Clasto-lactacystin Beta-lactone Research Articles

Abstract 517: Ubiquitylation of G Protein Coupled Receptor Kinase 4 (GRK4) Regulates its Localization and Abundance

The dopamine-1 receptor (D 1 R) is responsible for regulating up to 60% of natriuresis in the kidney under sodium loaded conditions. G protein coupled receptor kinase type 4 (GRK4) phosphorylates the D 1 R and reduces its membrane expression, but little is known about regulation of GRK4. We hypothesized that GRK4 is targeted for elimination through ubiquitylation. Using D 1 R and GRK4γ stably transfected human embryonic kidney cells (HEK293), we demonstrated GRK4 and ubiquitin coimmunoprecipitation. Addition of clasto-lactacystin beta-lactone (CLBL, an inhibitor of the 26S proteasome) increased GRK4 expression levels (western blot) using two different GRK4 fusion protein constructs. The addition of CLBL (10 μmol/L, 24 hrs) increased the expression of both a tandem affinity tagged (tap-tag) GRK4 fusion construct (2.65±0.19 fold over vehicle (VEH): VEH 4,010±404 RFU; CLBL 10,626±744 RFU, P<0.05, N=3) as well as a mCherry GRK4 fusion construct (6.98±0.50 fold increase over VEH: VEH 5,044±3229 RFU; CLBL 35,210±2547 RFU, P<0.05, N=3). The ubiquitin binding sites (AA 216,217; MYACK Ub K Ub LQKK) are located near the nuclear localization signal (AA 219-228; QKKRIKKRK) in GRK4. We therefore examined the subcellular localization of GRK4 after CLBL treatment. Nuclear accumulation of the GRK4-mCherry fusion protein fluorescence decreased markedly while cytoplasmic GRK4-mCherry fluorescence increased (nuclear to cytoplasmic GRK4 ratio: VEH 1.18±0.09 RFU; CLBL 0.78±0.03 RFU, P<0.01, N=12). In summary, GRK4 expression levels are negatively regulated by the ubiquitin proteasome system and ubiquitylation of GRK4 prevents nuclear import. We hypothesize that ubiquitylation is sterically hindering the nuclear localization sequence in GRK4 from interacting with the nuclear import machinery of the cell. Through manipulation of GRK4 ubiquitylation, one may be able to separate nuclear vs. cytoplasmic activity of GRK4 and reverse the negative impact overactive GRK4 has on dopaminergic signaling.

Two inhibitors of the ubiquitin proteasome system enhance long-term memory formation upon olfactory conditioning in the honeybee (Apis mellifera)

In honeybees (Apis mellifera), the proteasome inhibitor Z-Leu-Leu-Leu-CHO (MG132) enhances long-term memory (LTM) formation. Studies in vertebrates using different inhibitors of the proteasome demonstrate the opposite, namely an inhibition of memory formation. The reason for this contradiction remains unclear. MG132 is an inhibitor of the proteasome, but also blocks other proteases. Accordingly, one possible explanation might be that other proteases affected by MG132 are responsible for the enhancement of LTM formation. We test this hypothesis by comparing the effect of MG132 and the more specific proteasome inhibitor clasto-lactacystin beta-lactone (β-lactone). We show that these two inhibitors block the activity of the proteasome in honeybee brains to a similar extent, do not affect the animals' survival but do enhance LTM retention upon olfactory conditioning. Thus, the enhancement of LTM formation is not due to MG132-specific side effects, but to inhibition of a protease targeted by MG132 and β-lactone, i.e. the proteasome.

