass. For unit energy delivered to cooking vessels, the EFs ranged 0.42–0.77 (Pb), 0.79–1.16 (Cu), 0.01–0.03 (Cd), 1.93–5.09 (As), 0.08–0.19 mg/MJ (Ni) for pellets, and 0.30–0.56 (Pb), 0.41–1.86 (Cu), 0.04–0.06 (Cd), 3.25–5.49 (As), 0.12–0.26 (Ni) mg/MJ for raw biomass. This study found that moisture, volatile matter and modified combustion efficiency were the important factors affecting metal emissions. Comparisons of the mass-based and task-based EFs found that biomass pellets produced higher metal emissions than the same amount of raw biomass. However, metal emissions from pellets were not higher in terms of unit energy delivered.
doi:10.1038/srep05611
PMCID: PMC4085603  PMID: 25002204
7.  Clinically used antirheumatic agent auranofin is a proteasomal deubiquitinase inhibitor and inhibits tumor growth 
Oncotarget  2014;5(14):5453-5471.
Proteasomes are attractive emerging targets for anti-cancer therapies. Auranofin (Aur), a gold-containing compound clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer but its anti-cancer mechanism is poorly understood. Here we report that (i) Aur shows proteasome-inhibitory effect that is comparable to that of bortezomib/Velcade (Vel); (ii) different from bortezomib, Aur inhibits proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14 rather than the 20S proteasome; (iii) inhibition of the proteasome-associated DUBs is required for Aur-induced cytotoxicity; and (iv) Aur selectively inhibits tumor growth in vivo and induces cytotoxicity in cancer cells from acute myeloid leukemia patients. This study provides important novel insight into understanding the proteasome-inhibiting property of metal-containing compounds. Although several DUB inhibitors were reported, this study uncovers the first drug already used in clinic that can inhibit proteasome-associated DUBs with promising anti-tumor effects.
PMCID: PMC4170648  PMID: 24977961
cancer; deubiquitinase; proteasome; auranofin
8.  Autophagic-Lysosomal Inhibition Compromises Ubiquitin-Proteasome System Performance in a p62 Dependent Manner in Cardiomyocytes 
PLoS ONE  2014;9(6):e100715.
Intracellular protein degradation is primarily performed by the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). The interplay between these two pathways has been rarely examined in intact animals and the mechanism underlying the interplay remains unclear. Hence, we sought to test in vivo and in vitro the impact of inhibition of the ALP on UPS proteolytic performance in cardiomyocytes and to explore the underlying mechanism. Transgenic mice ubiquitously expressing a surrogate UPS substrate (GFPdgn) were treated with bafilomycin-A1 (BFA) to inhibit the ALP. Myocardial and renal GFPdgn protein levels but not mRNA levels were increased at 24 hours but not 3 hours after the first injection of BFA. Myocardial protein abundance of key proteasome subunits and the activities of proteasomal peptidases were not discernibly altered by the treatment. In cultured neonatal rat ventricular myocytes (NRVMs), the surrogate UPS substrate GFPu and a control red fluorescence protein (RFP) were co-expressed to probe UPS performance. At 12 hours or 24 hours after ALP inhibition by 3-methyladenine (3-MA) or BFA, GFPu/RFP protein ratios and the protein half-life of GFPu were significantly increased, which is accompanied by increases in p62 proteins. Similar findings were obtained when ALP was inhibited genetically via silencing Atg7 or Rab7. ALP inhibition-induced increases in GFPu and p62 are co-localized in NRVMs. siRNA-mediated p62 knockdown prevented ALP inhibition from inducing GFPu accumulation in NRVMs. We conclude that in a p62-dependent fashion, ALP inhibition impairs cardiac UPS proteolytic performance in cardiomyocytes in vitro and in vivo.
doi:10.1371/journal.pone.0100715
PMCID: PMC4069113  PMID: 24959866
9.  A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases 
Scientific Reports  2014;4:5240.
