Mitochondrial calpain 10 is degraded by Lon protease after oxidant injury

Calpain 10 is ubiquitously expressed and is one of four mitochondrial matrix proteases. We determined that over-expression or knock-down of mitochondrial calpain 10 results in cell death, demonstrating that mitochondrial calpain 10 is required for viability. Thus, we studied calpain 10 degradation in isolated mitochondrial matrix, mitochondria and in renal proximal tubular cells (RPTC) under control and toxic conditions. Using isolated renal cortical mitochondria and mitochondrial matrix, calpain 10 underwent rapid degradation at 37°C that was blocked with Lon inhibitors but not by calpain or proteasome inhibitors. While exogenous Ca(2+) addition, Ca(2+) chelation or exogenous ATP addition had no effect on calpain 10 degradation, the oxidants tert-butyl hydroperoxide (TBHP) or H(2)O(2) increased the rate of degradation. Using RPTC, mitochondrial and cytosolic calpain 10 increased in the presence of MG132 (Lon/proteasome inhibitor) but only cytosolic calpain 10 increased in the presence of epoxomicin (proteasome inhibitor). Furthermore, TBHP and H(2)O(2) oxidized mitochondrial calpain 10, decreased mitochondrial, but not cytosolic calpain 10, and pretreatment with MG132 blocked TBHP-induced degradation of calpain 10. In summary, mitochondrial calpain 10 is selectively degraded by Lon protease under basal conditions and is enhanced under and oxidizing conditions, while cytosolic calpain 10 is degraded by the proteasome.     

An internal segment (residues 58-119) of the hepatitis B virus X protein is sufficient to activate MAP kinase pathways in mouse liver

StAR, a protein synthesized in the cytoplasm and subsequently imported into mitochondria, regulates the rate-determining step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The active form of StAR is the 37 kDa pre-protein, which has a short half-life. To determine whether proteasomes participate in the turnover of StAR, we incubated primary cultures of preovulatory rat granulosa cells and immortalized human granulosa cells in the presence of MG132, a specific inhibitor to proteasome catalysis. This treatment caused accumulation of StAR in unstimulated cells. Moreover, incubation of the cells with MG132 in the presence of forskolin (FK), luteinizing hormone/chorionic gonadotropin or follicular stimulating hormone augmented the accumulation of both the 37 kDa cytoplasmic protein and the 30 kDa mature mitochondrial protein, compared to cells incubated with FK or the gonadotropic hormones alone. Concomitantly, progesterone production was enhanced. In contrast no elevation in the 37 kDa StAR intracellular levels or progesterone production was observed following incubation of the cells with the cysteine protease inhibitor E-64. The increase of the 37 kDa StAR protein was evident after 15 min and 30 min of incubation with MG132 (143% and 187% of control values, respectively) with no significant elevation of the 30 kDa protein. Accumulation of the intermediate mitochondrial 32 kDa protein was evident after 1-2 h and the accumulation of the 30 kDa protein was evident only after 4 h of incubation with MG132. In contrast, no elevation in adrenodoxin, a component of the cytochrome P450scc enzyme system, was found. These data suggest that StAR protein is either directly or indirectly degraded by the proteasome which may explain, in part, its short half-life. Moreover, it seems that the cytosolic 37 kDa protein, which is responsible for the steroidogenic activity of StAR, is the primary proteasomal substrate and that the inhibition of its degradation by MG132 causes the up-regulation of progesterone production.     

Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins

Steroidogenic acute regulatory protein (StAR) is a nuclear encoded mitochondrial protein that enhances steroid synthesis by facilitating the transfer of cholesterol to the inner membranes of mitochondria in hormonally regulated steroidogenic cells. It is currently assumed that StAR activity commences before or during StAR import into the mitochondrial matrix. The present study was designed to demonstrate that, once imported and becoming physiologically irrelevant, exhaustive accumulation of StAR must be limited by a rapid degradation of the protein to prevent potential damage to the organelles. The use of uncouplers and manipulation of the interior mitochondrial pH in hormone-induced ovarian granulosa cells and StAR-expressing COS cells suggests that StAR degradation is biphasic and involves two classes of proteases. During phase I, which normally lasts for the first approximately 2 h following import, StAR is rapidly degraded by a protease, or proteases, that can be arrested by a nonclassical action of proteasome inhibitors such as MG132. StAR molecules that evade phase I are subjected to a second class of protease(s), which is slower and MG132 resistant. A third proteolytic entity was revealed in studies with C-28 StAR, a loss-of-function mutant of StAR. Upon initiation of its import, C-28 StAR dissipates the inner membrane potential and causes swelling of the mitochondria. Degradation of C-28 StAR, probably by an intermembrane space protease, is extremely rapid and MG132 insensitive. Collectively, this study defines StAR as the first naturally occurring mitochondrial protein that can serve as a substrate to probe multiple proteolytic activities in mammalian mitochondria.     

Towards the control of intracellular protein turnover: mitochondrial Lon protease inhibitors versus proteasome inhibitors

Cellular protein homeostasis results from the combination of protein biogenesis processes and protein quality control mechanisms, which contribute to the functional state of cells under normal and stress conditions. Proteolysis constitutes the final step by which short-lived, misfolded and damaged intracellular proteins are eliminated. Protein turnover and oxidatively modified protein degradation are mainly achieved by the proteasome in the cytosol and nucleus of eukaryotic cells while several ATP-dependent proteases including the matrix protease Lon take part in the mitochondrial protein degradation. Moreover, Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. Specific inhibitors are commonly used to assess cellular functions of proteolytic systems as well as to identify their protein substrates. Here, we present and discuss known proteasome and Lon protease inhibitors. To date, very few inhibitors of Lon have been described and no specific inhibitors of this protease are available. The current knowledge on both catalytic mechanisms and inhibitors of these two proteases is first described and attempts to define specific non-peptidic inhibitors of the human Lon protease are presented.     

Cleavage site selection within a folded substrate by the ATP-dependent lon protease

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.     

Rapid turnover of tryptophan hydroxylase is driven by proteasomes in RBL2H3 cells, a serotonin producing mast cell line

Previously we demonstrated that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by ATP-dependent proteolysis in serotonin producing mast cells [Hasegawa et al. (1995) FEBS Lett. 368, 151-154]. We searched for the major proteases involved in the rapid degradation of TPH in RBL2H3 cells. Among various protease inhibitors tested, proteasome inhibitors MG115, MG101, MG132, and lactacystin effectively inhibited the intracellular degradation of TPH. Administration of the proteasome inhibitors to cultured cells caused more than a 5-fold accumulation of TPH. Administration of the inhibitors together with cycloheximide stabilized the amount of TPH with no appreciable increase or decrease. Although MG-series proteasome inhibitors could inhibit calpain, the involvement of calpain was excluded since the cysteine protease inhibitor E-64-d, which acts on calpain, had no effect. Extracts of RBL2H3 cells were shown to contain a protease that digests TPH in an ATP-dependent manner and is sensitive to proteasome inhibitors. The ubiquitination of TPH could be visualized by Western blot analysis using both anti-TPH and anti-ubiquitin antibodies. Based on these results, we conclude that 26S proteasomes are mainly involved in the degradation of TPH. In the reported amino acid sequences of TPH from various sources including human, rabbit, rat, and mouse, a PEST sequence that is widely shared among short-lived proteins has been recognized. It was noted that the PEST sequence lies within the most conserved portion of the enzyme, the pteridine binding site.     

Proteasome inhibitors impair RANKL‐induced NF‐κB activity in osteoclast‐like cells via disruption of p62, TRAF6, CYLD,and IκBα signaling cascades