Studying protein degradation pathways in vivo using a cranial window-based approach

Understanding how specific proteins are degraded by neurons in living animals is a fundamental question with relevance to many neurodegenerative diseases. Dysfunction in the ubiquitin–proteasome system (UPS) specifically has been implicated in several important neurodegenerative diseases including Alzheimer’s Disease, Parkinson’s Disease, and amyotrophic lateral sclerosis. Research in this area has been limited by the fact that many inhibitors of the UPS given systemically do not cross the blood–brain barrier (BBB) in appreciable levels. This limits the ability to easily test in vivo specific hypotheses generated in reduced systems, like brain slice or dissociated cell culture, about whether the UPS may degrade a particular protein of interest. Although several techniques including intracerebral application via direct syringe injection, catheter-pump systems and drug-eluting beads are available to introduce BBB-impermeant drugs into brain they each have certain limitations and new approaches could provide further insights into this problem. In order to test the role of the UPS in protein degradation in vivo we have developed a strategy to treat mouse cortex with the UPS inhibitor clasto-lactacystin beta-lactone (CLBL) via a “cranial window” and recover the treated tissue for immunoblot analysis. This approach can be used in several different cranial window configurations including single window and double hemi-window arrangements that are tailored for different applications. We have also developed two different strategies for recovering treated cortical tissue including a vibratome/laser capture microscopy (LCM)-based and a vibratome only-based approach, each with its own specific advantages. We have documented UPS inhibition >600μm deep into the cortex with this strategy. This set of techniques in the living mammalian brain is complementary to previously developed approaches and extends the repertoire of tools that can be used to the study protein degradation pathways relevant to neurodegenerative disease.

Turnover of Mitochondrial Steroidogenic Acute Regulatory (StAR) Protein by Lon Protease: The Unexpected Effect of Proteasome Inhibitors

Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein promoting transfer of cholesterol into steroid making mitochondria in specialized cells of the adrenal cortex and gonads. Our previous work has demonstrated that StAR is rapidly degraded upon import into the mitochondrial matrix. To identify the protease(s) responsible for this rapid turnover, murine StAR was expressed in wild-type Escherichia coli or in mutant strains lacking one of the four ATP-dependent proteolytic systems, three of which are conserved in mammalian mitochondria-ClpP, FtsH, and Lon. StAR was rapidly degraded in wild-type bacteria and stabilized only in lon (-)mutants; in such cells, StAR turnover was fully restored upon coexpression of human mitochondrial Lon. In mammalian cells, the rate of StAR turnover was proportional to the cell content of Lon protease after expression of a Lon-targeted small interfering RNA, or overexpression of the protein. In vitro assays using purified proteins showed that Lon-mediated degradation of StAR was ATP-dependent and blocked by the proteasome inhibitors MG132 (IC(50) = 20 microm) and clasto-lactacystin beta-lactone (cLbetaL, IC(50) = 3 microm); by contrast, epoxomicin, representing a different class of proteasome inhibitors, had no effect. Such inhibition is consistent with results in cultured rat ovarian granulosa cells demonstrating that degradation of StAR in the mitochondrial matrix is blocked by MG132 and cLbetaL but not by epoxomicin. Both inhibitors also blocked Lon-mediated cleavage of the model substrate fluorescein isothiocyanate-casein. Taken together, our former studies and the present results suggest that Lon is the primary ATP-dependent protease responsible for StAR turnover in mitochondria of steroidogenic cells.

Proteolytic Mechanisms Underlying Mitochondrial Degradation in the Ocular Lens

To remove light-scattering structures from the visual axis, all intracellular organelles are eliminated from cells in the center of the developing ocular lens. Organelle degradation is accompanied by an increase in VEIDase (caspase-6-like) activity, but data from caspase-null mice suggest that the lens VEIDase is not caspase-6. The goal of the present work was to identify the lens VEIDase and determine whether it plays a role in organelle breakdown. The approximate molecular mass of the lens VEIDase was determined by size-exclusion chromatography. Three proteasome inhibitors (NLVS, MG132, and clasto-lactacystin beta-lactone) were tested for their ability to inhibit lens VEIDase activity. Lens lysates were immunodepleted of proteasomes using an antibody against the 20S proteasome. To inhibit the ubiquitin-proteasome pathway (UPP) in vivo, lactacystin was injected into the vitreous humor of the developing chicken eye. The effect of lactacystin on mitochondrial degradation was assessed by examining the disappearance of succinate-ubiquinone oxidoreductase, an integral protein of the inner mitochondrial membrane. The lens VEIDase eluted at approximately 700 kDa from a size-exclusion column and was inhibited by the proteasome inhibitors NLVS, MG132, and clasto-lactacystin beta-lactone. In vivo, the trypsin-like activity of the proteasome was reduced by 60% to 70% after lactacystin injection. Proteasome inhibition was associated with the accumulation of ubiquitinated proteins and reversible opacification of the lens cortex. In lactacystin-injected eyes, the programmed degradation of succinate-ubiquinone oxidoreductase was inhibited in the central lens fiber cells. These data suggest that lens VEIDase activity is attributable to the proteasome and that the UPP may function in the removal of organelle components during lens fiber cell differentiation.