The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy.
doi:10.1038/srep05240
PMCID: PMC4050382  PMID: 24912524
11.  The proteasome function reporter GFPu accumulates in young brains of the APPswe/PS1dE9 Alzheimer’s disease mouse model 
Alzheimer’s disease (AD), the most common cause of dementia, is neuropathologically characterized by accumulation of insoluble fibrous inclusions in the brain in the form of intracellular neurofibrillary tangles and extracellular senile plaques. Perturbation of the ubiquitin-proteasome system (UPS) has long been considered an attractive hypothesis to explain the pathogenesis of AD. However, studies on UPS functionality with various methods and AD models have achieved non-conclusive results. To get further insight into UPS functionality in AD, we have crossed a well-documented APPswe/PS1dE9 AD mouse model with a UPS functionality reporter, GFPu, mouse expressing green fluorescence protein (GFP) fused to a constitutive degradation signal (CL-1) that facilitates its rapid turnover in conditions of a normal UPS. Our western blot results indicate that GFPu reporter protein was accumulated in the cortex and hippocampus but not striatum in the APPswe/PS1dE9 AD mouse model at 4 weeks of age, which is confirmed by fluorescence microscopy and elevated levels of p53, an endogenous UPS substrate. In accordance with this, the levels of ubiquitinated proteins were elevated in the AD mouse model. These results suggest that UPS is either impaired or functionally insufficient in specific brain regions in the APPswe/PS1dE9 AD mouse model at a very young age, long before senile plaque formation and the onset of memory loss. These observations may shed new light on the pathogenesis of AD.
doi:10.1007/s10571-013-0022-9
PMCID: PMC3954921  PMID: 24363091
Alzheimer disease; ubiquitin-proteasome system; proteasome function reporter; GFPu; protein degradation; ubiquitinated proteins
12.  Ubiquitin Receptors and Protein Quality Control 
Protein quality control (PQC) is essential to intracellular proteostasis and is carried out by sophisticated collaboration between molecular chaperones and targeted protein degradation. The latter is performed by proteasome-mediated degradation, chaperone-mediated autophagy (CMA), and selective macroautophagy, and collectively serve as the final line of defense of PQC. Ubiquitination and subsequently ubiquitin (Ub) receptor proteins (e.g., p62 and Ubiquilins) are important common factors for targeting misfolded proteins to multiple quality control destinies, including the proteasome, lysosomes, and perhaps aggresomes, as well as for triggering mitophagy to remove defective mitochondria. PQC inadequacy, particularly proteasome functional insufficiency, has been shown to participate in cardiac pathogenesis. Tremendous advances have been made in unveiling the changes of PQC in cardiac diseases. However, the investigation into the molecular pathways regulating PQC in cardiac (patho)physiology, including the function of most ubiquitin receptor proteins in the heart, has only recently been initiated. A better understanding of molecular mechanisms governing PQC in cardiac physiology and pathology will undoubtedly provide new insights into cardiac pathogenesis and promote the search for novel therapeutic strategies to more effectively battle heart disease.
doi:10.1016/j.yjmcc.2012.09.012
PMCID: PMC3571097  PMID: 23046644
ubiquitin; proteasome; Ubiquilin; p62; autophagy
13.  Post-translational Modification and Quality Control 
Circulation research  2013;112(2):367-381.
Protein quality control (PQC) functions to minimize the level and toxicity of misfolded proteins in the cell. PQC is performed by intricate collaboration among chaperones and target protein degradation. The latter is carried out primarily by the ubiquitin-proteasome system and perhaps autophagy. Terminally misfolded proteins that are not timely removed tend to form aggregates. Their clearance requires macroautophagy. Macroautophagy serves in intracellular quality control also by selectively segregating defective organelles (e.g., mitochondria) and targeting them for degradation by the lysosome. Inadequate PQC is observed in a large subset of failing human hearts with a variety of etiologies and its pathogenic role has been experimentally demonstrated. Multiple post-translational modifications (PTMs) can occur to substrate proteins and/or PQC machineries, promoting or hindering the removal of the misfolded proteins. This article highlights recent advances in PTMs-mediated regulation of intracellular quality control mechanisms and its known involvement in cardiac pathology.