Proteasome inhibitors represent a promising therapy for the treatment of relapsed and/or refractory multiple myeloma, a disease that is concomitant with osteolysis and enhanced osteoclast formation. While blockade of the proteosome pathway has been recently shown to influence osteoclast formation and function, the precise molecular cascade underlying these effects is presently unclear. Here, we provide evidence that proteasome inhibitors directly impair osteoclast formation and function via the disruption of key RANK‐mediated signaling cascades. Disruption of the proteosome pathway using selective inhibitors (MG‐132, MG‐115, and epoxomicin) resulted in the accumulation of p62 and CYLD, and altered the subcellular targeting and distribution of p62 and TRAF6 in osteoclast‐like cells. Proteosome inhibition also blocked RANKL‐induced NF‐κB activation, IκBα degradation and nuclear translocation of p65. The disruption in RANK‐signaling correlated dose‐dependently with an impairment in osteoclastogenesis, with relative potency epoxomicin > MG‐132 > MG‐115 based on equimolar concentrations. In addition, these inhibitors were found to impact osteoclastic microtubule organization and attenuate bone resorption. Based on these data we propose that deregulation of key RANK‐mediated signaling cascades (p62, TRAF6, CYLD, and IκBα) underscores proteasome‐mediated inhibition of osteolytic bone conditions. J. Cell. Physiol. 220: 450–459, 2009. © 2009 Wiley‐Liss, Inc.     

Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132

Ubiquitin-dependent proteolysis has been implicated in the recognition and selective elimination of paternal mitochondria and mitochondrial DNA (mtDNA) after fertilization in mammals. Initial evidence suggests that this process is contributed to by lysosomal degradation of the ubiquitinated sperm mitochondrial membrane proteins. The present study examined the role of the proteasome-dependent protein degradation pathway of the ubiquitin system, as opposed to lysosomal proteolysis of the ubiquitinated proteins, in the regulation of sperm mitochondrion elimination after fertilization. Boar spermatozoa prelabeled with vital fluorescent mitochondrial probes MitoTracker were used to trace the degradation of paternal mitochondria after in vitro fertilization (IVF) of porcine oocytes. The degradation of sperm mitochondria in the cytoplasm of fertilized oocytes started very rapidly, i.e., within 12-20 h after insemination. Four stages of paternal mitochondrial degradation were distinguished, ranging from an intact mitochondrial sheath (type 1) to complete degradation (type 4). At 27-30 h postinsemination, 96% of zygotes contained the partially (type 3) or completely (type 4) degraded sperm mitochondria. Highly specific peptide inhibitors of the ubiquitin-proteasome pathway, lactacystin (10 and 100 microM) and MG132 (10 microM), efficiently blocked the degradation of the sperm mitochondria inside the fertilized egg when applied 6 h after insemination. Using 10 microM MG132, only 13.6% of fertilized oocytes screened 27-30 h after IVF displayed type 3 sperm mitochondria, and there was no incidence of type 4, completely degraded mitochondria. Although lactacystin is not a reversible agent, the effect of MG132 was fully reversible: zygotes transferred to regular culture medium after 24 h of culture with 10 microM MG132 resumed development and degraded sperm mitochondria within the next cell cycle. Surprisingly, penetration of the zona pellucida (ZP) was also inhibited by MG-132 and lactacystin when the inhibitors were added at insemination. Altogether, these data provide the first evidence of the participation of proteasomes in the control of mammalian mitochondrial inheritance and suggest a new role of the ubiquitin-proteasome pathway in mammalian fertilization.     

Roles of the ubiquitin/proteasome pathway in pollen tube growth with emphasis on MG132-induced alterations in ultrastructure, cytoskeleton, and cell wall components

The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner, while hardly similar effects were detected when cysteine-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanate-phalloidin and beta-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.     

Transmission of cell stress from endoplasmic reticulum to mitochondria: enhanced expression of Lon protease

The rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial-encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP-dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (-/-) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX I-containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex.     