α,β-Unsaturated β-Silyl Imide Substrates for Catalytic, Enantioselective Conjugate Additions: A Total Synthesis of (+)-Lactacystin and the Discovery of a New Proteasome Inhibitor

Chiral (salen)Al mu-oxo dimer 1 catalyzes the highly enantioselective conjugate addition of carbon-centered nucleophiles to alpha,beta-unsaturated silyl imides. Allyldimethylsilane-substituted imide 4 was identified as an optimal substrate, undergoing addition reactions with a variety of nitrile nucleophiles in high yield and enantiomeric excess. The silicon-containing products are synthetically useful chiral building blocks, as demonstrated by their application to an enantioselective total synthesis of the potent proteasome inhibitor (+)-lactacystin (2). Elaboration of lactam 5a to the natural product was effected in 12 steps and in 11% overall yield and proceeded through an unusual spiro beta-lactone intermediate (11). This compound was found to inhibit the chymotrypsin-like site of the 26S proteasome at similar levels to known inhibitor clasto-lactacystin beta-lactone (omuralide).

Enantioselective total syntheses of (−)-clasto-lactacystin β-lactone and 7-epi-(−)-clasto-lactacystin β-lactone

An alkylidene carbene 1,5-CH insertion has been used as a key step in an efficient enantioselective total synthesis of (-)-clasto-lactacystin beta-lactone, and its C7-epimer. An additional noteworthy feature of the synthesis is the use of a novel oxidative deprotection procedure, utilizing DMDO, for the conversion of a late-stage benzylidene acetal into a primary alcohol and a secondary benzoate ester.

Utility of the Ammonia‐Free Birch Reduction of Electron‐Deficient Pyrroles: Total Synthesis of the 20S Proteasome Inhibitor, clasto‐Lactacystin β‐Lactone

A new synthesis of the 20S proteasome inhibitor clasto-lactacystin beta-lactone is described. Our route to this important natural product involves the partial reduction of an electron deficient pyrrole as a key step. By judicious choice of enolate counterion, we were able to exert complete control over the stereoselectivity of the reduction/aldol reaction. Early attempts to complete the synthesis by using a C-4 methyl substituted pyrrole are described in full, together with our attempts to promote regioselective elimination of a tertiary alcohol. The lessons learnt from this first approach led us to develop another, and ultimately successful, route that introduced the C-4 methyl group at a late stage in the synthesis. Our successful route is then described and this contains several highly stereoselective steps including a cis-dihydroxylation and an enolate methylation. The final synthesis proceeds in just 13 steps and in 15 % overall yield making it an extremely efficient route to this valuable compound.

A proteasomal stress response: pre‐treatment with proteasome inhibitors increases proteasome activity and reduces neuronal vulnerability to oxidative injury

