Characterization of a serine proteinase mediating encystation of Acanthamoeba

Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.     

Changes in sleep characteristics of rat preclinical model of Parkinson’s disease based on attenuation of the ubiquitin—proteasome system activity in the brain

Numerous experimental and epidemiological data indicate a high significance of environmental neurotoxins, specifically, inhibitors of the ubiquitin–proteasome system, in pathogenesis of Parkinson’s disease (PD). To develop a preclinical model of PD in rats we used a technique of intranasal administration of lactacystin, a natural proteasome inhibitor, into the brain. It was found that three weeks after the first lactacystin administration it induced a little degeneration of dopaminergic neurons in the olfactory bulb and substantia nigra pars compacta without any olfactory dysfunction and motor behavior disorders. Besides, its effect led to the appearance of some signs of sleep disorders: increased somnolence (especially in the dark, active daily phase), fragmentation of slow-wave sleep, decreased EEG delta rhythm during slow-wave sleep. These signs share some similarity with PD and could be useful in clinical studies for the quick search for polysomnographic markers of the early PD stage.     

Proteasome Activity Is Critical for the cAMP-Induced Differentiation of Neuroblastoma Cells

1. The ubiquitin–proteasome pathway is involved in a variety of cellular functions in mammalian cells. The role of proteasome, however, in the course of cell differentiation is not well characterized. We hypothesized that proteasome activity might be essential during neuronal cell differentiation.2. To investigate the role of proteasome during neuronal differentiation, we made use of a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons upon elevation of the intracellular level of adenosine 3,5-cyclic monophosphate (cAMP). To monitor proteasome activity in NBP2 cells, we integrated an expression cassette~for a short-lived green fluorescent protein (d2EGFP) into these cells, which were designated as NBP2-PN25. When NBP2-PN25 cells were treated with a proteasome inhibitor, lactacystin or MG132, a dose-dependent increase in the constitutive levels of d2EGFP expression was detected.3. We also found that proteasome inhibition by lactacystin during the cAMP-induced differentiation of NBP2-PN25 cells triggered cell death. Both lactacystin and cAMP induction reduced the expression of mRNA for the differentiation-associated genes, such as N-mycand cyclin B1. While cAMP-inducing agents decreased the level of N-myc and cyclin B1 proteins, lactacystin increased the level of these proteins.4. Our data suggest that a reduced level of N-myc and cyclin B1 proteins is critical to commence differentiation, and this can be blocked by a proteasome inhibitor, leading to cell death. Concomitant induction of differentiation and proteasome inhibition, may, therefore, be potentially useful for the treatment of human neuroblastomas.     

The ubiquitin-proteasome pathway plays an essential role in proteolysis during Trypanosoma cruzi remodeling

Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage. The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels. About 30 proteins in a range of 25-110 kDa were detected in the purified T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T. cruzi 20S core specifically recognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling. Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.     

Encystation process of Giardia lamblia: morphological and regulatory aspects

One important step in the life cycle of the pathogenic protozoan Giardia lamblia is the transformation of the proliferative form, the trophozoite, into the non-proliferative cyst. This process, known as encystation, can be triggered in vitro. Morphological analysis showed that during trophozoite-cyst transformation, major changes take place: modification of the protozoan shape, internalization of the flagella, fragmentation of the adhesive disk, and appearance of encystation vesicles (ESVs), which later on fuse with the plasma membrane forming the cell wall. Sites of attachment of these vesicles to the inner portion of the protozoan plasma membrane were observed 6 h after the beginning of the encystation process. These sites were only visible when we used high-resolution scanning electron microscopy to study Giardia surface. In order to analyze the involvement of protein kinases and phosphatases on the encystation process, inhibitors of these enzymes were added to the culture medium, and their effect on the differentiation process was determined using light, immunofluorescence, and electron microscopy. Significant inhibition was observed with LY294002, an inhibitor of PI3 kinase; genistein, an inhibitor of tyrosine kinase; and staurosporine, at concentrations, which inhibit protein kinase C. Okadaic acid, an inhibitor or protein phosphatase, and wortmannin, an inhibitor of PI3K, did not interfere with the encystation process. However, they induced the appearance of large and pleomorphic forms where several nuclei and disorganization of the peripheral vesicles were observed.     