doi:10.1161/CIRCRESAHA.112.268706
PMCID: PMC3566557  PMID: 23329792
chaperones; the ubiquitin proteasome system; autophagy; chaperone-mediated autophagy; neddylation; deubiquitination; phosphorylation
14.  The Carboxy-terminus of BAK1 regulates kinase activity and is required for normal growth of Arabidopsis 
Binding of brassinolide to the brassinosteroid-insenstive 1(BRI1) receptor kinase promotes interaction with its co-receptor, BRI1-associated receptor kinase 1 (BAK1). Juxtaposition of the kinase domains that occurs then allows reciprocal transphosphorylation and activation of both kinases, but details of that process are not entirely clear. In the present study we show that the carboxy (C)-terminal polypeptide of BAK1 may play a role. First, we demonstrate that the C-terminal domain is a strong inhibitor of the transphosphorylation activity of the recombinant BAK1 cytoplasmic domain protein. However, recombinant BAK1 lacking the C-terminal domain is unable to transactivate the peptide kinase activity of BRI1 in vitro. Thus, the C-terminal domain may play both a positive and negative role. Interestingly, a synthetic peptide corresponding to the full C-terminal domain (residues 576–615 of BAK1) interacted with recombinant BRI1 in vitro, and that interaction was enhanced by phosphorylation at the Tyr-610 site. Expression of a BAK1 C-terminal domain truncation (designated BAK1-ΔCT-Flag) in transgenic Arabidopsis plants lacking endogenous bak1 and its functional paralog, bkk1, produced plants that were wild type in appearance but much smaller than plants expressing full-length BAK1-Flag. The reduction in growth may be attributed to a partial inhibition of BR signaling in vivo as reflected in root growth assays but other factors are likely involved as well. Our working model is that in vivo, the inhibitory action of the C-terminal domain of BAK1 is relieved by binding to BRI1. However, that interaction is not essential for BR signaling, but other aspects of cellular signaling are impacted when the C-terminal domain is truncated and result in inhibition of growth. These results increase the molecular understanding of the C-terminal domain of BAK1 as a regulator of kinase activity that may serve as a model for other receptor kinases.
doi:10.3389/fpls.2014.00016
PMCID: PMC3912384  PMID: 24550926
brassinosteroid; BAK1; BRI1; domain; phosphotyrosine; protein–peptide interaction
15.  Clinicopathological features and prognosis of pseudomyxoma peritonei 
The aim of this study was to evaluate the effects of treatment and the factors influencing the postoperative recurrence and survival time for pseudomyxoma peritonei (PMP). A total of 39 patients with PMP who received treatment were analyzed in The General Hospital of PLA (Beijing, China) between 2002 and 2011. The patients received cytoreductive surgery (CRS) and 25 cases of PMP recurred. Seven patients received postoperative hyperthermic intraperitoneal chemoperfusion (HIPEC). The median follow-up was 40 months. There were eight mortalities in this period. The 5- and 10-year survival rates were 89.0 and 35.0%, respectively. The medians of overall survival (OS) and recurrence time were 37 and 4 months, respectively. Multivariate analyses revealed that pathological subtype was able to influence the recurrence (P=0.042) and OS (P=0.033) times, as an independent prognostic factor. HIPEC was significantly associated with postoperative recurrence time (P=0.017). Patients with disseminated peritoneal adenomucinosis had a more favorable prognosis. CRS combined with HIPEC was able to extend the postoperative recurrence time for patients with PMP.
doi:10.3892/etm.2013.1408
PMCID: PMC3861383  PMID: 24348787
pseudomyxoma peritonei; cytoreductive surgery; hyperthermic intraperitoneal chemoperfusion; prognosis
16.  Gambogic acid suppresses pressure overload cardiac hypertrophy in rats 
Cardiac hypertrophy is a common response of the heart to a variety of cardiovascular stimuli. Pathological cardiac hypertrophy eventually leads to heart failure. Gambogic acid (GA) is a main active ingredient isolated from the gamboge resin of Garcinia hanburyi trees and has potent anti-tumor and anti-inflammatory effects that are associated with inhibition of the NF-κB pathway. We and others recently reported that GA can significantly inhibit the function of the proteasome with much less toxicity than conventional proteasome inhibitors. The increasing lines of evidence indicate that the inhibition of the proteasome can promote the regression of cardiac hypertrophy induced by pressure overload through the blockade of the NF-κB pathway. In the present study, we examined the effect of GA on pressure overload or isoproterenol infusion induced cardiac hypertrophy and fibrosis, and changes in myocardial NF-κB signaling. We observed that the heart weight/body weight ratio, the size of cardiomyocytes, interstitial fibrosis, and the reactivation of fetal genes (α-SK-actin and BNP mRNA) were markedly increased by abdominal aorta constriction (AAC) or isoproterenol infusion (ISO), all of which were effectively inhibited by GA treatment. Furthermore, GA treatment abolished proteasome chymotrypsin-like activity increases induced by AAC or ISO, led to increased myocardial IκB protein, decreased NF-κB p65 subunit levels in the nuclear fraction, decreased NF-κB DNA-binding activity, and reduced IL2 levels in the myocardium of rats subject to AAC or ISO. In conclusion, GA treatment can suppress cardiac hypertrophy and fibrosis induced by pressure overload or isoproterenol possibly through the inhibition of the proteasome and the NF-κB pathway, suggesting that GA treatment may provide a new strategy to treat cardiac hypertrophy.