We report here that exposure to low concentrations of proteasome inhibitors (e.g. 10-100 nm MG-132, 0.1-3 nm epoxomicin or 10-30 nm clasto-lactacystin beta-lactone) resulted in an enhancement, rather than an inhibition, of proteasome activity in cultured neocortical neurons. Size-fractionation chromatography confirmed that the enhanced peptide cleavage activity was associated with proteasome-sized complexes. This sub toxic exposure reduced neuronal death caused by subsequent exposure to oxidative stress (100-200 microm H(2)O(2) for 30 min, 24-h exposure to 100 microm paraquat or 7.5 microm menadione), but did not alter vulnerability to excitotoxicity (5-min exposure to 30-100 microm NMDA or 24 exposure to 12 microm NMDA). Sub toxic proteasome inhibitor exposure caused an increase in levels of proteasome core subunit proteins and mRNAs, but not in levels of potentially cytoprotective heat shock proteins (hsp70, hsp90 and hsp40). The neuroprotective effects of proteasome inhibitor pre-treatment were blocked by coapplication of proteasome inhibitors during the oxidative insult. These findings support a model in which sublethal proteasome inhibition induces neurons to increase proteasome activity and promotes resistance to oxidative injury and suggests that enhancement of proteasome activity is a potential therapeutic target for diseases in which oxidative stress has been implicated.

Intracellular degradation of beta4 integrin in lethal junctional epidermolysis bullosa with pyloric atresia

Junctional epidermolysis bullosa with pyloric atresia (PA-JEB) is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous blistering and gastric outlet obstruction. The disease, which is caused by mutations in the alpha6beta4 integrin genes (ITGA6, ITGB4), is usually lethal. However, nonlethal cases have also been reported. Mutation database analysis has suggested that premature termination codons predominantly result in lethal forms while missense mutations frequently associate with nonlethal variants. Nevertheless, it is becoming more and more evident that the disease phenotype is also influenced by the position of the mutation in the protein functional domains. To investigate the molecular basis of a novel PA-JEB lethal case. Reverse transcriptase-polymerase chain reaction and direct sequencing-based mutation screening were performed. Mutation consequences in the patient's keratinocytes were then analysed by Northern blot and immunoprecipitation. Immunofluorescence analysis of cultured keratinocytes treated with protein intracellular degradation pathway inhibitors was also carried out. The phenotype was caused by the presence, in the homozygous state, of a novel 33 bp in-frame deletion (nucleotides 175-207) in the ITGB4 coding sequence. Despite the normal steady-state level of integrin beta4 mRNA, the mutation, designated DeltaR59-A69, results in the almost complete absence of alpha6beta4 integrin in the patient's skin and cultured keratinocytes. Exposure of the patient's keratinocytes to the proteasomal inhibitor clasto-lactacystin beta-lactone increased the expression of the mutated beta4 integrin chains indicating that the proteasome complex is involved in the degradation of the internally deleted beta4 polypeptides. We report for the first time a homozygous in-frame deletion in the ITGB4 gene. Our results suggest that the deletion of amino acids R59-A69 interferes with the biosynthetic folding of the protein, leading to a rapid degradation of the mutated beta4 chains. These findings provide new insight into the pathogenic effects of mutations affecting different functional domains of the beta4 integrin molecule and their prognostic implications in PA-JEB patients.

Host cell gene expression during human immunodeficiency virus type 1 latency and reactivation and effects of targeting genes that are differentially expressed in viral latency.

The existence of reservoirs of cells latently infected with human immunodeficiency virus (HIV) is a major obstacle to the elimination of HIV infection. We studied the changes in cellular gene expression that accompany the reactivation and completion of the lytic viral cycle in cell lines chronically infected with HIV-1. We found that several genes exhibited altered expression in the chronically infected cells compared to the uninfected parental cells prior to induction into lytic replication. A number of gene classes showed increased expression in the chronically infected cells, notably including genes encoding proteasomes, histone deacetylases, and many transcription factors. Following induction of the lytic replication cycle, we observed ordered, time-dependent changes in the cellular gene expression pattern. Approximately 1,740 genes, many of which fall into 385 known pathways, were differentially expressed (P < 0.001), indicating that completion of the HIV replication cycle is associated with distinct, temporally ordered changes in host cell gene expression. Maximum changes were observed in the early and intermediate phases of the lytic replication cycle. Since the changes in gene expression in chronically infected cells suggested that cells latently infected with HIV have a different gene expression profile than corresponding uninfected cells, we studied the expression profiles of three different chronically infected cell lines to determine whether they showed similar changes in common cellular genes and pathways. Thirty-two genes showed significant differential expression in all cell lines studied compared to their uninfected parental cell lines. Notable among them were cdc42 and lyn, which were downregulated and are required for HIV Nef binding and viral replication. Other genes previously unrelated to HIV latency or pathogenesis were also differentially expressed. To determine the effects of targeting products of the genes that were differentially expressed in latently infected cells, we treated the latently infected cells with a proteasome inhibitor, clastolactacystin-beta-lactone (CLBL), and an Egr1 activator, resveratrol. We found that treatment with CLBL and resveratrol stimulated lytic viral replication, suggesting that treatment of cells with agents that target cellular genes differentially expressed in latently infected cells can stimulate lytic replication. These findings may offer new insights into the interaction of the latently infected host cell and HIV and suggest therapeutic approaches for inhibiting HIV infection and for manipulating cells latently infected with HIV so as to trigger lytic replication.