Nitric oxide induces neutral ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures

The neutral ceramidase is a key enzyme in the regulation of cellular ceramide levels. Previously we have reported that stimulation of rat renal mesangial cells with nitric oxide (NO) donors leads to an inhibition of neutral ceramidase activity which is due to increased degradation of the enzyme. This and the concomitant activation of the sphingomyelinase results in an amplification of ceramide levels. Here, we show that the NO-triggered degradation of neutral ceramidase involves activation of the ubiquitin/proteasome complex. The specific proteasome inhibitor lactacystin completely reverses the NO-induced degradation of ceramidase protein and neutral ceramidase activity. As a consequence, the cellular amount of ceramide, which drastically increases by NO stimulation, is reduced in the presence of lactacystin. Furthermore, ubiquitinated neutral ceramidase accumulates after NO stimulation. In summary, our data clearly show that the ubiquitin/proteasome complex is an important determinant of neutral ceramidase activity and thereby regulates the availability of ceramide.     

Expression of the ubiquitin proteasome system in neonatal rat gonocytes and spermatogonia: role in gonocyte differentiation

The ubiquitin proteasome system (UPS) consists of a cascade of enzymatic reactions leading to the ubiquitination of proteins, with consequent degradation or altered functions of the proteins. Alterations in UPS genes have been associated with male infertility, suggesting the role of UPS in spermatogenesis. In the present study, we questioned whether UPS is involved in extensive remodeling and functional changes occurring during the differentiation of neonatal testicular gonocytes to spermatogonia, a step critical for the establishment of the spermatogonial stem cell population. We found that addition of the proteasome inhibitor lactacystin to isolated gonocytes inhibited their retinoic acid-induced differentiation in a dose-dependent manner, blocking the induction of the spermatogonial gene markers Stra8 and Dazl. We then compared the UPS gene expression profiles of Postnatal Day (PND) 3 gonocytes and PND8 spermatogonia, using gene expression arrays and quantitative real-time PCR analyses. We identified 205 UPS genes, including 91 genes expressed at relatively high levels. From those, 28 genes were differentially expressed between gonocytes and spermatogonia. While ubiquitin-activating enzymes and ligases showed higher expression in gonocytes, most ubiquitin conjugating and deubiquitinating enzymes were expressed at higher levels in spermatogonia. Concomitant with the induction of spermatogonial gene markers, retinoic acid altered the expression of many UPS genes, suggesting that the UPS is remodeled during gonocyte differentiation. In conclusion, these studies identified novel ubiquitin-related genes in gonocytes and spermatogonia and revealed that proteasome function is involved in gonocyte differentiation. Considering the multiple roles of the UPS, it will be important to determine which UPS genes direct substrates to the proteasome and which are involved in proteasome-independent functions in gonocytes and to identify their target proteins.     

Characterization and role of protozoan parasite proteasomes

The proteasome, a large non-lysosomal multi-subunit protease complex, is ubiquitous in eukaryotic cells. In protozoan parasites, the proteasome is involved in cell differentiation and replication, and could therefore be a promising therapeutic target. This article reviews the present knowledge of proteasomes in protozoan parasites of medical importance such as Giardia, Entamoeba, Leishmania, Trypanosoma, Plasmodium and Toxoplasma spp.     

Atg3-mediated lipidation of Atg8 is involved in encystation of Acanthamoeba

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.     

Cell cycle events mediate lactacystin‐induced apoptotic death of neuronal PC12 cells

Dysfunction of the UPS (ubiquitin—proteasome system) has been implicated in dopaminergic neuronal death in PD (Parkinson's disease). Recent studies suggest that unregulated cell cycle events play a key role in neuronal death. In this study, the effects of UPS dysfunction on cell cycle events in neuronal differentiated PC12 cells were analysed using a specific inhibitor of proteasome, lactacystin. Lactacystin induced apoptosis, G₂/M cell cycle arrest and sustained the phosphorylation of the pRB (retinoblastoma protein), the key molecular process of G₁/S transition, in neuronal PC12 cells. Furthermore, inhibition of cell cycle progression protected against lactacystin‐induced cell apoptosis. Finally, we determined that lactacystin activated the ERK signalling pathway. Inhibition of ERK1/2 activation by MEK‐1 inhibitor PD98059 decreased cell cycle aberrant and prevented apoptosis induced by lactacystin. These results indicate that aberrant cell cycle events contribute to apoptotic death induced by UPS dysfunction.     