PMCID: PMC3819582  PMID: 24224134
Gambogic acid; cardiac hypertrophy; pressure overload; isoproterenol; proteasome; NF-κB
17.  Bortezomib, a Proteasome Inhibitor, Attenuates Angiotensin II-Induced Hypertension and Aortic Remodeling in Rats 
PLoS ONE  2013;8(10):e78564.
Background
Hypertension is a highly prevalent disorder and a major risk factor for cardiovascular diseases. Hypertensive vascular remodeling is the pathological mal-adaption of blood vessels to the hypertensive condition that contributes to further development of high blood pressure and end-organ damage. Hypertensive remodeling involves, at least in part, changes in protein turnover. The ubiquitin proteasome system (UPS) is a major protein quality and quantity control system. This study tested the hypothesis that the proteasome inhibitor, bortezomib, would attenuate AngII-induced hypertension and its sequelae such as aortic remodeling in rats.
Methodology/Principal Findings
Male Sprague Dawley rats were subjected to AngII infusion for two weeks in the absence or presence of bortezomib. Mean arterial pressure was measured in conscious rats. Aortic tissue was collected for estimation of wall area, collagen deposition and expression of tissue inhibitors of matrix metalloproteases (TIMP), Ki67 (a marker of proliferation), reactive oxygen species (ROS) and VCAM-1 (a marker of inflammation). AngII infusion increased arterial pressure significantly (160±4 mmHg vs. vehicle treatment 133±2 mmHg). This hypertensive response was attenuated by bortezomib (138±5 mmHg). AngII hypertension was associated with significant increases in aortic wall to lumen ratio (∼29%), collagen deposition (∼14%) and expression of TIMP1 and TIMP2. AngII also increased MMP2 activity, proteasomal chymotrypsin-like activity, Ki67 staining, ROS generation and VCAM-1 immunoreactivity. Co-treatment of AngII-infused rats with bortezomib attenuated these AngII-induced responses.
Conclusions
Collectively, these data support the idea that proteasome activity contributes to AngII-induced hypertension and hypertensive aortic vascular remodeling at least in part by modulating TIMP1/2 and MMP2 function. Preliminary observations are consistent with a role for ROS, inflammatory and proliferative mechanisms in this effect. Further understanding of the mechanisms by which the proteasome is involved in hypertension and vascular structural remodeling may reveal novel targets for pharmacological treatment of hypertension, hypertensive remodeling or both.
doi:10.1371/journal.pone.0078564
PMCID: PMC3813683  PMID: 24205262
18.  Genetically Induced Moderate Inhibition of the Proteasome in Cardiomyocytes Exacerbates Myocardial Ischemia-Reperfusion Injury in Mice 
Circulation research  2012;111(5):532-542.
Rationale
Both cardiomyocyte-restricted proteasome functional enhancement and pharmacological proteasome inhibition (PSMI) were shown to attenuate myocardial ischemia/reperfusion (I/R) injury. The role of cardiac proteasome dysfunction during I/R and the perspective to diminish I/R injury by manipulating proteasome function remain unclear.
Objectives
We sought to determine proteasome adequacy in I/R hearts, create a mouse model of cardiomyocyte-restricted PSMI (CR-PSMI), and test CR-PSMI impact on I/R injury.