Proteasome inhibitors against amelanotic melanoma

The incidence of malignant melanoma, the most aggressive skin cancer, is increasing constantly. Despite new targeted therapies, the prognosis for patients with metastatic disease remains poor. Thus, there is a need for new combinational treatments, and antineoplastic agents potentially valuable in this approach are inhibitors of the ubiquitin-proteasome system (UPS). In this work, we analyze the cytotoxicity mechanisms of proteasome inhibitors (MG-132, epoxomicin, and lactacystin) in a specific form of melanoma which does not synthesize melanin—the amelanotic melanoma (Ab cells). We found that the most cytotoxic of the compounds tested was epoxomicin. Caspase-9 activation as well as cytochrome C and AIF release from mitochondria indicated that exposure to epoxomicin induced the mitochondrial pathway of apoptosis. Epoxomicin treatment also resulted in accumulation of Bcl-2 family members—proapoptotic Noxa and antiapoptotic Mcl-1, which were postulated as the targets for bortezomib in melanoma. Inhibition of caspases by BAF revealed that cell death was partially caspase-independent. We observed no cell cycle arrest preceding the apoptosis of Ab cells, even though cdk inhibitors p21^Cip1/Waf1 and p27^Kip1 were up-regulated. The cell cycle was blocked only after inactivation of caspases by the pan-caspase inhibitor BAF. In summary, this is the first study exploring molecular mechanisms of cell death induced by epoxomicin in melanoma. We found that Ab cells died on the mitochondrial pathway of apoptosis and also partially by the caspase-independent way of death. Apoptosis induction was fast and efficient and was not preceded by cell cycle arrest.     

Identification of the proteasome inhibitor MG262 as a potent ATP-dependent inhibitor of the Salmonella enterica serovar Typhimurium Lon protease

Lon is a homo-oligomeric ATP-dependent serine protease which functions in the degradation of damaged and certain regulatory proteins. The importance of Lon activity in bacterial pathogenicity has led to its emergence as a target in the development of novel antibiotics. As no potent inhibitors of Lon activity have been reported to date, we sought to identify an inhibitor which could serve as a lead compound in the development of a potent Lon-specific inhibitor. To determine whether a nucleotide- or peptide-based inhibitor would be more effective, we evaluated the steady-state kinetic parameters associated with both ATP and peptide hydrolysis by human and Salmonella enterica serovar Typhimurium Lon. Although the ATP hydrolysis activities of both homologues are kinetically indistinguishable, they display marked differences in peptide substrate specificity. This suggests that a peptide-based inhibitor could be developed which would target bacterial Lon, thereby decreasing side-effects due to cross-reactivity with human Lon. Using Salmonella enterica serovar Typhimurium Lon as a model, we evaluated the IC50 values of a series of commercially available peptide-based inhibitors. Those inhibitors which behave as transition state analogues were the most useful in inhibiting Lon activity. The peptidyl boronate, MG262, was the most potent inhibitor tested (IC50 = 122 +/- 9 nM) and required binding, but not hydrolysis, of ATP to initiate inhibition. We hope to use MG262 as a lead compound in the development of future Lon-specific inhibitors.     

Oxidative stress and protease dysfunction in the yeast model of Friedreich ataxia

Friedreich ataxia has frequently been associated with an increased susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich ataxia to study the physiological consequences of a shift from anaerobiosis to aerobiosis. Cells lacking frataxin (Deltayfh1) showed no growth defect when cultured anaerobically. Under these conditions, a significant amount of aconitase was functional, with an intact 4 Fe/4 S cluster. When shifted to aerobic conditions, aconitase was rapidly degraded, and oxidatively modified proteins (carbonylated and HNE-modified proteins) accumulated in both the cytosol and the mitochondria. The ATP-dependent mitochondrial protease Pim1 (Lon) was strongly activated, although its expression level remained unchanged, and the cytosolic activity of the 20S proteasome was greatly decreased, compared to that in wild-type cells. Analysis of the purified proteasome revealed that the decrease in proteasome activity was likely due to both direct inactivation of the enzyme and inhibition by cytosolic oxidized proteins. These features indicate that the cells were subjected to major oxidative stress triggered by oxygen. Accumulation of oxidatively modified proteins, activation of Pim1, and proteasome inhibition did not directly depend on the amount of mitochondrial iron, because these phenotypes remained unchanged when the cells were grown under iron-limiting conditions, and these phenotypes were not observed in another mutant (Deltaggc1) which overaccumulates iron in its mitochondrial compartment. We conclude that oxygen is primarily involved in generating the deleterious phenotypes that are observed in frataxin-deficient yeast cells.     

