L-dopa and dopamine enhance the formation of aggregates under proteasome inhibition in PC12 cells

The formation of inclusion bodies in dopaminergic neurons is associated with the pathogenesis of Parkinson's disease. In order to clarify the role of dopamine/L-dopa in the formation of protein aggregates, we investigated dopamine/L-dopa-related aggregation using an experimental inclusion model. The inhibition of tyrosine hydroxylase (TH) by alpha-methyltyrosine dramatically decreased MG132-induced aggregate formation. In addition, the inhibition of TH caused the upregulation of proteasomes in cultured cells and the dopamine/L-dopa induced non-enzymatic polymerization of ubiquitin. This inhibition did not affect cell viability. These results suggest that dopamine/L-dopa might enhance aggregate formation, and that intracellular aggregates may not be toxic to cells.     

Calcium carbonate prevents Botryococcus braunii growth inhibition caused by medium acidification

Journal of Applied Phycology - Microalgae, Botryococcus braunii in particular, have received increasing interest owing to their potential as biofuel sources. Although the fertilizer components...     

Vasohibin prevents arterial neointimal formation through angiogenesis inhibition

Vasohibin is a VEGF-inducible angiogenesis inhibitor in vascular endothelium. Here we examined the presence of vasohibin in human arterial wall, and found it in endothelium of adventitial microvessels in atherosclerotic lesion. Adventitial angiogenesis is involved in the progression of neointimal formation. Even in the presence of endogenous angiogenesis inhibitors, pathological angiogenesis persists. However, the supplementation of exogenous angiogenesis inhibitors can prevent pathological angiogenesis. We evaluated the potential role of vasohibin in neointimal formation. Adenovirus-mediated human vasohibin gene transfer in mouse liver resulted in the release of vasohibin in plasma and exhibited anti-angiogenic effects at remote sites. This gene transfer inhibited adventitial angiogenesis, macrophage infiltration, and neointimal formation after cuff placement on mouse femoral artery. Vasohibin exhibited no direct effect on migration and proliferation of smooth muscle cells. Thus, vasohibin has an activity to prevent neointimal formation by inhibiting adventitial angiogenesis.     

SIRT2 interferes with autophagy-mediated degradation of protein aggregates in neuronal cells under proteasome inhibition

Abnormal protein aggregates have been suggested as a common pathogenesis of many neurodegenerative diseases. Two well-known protein degradation pathways are responsible for protein homeostasis by balancing protein biosynthesis and degradative processes: the ubiquitin–proteasome system (UPS) and autophagy-lysosomal system. UPS serves as the primary route for degradation of short-lived proteins, but large-size protein aggregates cannot be degraded by UPS. Autophagy is a unique cellular process that facilitates degradation of bulky protein aggregates by lysosome. Recent studies have demonstrated that autophagy plays a crucial role in the pathogenesis of neurodegenerative diseases characterized by abnormal protein accumulation, suggesting that regulation of autophagy may be a valuable therapeutic strategy for the treatment of various neurodegenerative diseases. Sirtuin-2 (SIRT2) is a class III histone deacetylase that is expressed abundantly in aging brain tissue. Here, we report that SIRT2 increases protein accumulation in murine cholinergic SN56 cells and human neuroblastoma SH-SY5Y cells under proteasome inhibition. Overexpression of SIRT2 inhibits lysosome-mediated autophagic turnover by interfering with aggresome formation and also makes cells more vulnerable to accumulated protein-mediated cytotoxicity by MG132 and amyloid beta. Moreover, MG132-induced accumulation of ubiquitinated proteins and p62 as well as cytotoxicity are attenuated in siRNA-mediated SIRT2-silencing cells. Taken together, these results suggest that regulation of SIRT2 could be a good therapeutic target for a range of neurodegenerative diseases by regulating autophagic flux.     

Cysteamine prevents and reverses the inhibition of pyruvate kinase activity caused by cystine in rat heart

Cystinosis is a disorder associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending on the involved tissues. Accumulation of cystine in myocardium may lead to heart failure. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that thiolic enzymes like pyruvate kinase (PK) may be altered by disulfides like cystine, the main objective of the present study was to investigate the effect of cystine on PK activity in the heart of developing rats. We performed kinetic studies and investigated the effects of reduced glutathione (GSH), a biologically occurring thiol groups protector, and cysteamine, the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits PK activity by oxidation of the sulfhydryl groups of the enzyme. Although there is no definite proof of cystine within cytoplasm, there is indirect proof t it is able to escape lysosomes and come in contact with PK. Considering that cysteamine is used in patients with cystinosis because it causes parenchymal organ cystine depletion, the present data provide a possible new effect for this drug.     

Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene

Mutations in LMNA, which encodes A-type nuclear lamins, cause disorders of striated muscle that have as a common feature dilated cardiomyopathy. We have demonstrated an abnormal activation of both the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) branches of the mitogen-activated protein kinase signaling cascade in hearts from Lmna^H222P/H222P mice that develop dilated cardiomyopathy. We previously showed that pharmacological inhibition of cardiac ERK signaling in these mice delayed the development of left ventricle dilatation and deterioration in ejection fraction. In the present study, we treated Lmna^H222P/H222P mice with SP600125, an inhibitor of JNK signalling. Systemic treatment with SP600125 inhibited JNK phosphorylation, with no detectable effect on ERK. It also blocked increased expression of RNAs encoding natriuretic peptide precursors and proteins involved in the architecture of the sarcomere that occurred in placebo-treated mice. Furthermore, treatment with SP600125 significantly delayed the development of left ventricular dilatation and prevented decreases in cardiac ejection fraction and fibrosis. These results demonstrate a role for JNK activation in the development of cardiomyopathy caused by LMNA mutations. They further provide proof-of-principle for JNK inhibition as a novel therapeutic option to prevent or delay the cardiomyopathy in humans with mutations in LMNA.     

Cell-specific inhibition of paramyxovirus maturation by proteasome inhibitors

Effects of proteasome inhibitors on the replication of a paramyxovirus in comparison with the effects on replication of an orthomyxovirus and rhabdovirus were investigated. Treatment of Sendai virus (SeV)-infected LLC-MK2 cells with 50 microM MG132 reduced virus growth to ca. 1/10,000, and treatment with different concentrations of MG132 reduced virus growth in a dose-dependent manner. Released amounts of viral proteins were reduced in correspondence with decrease in infectivity. The inhibition of virus maturation was confirmed by an SeV-like particle formation system. Lactacystin also impaired SeV growth and zLL impaired the growth to a lesser extent, suggesting involvement of proteasomes in the restriction of virus growth. In the presence of MG132, localizations of the M protein and viral F and HN glycoproteins on the cell membrane appeared to be partly dissociated, although the viral glycoproteins were normally transported to the cell surface. These results suggest that an early step of SeV assembly was disturbed by proteasome inhibitors. The relationship of the results with ubiquitin is also discussed. SeV maturation was less susceptible and resistant to MG132 in CV1 cells and A549 cells, respectively, indicating cell specificity of the drug effect. Release of vesicular stomatitis virus also showed high susceptibility to MG132 and release of influenza virus A/WSN/33 was only mildly susceptible to the drug in LLC-MK2 cells. Effects of proteasome inhibitors on virus maturation are thus highly cell-specific and partly virus-specific.     

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.     

ClC-2 inhibition prevents macrophage foam cell formation by suppressing Nlrp3 inflammasome activation

ABSTRACT

Macrophage foam cell formation and inflammation are a pathological hallmark of atherosclerosis. ClC-2 has been implicated in various pathological processes, including inflammation and lipid metabolic disorder. However, the functional role of ClC-2 in macrophage foam cell formation and inflammation is unclear. Here, we found that ClC-2 was dominantly expressed in macrophages of atherosclerotic plaque and increased in atherogenesis. Knockdown of ClC-2 inhibited ox-LDL -induced lipid uptake and deposition in macrophages. The increase in CD36 expression and the decrease in ABCA1 expression induced by ox-LDL were alleviated by ClC-2 downregulation. Further, ClC-2 lacking limited the ox-LDL-induced secretion of inflammatory cytokines and chemokine, and suppressed Nlrp3 inflammasome activation. Restoration of Nlrp3 expression reversed the effect of ClC-2 downregulation on macrophage lipid accumulation and inflammation. Collectively, our study demonstrates that ClC-2 knockdown ameliorates ox-LDL-induced macrophage foam cell formation and inflammation by inhibiting Nlrp3 inflammasome activation.     

Autophagy induction rescues toxicity mediated by proteasome inhibition

The ubiquitin-proteasome and macroautophagy-lysosome pathways are major routes for intracytosolic protein degradation. In many systems, proteasome inhibition is toxic. A Nature article by Pandey et al. shows that this toxicity can be modulated by altering autophagic activity. Their tantalizing results suggest that overexpression of HDAC6 may increase flux through the autophagy pathway, thereby attenuating the toxicity resulting from proteasome inhibition.     

Melatonin prevents the increase in hydroxyl radical-spin trap adduct formation caused by the addition of cisplatin in vitro

We studied the generation of reactive oxygen species (ROS) caused by cisplatin administration and the preventive effect of melatonin, the main secretory product of the pineal gland, on the reaction in vitro using electron spin resonance spectroscopy. Cisplatin induced generation of the hydroxyl radical (OH*) in phosphate buffer in pH 7.4 as a dose-dependent manner. However, OH* was not generated in phosphate buffer containing chloride ions at concentration exceeding 120 mM. The induction of OH. production by cisplatin was completely inhibited by the addition of melatonin, but not by the addition of 6-hydroxymelatonin, which is a hepatic metabolite of melatonin. Furthermore, melatonin was the most effective agent for preventing of OH* formation among various well-known antioxidants including mannitol and reduced glutathione. These results indicate that melatonin may scavenge OH. directly and thereby prevent renal tissue damage caused by OH* produced in response to cisplatin treatment.     