Methods and Results
Myocardial I/R were modeled by ligation (30min) and subsequent release of the left anterior descending artery in mice overexpressing GFPdgn, a validated surrogate proteasome substrate. At 24h of reperfusion, myocardial proteasome activities were significantly lower while total ubiquitin conjugates and GFPdgn protein levels were markedly higher in all regions of the I/R hearts than the sham controls, indicative of proteasome functional insufficiency. CR-PSMI in intact mice was achieved by transgenic (tg) overexpression of a peptidase-disabled mouse β5 subunit (T60A-β5) driven by an attenuated mouse mhc6 promoter. Overexpressed T60A-β5 can replace endogenous β5 and inhibits proteasome chymotrypsin-like activities in the heart. Mice with moderate CR-PSMI showed no abnormalities at the baseline but displayed markedly more pronounced structural and functional damage during I/R, compared with non-tg littermates. The exacerbation of I/R injury by moderate CR-PSMI was associated with significant increases in the protein level of PTEN and protein kinase Cδ (PKCδ), decreased Akt activation, and reduced PKCε.
Conclusions
Myocardial I/R causes proteasome functional insufficiency in cardiomyocytes and moderate CR-PSMI augments PTEN and PKCδ, suppresses Akt and PKCε, increases cardiomyocyte apoptosis, and aggravates I/R injury in mice.
doi:10.1161/CIRCRESAHA.112.270983
PMCID: PMC3426260  PMID: 22740087
proteasome inhibition; myocardial reperfusion injury; transgenic mouse; Akt; PKCδ
19.  Gambogic acid moderates cardiac responses to chronic hypoxia likely by acting on the proteasome and NF-κB pathway 
Gambogic acid (GA) is the principal active ingredient of gamboges. GA was reported to exert anti-tumor and anti-inflammatory effects both in vitro and in vivo. Previously, we have shown that GA is a more tissue-specific proteasome inhibitor than bortezomib and it is less toxic to peripheral white blood cells compared to bortezomib. Ubiquitous proteasome inhibition was shown by some reports, but not by others, to prevent cardiac remodeling in response to pressure overload by blocking the NF-κB pathway; however, whether GA modulates the development of chronic hypoxia-induced right ventricular hypertrophy has not been investigated yet. Here we report that GA can significantly attenuate right ventricular hypertrophy induced by chronic hypoxia, reduce cardiac fibrosis, and remarkably block the reactivation of bona fide fetal genes in the cardiac tissue. Furthermore, we also investigated the potential molecular targets of GA on right ventricular hypertrophy. The results showed that GA could accumulate the IκB levels associated with decreased proteasomal activity, block the translocation of NF-κB from the cytoplasm to the nucleus, decrease NF-κB DNA-binding activity, and reduce IL-2 levels. In conclusion, GA is capable of preventing the development of chronic hypoxia-induced right ventricular hypertrophy. GA has great potential to be developed into an effective anti-hypertrophy agent.
PMCID: PMC3751679  PMID: 23991348
Gambogic acid; chronic hypoxia; right ventricular hypertrophy; NF-κB
20.  The Ubiquitin–Proteasome System and Cardiovascular Disease 
Over the past decade, the role of the ubiquitin–proteasome system (UPS) has been the subject of numerous studies to elucidate its role in cardiovascular physiology and pathophysiology. There have been many advances in this field including the use of proteomics to achieve a better understanding of how the cardiac proteasome is regulated. Moreover, improved methods for the assessment of UPS function and the development of genetic models to study the role of the UPS have led to the realization that often the function of this system deviates from the norm in many cardiovascular pathologies. Hence, dysfunction has been described in atherosclerosis, familial cardiac proteinopathies, idiopathic dilated cardiomyopathies, and myocardial ischemia. This has led to numerous studies of the ubiquitin protein (E3) ligases and their roles in cardiac physiology and pathophysiology. This has also led to the controversial proposition of treating atherosclerosis, cardiac hypertrophy, and myocardial ischemia with proteasome inhibitors. Furthering our knowledge of this system may help in the development of new UPS-based therapeutic modalities for mitigation of cardiovascular disease.
doi:10.1016/B978-0-12-397863-9.00009-2
PMCID: PMC3743449  PMID: 22727426
Ubiquitin–proteasome system; Heart; Vascular; Atherosclerosis; Ubiquitin protein ligases; Protein quality control; Heart failure; Cardiomyopathy; Myocardial ischemia; Preconditioning
21.  Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers 
PLoS ONE  2013;8(7):e67793.