Proteasomal activity in mammalian spermatozoa.

The proteasome is a multicatalytic cellular complex, which possess three different enzymatic activities, trypsin-like, chymotrypsin-like, and peptidylglutamyl peptidase. Its function is to remove abnormal or aged proteins. Recently, it has been suggested the participation of the sperm proteasome during mammalian fertilization. In this study, we present evidence that indicates that sperm extracts from several mammalian species, including hamster, mice, rats, bovine, rabbits, and humans all possess proteasome activity. We characterized the three specific activities of the proteasome using specific synthetic substrates and specific proteasome inhibitors. The results indicates that the highest specific activity detected was in mouse sperm toward the trypsin substrates and it was 1,114% of the activity of human sperm toward the chymotrypsin substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC, which was considered as 100%). In all cases, the lowest activity was toward substrates for the peptidylglutamyl peptidase hydrolyzing activity, and it was lowest for rabbit sperm (1.7% of the activity of human sperm toward the chymotrypsin substrate SLLVY-AMC). In addition, specific proteasome inhibitors were able to block all proteasome activities almost 100%, with the exception of clasto-Lactacystin beta-lactone upon rat sperm. All sperm extracts tested evidenced bands of about 29-32 kDa by Western blots using a monoclonal antibody against proteasome subunits alpha 1, 2, 3, 5, 6, and 7. In conclusion, sperm from several mammals possess enzymatic activities that correspond to the proteasome. The proteasome from the different species hold similar but distinctive enzymatic characteristics.

Extracellular localization of proteasomes in human sperm

The proteasome, a multienzymatic protease complex is present in human sperm. Here we present evidence indicating that the proteasome has an extracellular localization, on the plasma membrane of the sperm head. Motile sperm (>90%) in PBS were incubated with the proteasome inhibitors clasto-lactacystin beta-lactone or epoxomicin. Then, the substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC) was added and the enzyme activity evaluated in a spectrofluorometer. Other aliquots were resuspended in Tyrode's medium and incubated at different concentrations for various times with or without inhibitors in the presence of 0.4% azocasein. Hydrolysis of azocasein was evaluated at 440 nm. In addition, sperm membrane proteins were obtained incubating the sperm with Triton X-114 or with 0.5 M KCl plus Triton X-100 and removing insoluble material by centrifugation at 5,000g for 40 min. Proteasomal activity was evaluated with SLLVY-AMC and its presence corroborated by Western blotting. Formaldehyde fixed, unpermeabilized sperm were incubated with anti-proteasome monoclonal antibodies and evaluated using indirect immunofluorescence. The effect of proteasome inhibitors upon the progesterone-induced acrosome reaction was also evaluated. Results indicated that (a) whole, intact sperm were able to hydrolyze the proteasome substrates SLLVY-AMC and azocasein; this activity was inhibited by proteasome inhibitors; (b) proteasomal activity was detected in soluble sperm membrane protein preparations and Western blotting revealed the presence of the proteasome in these fractions; (c) indirect immunofluorescence revealed staining of the head region, particularly of the post acrosomal region; and (d) the proteasome plays an important role during the acrosome reaction.

Participation of the sperm proteasome in human fertilization.