Proteasome inhibition by lactacystin in primary neuronal cells induces both potentially neuroprotective and pro-apoptotic transcriptional responses: a microarray analysis

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat-shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up-regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor-mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up-regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor-mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor-mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up-regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro-apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.     

Effects of proteasome inhibitor (lactacystin) and cysteine protease inhibitor (E-64-d) on processes of mitosis in Xenopus embryonic cells

At least two different protease pathways have been implicated in the degradation that is required to control the eukaryotic cell cycle; these two pathways center on the activities of ubiquitin/proteasome and cysteine protease. The proteasome inhibitors, lactacystin and AcLLnL and the cysteine protease inhibitor E-64-d were tested for their ability to inhibit the cell cycles of Xenopus embryos. Lactacystin, AcLLnL and E-64-d all caused the complete arrest of the cell cycle. To define the specific cell cycle processes that were affected by the two inhibitors, we performed a cytological analysis. Inhibition of the cell cycle by lactacystin and E-64-d occurred during prophase and metaphase. The number of cells that arrested in prophase was 1.4-times higher in the E-64-d-treated group than in the control group and the number of arrested cells in the lactacystin-treated group was 1.4-times higher than in the E-64-d-treated group. The number of cells that arrested in metaphase was 3-to-4-times higher in the E-64-d and lactacystin groups than in the control group. These results indicate that both cysteine protease(s) and proteasomes are involved in the prophase and metaphase stages of cell division.     

Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani

Pteridine reductase 1 (PTR1) is an essential enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. The present work is focused on the degradation of PTR1 during growth phase in Leishmania donovani. Western blot analysis with PTR1-GFP transfected promastigotes revealed that PTR1 protein was degraded in the stationary phase of growth at the time when the parasites were undergoing metacyclogenesis. Fluorescence microscopy revealed cytoplasmic localization of GFP tagged protein extending to the flagellum in these stationary phase promastigotes, implying that degradation of the protein was not by the usual multivesicular tubule lysosome (MVT) pathway. A probable destruction box of nine amino acids Q63ADLSNVAK71 and possible lysine residue K156 was identified in L. donovani PTR1 to be the site for ubiquitin conjugation. This suggests that PTR1 degradation during the stationary phase of growth is mediated by the proteasome. This leads to lower levels of H4-biopterin, which favors metacyclogenesis, and subsequently results in a highly infective stage of the parasite. Therefore, this finding has importance to identify new target molecule like the proteasome for therapeutic intervention.     

Effect of proteasome inhibition on cellular oxidative damage, antioxidant defences and nitric oxide production

The ubiquitin/proteasome pathway plays an essential role in protein turnover in vivo, and contributes to removal of oxidatively damaged proteins. We examined the effects of proteasome inhibition on viability, oxidative damage and antioxidant defences in NT-2 and SK-N-MC cell lines. The selective proteasome inhibitor, lactacystin (1 microM) caused little loss of viability, but led to significant increases in levels of oxidative protein damage (measured as protein carbonyls), ubiquitinated proteins, lipid peroxidation and 3-nitrotyrosine, a biomarker of the attack of reactive nitrogen species (such as peroxynitrite, ONOO(-)) upon proteins. Higher levels (25 microM) of lactacystin did not further increase the levels of carbonyls, lipid peroxidation, 3-nitrotyrosine, or ubiquitinated proteins, but produced increases in the levels of 8-hydroxyguanine (a biomarker of oxidative DNA damage) and falls in levels of GSH. Lactacystin (25 microM) caused loss of viability, apparently by apoptosis, and also increased production of nitric oxide (NO.) (measured as levels of NO2- plus NO3-) by the cells; this was inhibited by N-nitro-L-arginine methyl ester (L-NAME), which also decreased cell death induced by 25 microM lactacystin and decreased levels of 3-nitrotyrosine. The NO. production appeared to involve nNOS; iNOS or eNOS were not detectable in either cell type. Another proteasome inhibitor, epoxomicin, had similar effects.