The COP9 signalosome (CSN), an evolutionally highly conserved protein complex composed of 8 unique subunits (CSN1 through CSN8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (UPS) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. Hepatocyte-specific knockout of the Cops8 gene (HS-Csn8KO) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. Here, we report that Csn8/CSN exerts profound impacts on hepatic UPS function and is critical to the stability of the pro-apoptotic protein Bim. Significant decreases in CIS (cytokine-inducible Src homology 2 domain-containing protein), a Bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of Bim proteins. Csn8 deficiency also significantly decreased 19S proteasome subunit Rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. The use of a surrogate UPS substrate further reveals severe impairment of UPS-mediated proteolysis in HS-Csn8KO livers. Inclusion body-like materials were accumulated in Csn8 deficient hepatocytes. In addition to Bim, massive hepatocyte apoptosis in HS-Csn8KO livers is also associated with elevated expression of other members of the Bcl2 family, including pro-apoptotic Bax as well as anti-apoptotic Bcl2 and Bcl-XL. Increased interaction between Bcl2 and Bim, but not between Bcl2 and Bax, was detected. Hence, it is concluded that hepatic CSN8 deficiency impairs the UPS in the liver and the resultant Bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering Bcl2 away from Bax.
doi:10.1371/journal.pone.0067793
PMCID: PMC3698095  PMID: 23840878
22.  Reductions in Emissions of Carbonaceous Particulate Matter and Polycyclic Aromatic Hydrocarbons from Combustion of Biomass Pellets in Comparisonwith Raw Fuel Burning 
Environmental science & technology  2012;46(11):6409-6416.
Biomass pellets are emerging as a cleaner alternative to traditional biomass fuels. The potential benefits of using biomass pellets include improving energy utilization efficiency and reducing emissions of air pollutants. To assess the environmental, climate, and health significance of replacing traditional fuels with biomass pellets, it is critical to measure the emission factors (EFs) of various pollutants from pellet burning. However, only a few field measurements have been conducted on the emissions of carbon monoxide (CO), particulate matter (PM), and polycyclic aromatic hydrocarbons (PAHs) from the combustion of pellets. In this study, pine wood and corn straw pellets were burned in a pellet burner (2.6 kW) and the EFs of CO, organic carbon, elemental carbon, PM, and PAHs (EFCO, EFOC, EFEC, EFPM, and EFPAH) were determined. The average EFCO, EFOC, EFEC, and EFPM were 1520±1170, 8.68±11.4, 11.2±8.7, and 188±87 mg/MJ for corn straw pellets, and 266±137, 5.74±7.17, 2.02±1.57, and 71.0±54.0 mg/MJ for pine wood pellets, respectively. Total carbonaceous carbon constituted 8 to 14% of the PM mass emitted. The measured values of EFPAH for the two pellets were 1.02±0.64 and 0.506±0.360 mg/MJ, respectively. The secondary side air supply in the pellet burner did not change the EFs of most pollutants significantly (p > 0.05). The only exceptions were EFOC and EFPM for pine wood pellets because of reduced combustion temperatures with the increased air supply. In comparison with EFs for the raw pine wood and corn straw, EFCO, EFOC, EFEC, and EFPM for pellets were significantly lower than those for raw fuels (p < 0.05). However, the differences in EFPAH were not significant (p > 0.05). Based on the measured EFs and thermal efficiencies, it was estimated that 95, 98, 98, 88, and 71% reductions in the total emissions of CO, OC, EC, PM, and PAHs could be achieved by replacing the raw biomass fuels combusted in traditional cooking stoves with pellets burned in modern pellet burners.
doi:10.1021/es300369d
PMCID: PMC3377013  PMID: 22568759
23.  Emission Factors, Size Distributions and Emission Inventories of Carbonaceous Particulate Matter from Residential Wood Combustion in Rural China 
Environmental science & technology  2012;46(7):4207-4214.
