HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: a potential mechanism for protease inhibitor-induced hyperlipidemia.

Highly active anti-retroviral therapies, which incorporate HIV protease inhibitors, resolve many AIDS-defining illnesses. However, patients receiving protease inhibitors develop a marked lipodystrophy and hyperlipidemia. Using cultured human and rat hepatoma cells and primary hepatocytes from transgenic mice, we demonstrate that protease inhibitor treatment inhibits proteasomal degradation of nascent apolipoprotein B, the principal protein component of triglyceride and cholesterol-rich plasma lipoproteins. Unexpectedly, protease inhibitors also inhibited the secretion of apolipoprotein B. This was associated with inhibition of cholesteryl-ester synthesis and microsomal triglyceride transfer-protein activity. However, in the presence of oleic acid, which stimulates neutral-lipid biosynthesis, protease-inhibitor treatment increased secretion of apolipoprotein B-lipoproteins above controls. These findings suggest a molecular basis for protease-inhibitor-associated hyperlipidemia, a serious adverse effect of an otherwise efficacious treatment for HIV infection.     

The nucleolar SUMO-specific protease SENP3 reverses SUMO modification of nucleophosmin and is required for rRNA processing

The ubiquitin-like SUMO system functions by a cyclic process of modification and demodification, and recent data suggest that the nucleolus is a site of sumoylation-desumoylation cycles. For example, the tumour suppressor ARF stimulates sumoylation of nucleolar proteins. Here, we show that the nucleolar SUMO-specific protease SENP3 is associated with nucleophosmin (NPM1), a crucial factor in ribosome biogenesis. SENP3 catalyses desumoylation of NPM1-SUMO2 conjugates in vitro and counteracts ARF-induced modification of NPM1 by SUMO2 in vivo. Intriguingly, depletion of SENP3 by short interfering RNA interferes with nucleolar ribosomal RNA processing and inhibits the conversion of the 32S rRNA species to the 28S form, thus phenocopying the processing defect observed on depletion of NPM1. Moreover, mimicking constitutive modification of NPM1 by SUMO2 interferes with 28S rRNA maturation. These results define SENP3 as an essential factor for ribosome biogenesis and suggest that deconjugation of SUMO2 from NPM1 by SENP3 is critically involved in 28S rRNA maturation.     

Oxidative stress induces neuronal death by recruiting a protease and phosphatase-gated mechanism.

Reactive oxygen species (ROS) cause death of cerebellar granule neurons. Here, a 15-min pulse of H(2)O(2) (100 microm) induced an active process of neuronal death distinct from apoptosis. Oxidative stress activated a caspase-independent but calpain-dependent decline of calcium/calmodulin-dependent protein kinase IV and cAMP- responsive element-binding protein (CREB). Calpain inhibitors restored calcium/calmodulin-dependent protein kinase IV and CREB but did not influence phosphorylated CREB levels or survival, indicating recruitment of an additional dephosphorylation process. Co-treatment with calpain and serine/threonine phosphatase inhibitors restored pCREB levels and rescued neurons. This phosphatase-activated signaling pathway was shown to be dependent on de novo protein synthesis. Further, gene transfer studies revealed that CREB is a common final effector of both apoptosis and ROS-induced death. Our data indicate that dephosphorylation and proteolytic signaling mechanisms underlie ROS-induced programmed cell death.     

Bacterial conjugation: a two-step mechanism for DNA transport

Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB. DsbB generates disulphides de novo from oxidized quinones. These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport. Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing. Two isomerases exist in Escherichia coli, DsbC and DsbG. The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction.     

A neuronal nucleolar trigger mechanism traceable by a routine staining method.

In experimental Wilson's disease produced by the intracardiac injection of copper sulphate, it is seen that the glia-neuronal interactions play an important role in the pathogenesis of neural lesions. A "trigger" copper-RNA particle is transferred from the oligodendroglial nucleus to activate the neuronal nucleolus causing a flooding of the neurone with RNA. This trigger particle stains differently with Mason's trichrome, giving a bright red colour. This altered reaction gives an insight into the feedback process for the formation of ribosomal RNA in the neurones. The alteration appears to lead to a block in the repressor activity of the neuronal RNA formation.     