Fertilization in mammals comprises the sequential interactions of the sperm with the cumulus oophorus, zona pellucida, and oocyte plasma membrane. Here we investigate proteasome activity in human sperm and its possible involvement during the fertilization process. Proteasome activity was measured in intact sperm and in sperm extracts using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC, in the presence or absence of the specific proteasome inhibitor, clasto-lactacystin beta-lactone. The participation of the proteasome was evaluated during (i) sperm-zona binding using the hemizona assay; (ii) zona pellucida-induced acrosome reactions with a pulse and chase design; (iii) progesterone-induced acrosome reactions incubating overnight capacitated sperm with progesterone; and (iv) progesterone-induced Ca(2+) influx using fura-2AM. Intact sperm and sperm extracts possessed proteasome activity, which was susceptible to inhibition by clasto-lactacystin beta-lactone. Sperm-zona binding was not inhibited by clasto-lactacystin beta-lactone. However, both zona pellucida- and progesterone-induced acrosome reactions were inhibited by clasto-lactacystin beta-lactone. The proteasome inhibitor also blocked the sustained phase of the Ca(2+) influx provoked by progesterone but not the peak. The human sperm proteasome is involved in the exocytosis of the acrosome, perhaps in events upstream of the plateau phase of the Ca(2+) influx.

BcR-induced Apoptosis Involves Differential Regulation of C16 and C24-Ceramide Formation and Sphingolipid-dependent Activation of the Proteasome

In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C(16)- ceramide predominated, was observed 6 h after BcR triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BcR triggering. The formation of these very long-chain ceramide species, in which C(24)-ceramide predominated, required the activation of effector caspases. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which long-chain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta-lactone, an irreversible inhibitor of the proteasome, prevented BcR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BcR cross-linking-induced activation of caspases. The formation of C(16)-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C(24)-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and important insights into the significance of specific ceramide species in defined stages of apoptosis.

Abnormally high expression of proteasome activator-gamma in thyroid neoplasm.

PA28-gamma is the activator of 20S proteasome, the ATP-dependent proteolytic system that plays an important role in cell cycle progression in various cell types. In this paper, we show the abnormally high expression of PA28-gamma in various thyroid neoplasms. Thyroid samples were obtained from patients with normal thyroid (4 cases) and with the following diseases: papillary adenocarcinoma (13 cases), multinodular goiter (4 cases), and anaplastic carcinoma (1 case). PA28-gamma expression was estimated by immunohistochemical staining and Western blotting. In all of the papillary adenocarcinoma samples, PA28-gamma was abnormally overexpressed, especially in cancer cells existing at the peripheral region of the cancer mass or in cancer cells invading the capsular region surrounding the cancer mass. In cancer cells of these areas, PA28-gamma was predominantly distributed in nucleus rather than in the cytoplasm of cancer cells. On the other hand, no obvious PA28-gamma expression was observed in the adjacent normal thyroid follicular cells. In multinodular goiter, the expression of PA28-gamma was relatively low compared with papillary adenocarcinoma. In anaplastic carcinoma, PA28-gamma was expressed at the highest level, especially in poorly differentiated regions such as squamous metaplasia of anaplastic cancer tissue. Therefore, the PA28-gamma expression seems to be restricted to thyroid cancer cells, especially in the region where the growth rate of cancer cells is accelerated. This result is further confirmed by the fact that C2, alpha-subunit of 20S proteasome, and proliferating cell nuclear antigen are similarly overexpressed in this region. Thus, PA28-gamma might be involved in the regulatory system for the cell cycle. Moreover, the growth of thyroid cancer cell lines was affected by the proteasome inhibitor, clasto-lactacystin beta-lactone. These results demonstrate that PA28-gamma is overexpressed in thyroid cancer, especially in its growth-accelerated cells.

Effect of proteasome inhibitors on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens.