Published emission factors (EFs) often vary significantly, leading to high uncertainties in emission estimations. There are few reliable EFs from field measurements of residential wood combustion in China. In this study, 17 wood fuels and one bamboo were combusted in a typical residential stove in rural China to measure realistic EFs of particulate matter (PM), organic carbon (OC) and elemental carbon (EC), as well as to investigate the influence of fuel properties and combustion conditions on the EFs. Measured EFs of PM, OC, and EC (EFPM, EFOC, and EFEC, respectively) were in the range of 0.38~6.4, 0.024~3.0 and 0.039~3.9 g/kg (dry basis), with means and standard derivation of 2.2±1.2, 0.62±0.64 and 0.83±0.69 g/kg, respectively. Shrubby biomass combustion produced higher EFs than tree woods, and both species had lower EFs than those of indoor crop residue burning (p<0.05). Significant correlations between EFPM, EFOC and EFEC were expected. By using a nine-stage cascade impactor, it was shown that size distributions of PM emitted from tree biomass combustions were unimodal with peaks at a diameter less than 0.4 µm (PM0.4), much finer than the PM from indoor crop residue burning. Approximately 79.4% of the total PM from tree wood combustion was PM with a diameter less than 2.1µm (PM2.1). PM size distributions for shrubby biomasses were slightly different from those for tree fuels. Based on the measured EFs, total emissions of PM, OC, and EC from residential wood combustion in rural China in 2007 were estimated at about 303, 75.7, and 92.0 Gg.
doi:10.1021/es203957u
PMCID: PMC3352236  PMID: 22380753
24.  Therapeutic Potential of Umbilical Cord Mesenchymal Stromal Cells Transplantation for Cerebral Palsy: A Case Report 
Cerebral palsy is the most common motor disability in childhood. In current paper, we first report our clinical data regarding administration of umbilical cord mesenchymal stem cells (MSCs) transplantation in treatment of cerebral palsy. A 5-year-old girl with cerebral palsy was treated with multiple times of intravenous and intrathecal administration of MSCs derived from her young sister and was followed up for 28 months. The gross motor dysfunction was improved. Other benefits included enhanced immunity, increased physical strength, and adjusted speech and comprehension. Temporary low-grade fever was the only side effect during the treatment. MSCs may be a safe and effective therapy to improve symptoms in children with cerebral palsy.
doi:10.1155/2013/146347
PMCID: PMC3603664  PMID: 23533920
25.  The COP9 Signalosome Regulates Autophagosome Maturation 
Circulation  2011;124(19):2117-2128.
Background
Autophagy is essential to intracellular homeostasis and involved in the pathophysiology of a variety of diseases. Mechanisms regulating selective autophagy remain poorly understood. The COP9 signalosome (CSN) is a conserved protein complex consisting of 8 subunits (CSN1 through CSN8) and known to regulate the ubiquitin-proteasome system (UPS). However, it is unknown whether CSN plays a role in autophagy.
Methods and Results
Marked increases in LC3-II and p62 proteins were observed upon Csn8 depletion in the cardiomyocytes of mouse hearts with cardiomyocyte-restricted knockout of the gene encoding CSN subunit 8 (CR-Csn8KO). The increases in autophagosomes were confirmed by probing with GFP-LC3 and electron microscopy. Autophagic flux assessments revealed that defective autophagosome removal was the cause of autophagosome accumulation and occurred prior to a global UPS impairment in Csn8-deficient hearts. Analyzing the prevalence of different stages of autophagic vacuoles revealed defective autophagosome maturation. Down-regulation of Rab7 was found to strikingly co-localize with the autophagosome accumulation at the individual cardiomyocyte level. A significantly higher percent of cardiomyocytes with autophagosome accumulation underwent necrosis in CR-Csn8KO hearts. Chronic lysosomal inhibition with Chloroquine induced cardiomyocyte necrosis in mice. Rab7 knockdown impaired autophagosome maturation of non-selective and selective autophagy and exacerbated cell death induced by proteasome inhibition in cultured cardiomyocytes.
Conclusions: (1)
Csn8/CSN is a central regulator in not only the proteasomal proteolytic pathway but also selective autophagy; (2) likely through regulating the expression of Rab7, Csn8/CSN plays a critical role in autophagosome maturation; and (3) impaired autophagosome maturation causes cardiomyocytes to undergo necrosis.
doi:10.1161/CIRCULATIONAHA.111.048934
PMCID: PMC3211066  PMID: 21986281
the COP9 signalosome; autophagy; lysosome; Rab7; necrosis

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