Catalytic site-specific inhibition of the 20S proteasome by 4-hydroxynonenal

The proteasome is responsible for most intracellular protein degradation and is essential for cell survival. Previous research has shown that the proteasome can be inhibited by a number of oxidants, including 4-hydroxynonenal (HNE). The present study demonstrates that HNE rapidly inhibits the chymotrypsin-like activity of the 20S proteasome purified from liver. Subunits containing HNE-adducts were identified following 2D gel electrophoresis, Western immunoblotting, and analysis by MALDI-TOF MS. At a time when only the chymotrypsin-like activity was inhibited, the alpha 6/C2 subunit was uniquely modified. These results provide important molecular details regarding the catalytic site-specific inhibition of proteasome by HNE.     

Dissociation of surfactant protein B from canine surfactant large aggregates during formation of small surfactant aggregates by in vitro surface area cycling.

Pulmonary surfactant isolated by lavage can be separated into large aggregates (LA) and small aggregates (SA). Pulse labeling experiments have shown that the LA subtype is the precursor of the SA subtype. Conversion of LA to SA can be demonstrated in vitro using the technique of surface area cycling. The precise mechanisms of surfactant subtype conversion remain unknown. We have previously reported a decline in surfactant-associated protein B (SP-B) during in vitro subtype conversion of canine surfactant. This led to the hypothesis that SP-B may be degraded by a serine protease 'convertase' during cycling. The current studies used a quantitative slot-blot assay to investigate the fates of SP-A and SP-B during in vitro cycling. These studies confirmed some SP-A is present in SA, but SP-B is confirmed to LA. Conversion leads to an apparent loss of SP-B during cycling. However, SP-B can be recovered from the walls of polypropylene and Teflon tubes by washing with chloroform:methanol. Recovered SP-B migrated on non-reducing tricine gels as a single band with an apparent molecular weight of 17 kDa, corresponding to intact SP-B dimer. Reconstitution studies demonstrated that the recovered SP-B retained its surface active properties as determined on a pulsating bubble surfactometer. We conclude in vitro surface area cycling of canine LA results in the dissociation of SP-B from surfactant lipids resulting in an apparent decline in SP-B levels.     

The effect of divalent metals and laminar shear on the formation of large freshwater aggregates

Aquatic sediment from Hamilton Harbor were suspended under controlled Couette shear to measure the changes in particle size distribution when the bulk concentration of divalent cations Cd²⁺, Cu²⁺, Ni²⁺ and Zn²⁺ was increased 500 ppb above ambient values. The size distribution of particles followed a bimodal distribution, at diameters of 20 and 200 m, and was modeled with a curvilinear collision model, using a logarithmic size scale to compensate for the decreasing density of larger aggregates. Although collision frequencies decreased with particle size, there was a limit (160 m) above which shear no longer affected collision. Addition of divalent metals caused formation of non-porous large aggregates greater than 300 m, at shears lower than 3 dynes cm^–2. The sharp increase in aggregate volume that resulted from metal addition indicated that a partitioning threshold exists in the harbor, coinciding with an imaginary line along the shore, where wind driven agitation causes a bottom shear of 3 dynes cm^–2. This threshold can be visualized as the area near shore where bottom sediments consist of sands with nominal size greater than 250 m. Calculations, using Stoke's settling, predict settling of large aggregates near thermocline depth, coincident with the appearance of fine clays on the sediment surface.     

Apoptosis induced by proteasome inhibition in human myoblast cultures

Dysfunction of the ubiquitin-proteasome system has recently been implicated in the pathogenesis of some untreatable myodegenerative diseases characterized by the formation of ubiquitinated inclusions in skeletal muscles. We have developed an in vitro model of proteasomal dysfunction by applying inhibitors of the proteasome to primary adult human skeletal muscle cultures. Our data show that proteasome inhibition causes both cytoplasmic accumulation of ubiquitinated inclusions and apoptotic death, the latter through accumulation of active caspase-3.     

Activation and inhibition of the proteasome by betulinic acid and its derivatives

This study discovered that betulinic acid (BA) is a potent proteasome activator that preferentially activates the chymotrypsin-like activity of the proteasome. Chemical modifications can transform BA into proteasome inhibitors. Chemical modifications at the C-3 position of BA resulted in compounds, such as dimethylsuccinyl BA (DSB), with various inhibitory activities against the human 20S proteasome. Interestingly, the proteasomal activation by BA and the inhibitory activity of DSB could be abrogated by introducing a side chain at the C-28 position. In summary, this study discovered a class of small molecules that can either activate or inhibit human proteasome activity depending on side chain modifications.     

Sensitization of tumor cells to NK cell-mediated killing by proteasome inhibition

Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.