The effect of three proteasome inhibitors, lactacystin, clasto-lactacystin beta-lactone, and MG-132, on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens was examined. All of these drugs blocked E. histolytica growth in a concentration-dependent manner; lactacystin was most potent for the inhibition and MG-132 showed the inhibitory effect only at higher concentrations. E. invadens was more resistant to these drugs than E. histolytica. Encystation of E. invadens was also inhibited and was more sensitive to the drugs than was growth. Beta-lactone was the most potent encystation inhibitor. The inhibitory effect of lactacystin and the beta-lactone on encystation was slightly and little abrogated by the removal of the drug, respectively. Multinucleation occurred in E. histolytica trophozoites treated with these drugs, being most marked with lactacystin. In contrast, no multinucleation was observed in E. invadens treated with the drugs. Electron microscopy revealed that the treatment of E. histolytica trophozoites with lactacystin led to an increase in the number of cells with many glycogen granules in the cytoplasm. Lactacystin, beta-lactone and MG-132 had no or little effect on the excystation and metacystic development of E. invadens. These results suggest that proteasome function plays an important role for Entamoeba growth and encystation, but has no obvious effect on excystation or metacystic development.

Ubiquitination of soluble and membrane-bound tyrosine hydroxylase and degradation of the soluble form.

Tyrosine hydroxylase (TH) demonstrates by two-dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH (hTH1) and as a membrane-bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type (wt)-hTH1 was found to be a substrate for the ubiquitin (Ub) conjugating enzyme system in a reconstituted in vitro system. When wt-hTH1 was expressed in a coupled transcription-translation TnT(R)-T7 reticulolysate system 35S-labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high-molecular-mass forms increased by time and was enhanced in the presence of Ub and clasto-lactacystin beta-lactone. In pulse-chase experiments the amount of full-length hTH1 decreased by first-order kinetics with a half-time of 7.4 h and 2.1 h in the absence and presence of an ATP-regenerating system, respectively. The ATP-dependent degradation was inhibited by clasto-lactacystin beta-lactone. Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated. Additional Ub-conjugates of this membrane, detected by Western blot analysis, have not yet been identified.

The Low Lysine Content of Ricin A Chain Reduces the Risk of Proteolytic Degradation after Translocation from the Endoplasmic Reticulum to the Cytosol

Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells by endocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmic reticulum (ER) membrane. To achieve this export, such toxins exploit the ER-associated protein degradation (ERAD) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates. Toxins that translocate from the ER have an unusually low lysine content. Since lysyl residues are potential ubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitination and subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residues within RTA were changed to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitin attachment. However, when four additional lysines were introduced into RTA in a way that did not compromise the activity, structure, or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasome inhibitor clasto-lactacystin beta-lactone increased the cytotoxicity of the lysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysyl residues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased the cytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomal inhibition. Our data support the hypothesis that the evolution of a low lysine content is a degradation-avoidance strategy for toxins that retrotranslocate from the ER.

Contrasting requirements for ubiquitylation during Fc receptor-mediated endocytosis and phagocytosis.

Fc receptors on leukocytes mediate internalization of antibody-containing complexes. Soluble immune complexes are taken up by endocytosis, while large antibody-opsonized particles are internalized by phagocytosis. We investigated the role of ubiquitylation in internalization of the human FcgammaRIIA receptor by endocytosis and phagocytosis. A fusion of FcgammaRIIA to green fluorescent protein (GFP) was expressed in ts20 cells, which bear a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme. Uptake of soluble IgG complexes mediated by FcgammaRIIA-GFP was blocked by incubation at the restrictive temperature, indicating that endocytosis requires ubiquitylation. In contrast, phagocytosis and phagosomal maturation were largely unaffected when ubiquitylation was impaired. FcgammaRIIA-GFP was ubiquitylated in response to receptor cross-linking. Elimination of the lysine residues present in the cytoplasmic domain of FcgammaRIIA impaired endocytosis, but not phagocytosis. The proteasomal inhibitor clasto-lactacystin beta-lactone strongly inhibited endocytosis, but did not affect phagocytosis. These studies demonstrate a role for ubiquitylation in the endocytosis of immune receptors, and reveal fundamental differences in the mechanisms underlying internalization of a single receptor depending on the size or multiplicity of the ligand complex.