Role of the novel metallopeptidase Mop112 and saccharolysin for the complete degradation of proteins residing in different subcompartments of mitochondria

Mitochondria harbor a conserved proteolytic system that mediates the complete degradation of organellar proteins. ATP-dependent proteases, like a Lon protease in the matrix space and m- and i-AAA proteases in the inner membrane, degrade malfolded proteins within mitochondria and thereby protect the cell against mitochondrial damage. Proteolytic breakdown products include peptides and free amino acids, which are constantly released from mitochondria. It remained unclear, however, whether the turnover of malfolded proteins involves only ATP-dependent proteases or also oligopeptidases within mitochondria. Here we describe the identification of Mop112, a novel metallopeptidase of the pitrilysin family M16 localized in the intermembrane space of yeast mitochondria. This peptidase exerts important functions for the maintenance of the respiratory competence of the cells that overlap with the i-AAA protease. Deletion of MOP112 did not affect the stability of misfolded proteins in mitochondria, but resulted in an increased release from the organelle of peptides, generated upon proteolysis of mitochondrial proteins. We find that the previously described metallopeptidase saccharolysin (or Prd1) exerts a similar function in the intermembrane space. The identification of peptides released from peptidase-deficient mitochondria by mass spectrometry indicates a dual function of Mop112 and saccharolysin: they degrade peptides generated upon proteolysis of proteins both in the intermembrane and matrix space and presequence peptides cleaved off by specific processing peptidases in both compartments. These results suggest that the turnover of mitochondrial proteins is mediated by the sequential action of ATP-dependent proteases and oligopeptidases, some of them localized in the intermembrane space.     

Overexpression of Lon contributes to survival and aggressive phenotype of cancer cells through mitochondrial complex I-mediated generation of reactive oxygen species

Lon protease is a multifunction protein and operates in protein quality control and stress response pathways in mitochondria. Human Lon is upregulated under oxidative and hypoxic stresses that represent the stress phenotypes of cancer. However, little literature undertakes comprehensive and detailed investigations on the tumorigenic role of Lon. Overexpression of Lon promotes cell proliferation, apoptotic resistance to stresses, and transformation. Furthermore, Lon overexpression induces the production of mitochondrial reactive oxygen species (ROS) that result from Lon-mediated upregulation of NDUFS8, a mitochondrial Fe-S protein in complex I of electron transport chain. Increased level of mitochondrial ROS promotes cell proliferation, cell survival, cell migration, and epithelial–mesenchymal transition through mitogen-activated protein kinase (MAPK) and Ras-ERK activation. Overall, the present report for the first time demonstrates the role of Lon overexpression in tumorigenesis. Lon overexpression gives an apoptotic resistance to stresses and induces mitochondrial ROS production through Complex I as signaling molecules to activate Ras and MAPK signaling, giving the survival advantages and adaptation to cancer cells. Finally, in silico and immunohistochemistry analysis showed that Lon is overexpressed specifically in various types of cancer tissue including oral cancer.     

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.     

Cellular proteasome activity facilitates herpes simplex virus entry at a postpenetration step

Herpes simplex virus (HSV) entry into cells is a multistep process that engages the host cell machinery. The proteasome is a large, ATP-dependent, multisubunit protease that plays a critical role in the maintenance of cell homeostasis. A battery of assays were used to demonstrate that proteasome inhibitors blocked an early step in HSV entry that occurred after capsid penetration into the cytosol but prior to capsid arrival at the nuclear periphery. Proteasome-dependent viral entry was not reliant on host or viral protein synthesis. MG132, a peptide aldehyde that competitively inhibits the degradative activity of the proteasome, had a reversible inhibitory effect on HSV entry. HSV can use endocytic or nonendocytic pathways to enter cells. These distinct entry routes were both dependent on proteasome-mediated proteolysis. In addition, HSV successfully entered cells in the absence of a functional host ubiquitin-activating enzyme, suggesting that viral entry is ubiquitin independent. We propose that proteasomal degradation of virion and/or host proteins is required for efficient delivery of incoming HSV capsids to the nucleus.