A basis for SUMO protease specificity provided by analysis of human Senp2 and a Senp2-SUMO complex

Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear metabolism and cell cycle progression in yeast. X-ray structures of the human Senp2 catalytic protease domain and of a covalent thiohemiacetal transition-state complex obtained between the Senp2 catalytic domain and SUMO-1 revealed details of the respective protease and substrate surfaces utilized in interactions between these two proteins. Comparative biochemical and structural analysis between Senp2 and the yeast SUMO protease Ulp1 revealed differential abilities to process SUMO-1, SUMO-2, and SUMO-3 in maturation and deconjugation reactions. Further biochemical characterization of the three SUMO isoforms into which an additional Gly-Gly di-peptide was inserted, or whereby the respective SUMO tails from the three isoforms were swapped, suggests a strict dependence for SUMO isopeptidase activity on residues C-terminal to the conserved Gly-Gly motif and preferred cleavage site for SUMO proteases.     

Update on sumoylation: defining core components of the plant SUMO conjugation system by phylogenetic comparison

The conjugation of the small ubiquitin-related modifier, SUMO, to substrate proteins is a reversible and dynamic process, and an important response of plants to environmental challenges. Nevertheless, reliable data have so far been restricted largely to the model plant Arabidopsis thaliana. The increasing availability of genome information for other plant species offers the possibility to identify a core set of indispensable components, and to discover species-specific features of the sumoylation pathway. We analyzed the enzymes responsible for the conjugation of SUMO to substrates for their conservation between dicots and monocots. We thus assembled gene sets that relate the Arabidopsis SUMO conjugation system to that of the dicot species tomato, grapevine and poplar, and to four plant species from the monocot class: rice, Brachypodium distachyon, Sorghum bicolor and maize. We found that a core set of genes with clear assignment in Arabidopsis had highly conserved homologs in all tested plants. However, we also observed a variation in the copy number of homologous genes, and sequence variations that suggested monocot-specific variants. Generally, SUMO ligases and proteases showed the most pronounced differences. Finally, we identified potential SUMO chain-binding ubiquitin ligases, pointing to an in vivo function of SUMO chains as degradation signals in plants.     

A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1

E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast. In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets. Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 A reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins. Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IkappaBalpha, and RanGAP1.     

Targeting SUMO E1 to ubiquitin ligases: a viral strategy to counteract sumoylation

SUMO-1 (small ubiquitin-related modifier-1) is a ubiquitin-like family member that is conjugated to its substrates through three discrete enzymatic steps, activation (involving the E1 enzyme (SAE1/SAE2)), conjugation (involving the E2 enzyme), and substrate modification (through the cooperation of the E2 and E3 protein ligases). The adenoviral protein Gam1 inactivates E1, both in vitro and in vivo, followed by SAE1/SAE2 degradation. We have shown here that Gam1 possesses a C-terminal SOCS domain that allows its interaction with two cellular cullin RING (really interesting new gene) ubiquitin ligases. We demonstrate that Gam1 is necessary for the recruitment of SAE1/SAE2 into Cul2/5-EloB/C-Roc1 ubiquitin ligase complexes and for subsequent SAE1 ubiquitylation and degradation. The degradation of SAE2 is not tightly related to Gam1 but is a consequent effect of SAE1 disappearance. These results reveal the mechanism by which a viral protein inactivates and subsequently degrades an essential cellular enzyme, arresting a key regulatory pathway.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection

The conjugation of small ubiquitin-like modifiers SUMO-1, SUMO-2 and SUMO-3 onto target proteins requires the concerted action of the specific E1-activating enzyme SAE1/SAE2, the E2-conjugating enzyme Ubc9, and an E3-like SUMO ligase. NMR chemical shift perturbation was used to identify the surface of Ubc9 that interacts with the SUMO ligase RanBP2. Unlike known ubiquitin E2-E3 interactions, RanBP2 binds to the beta-sheet of Ubc9. Mutational disruption of Ubc9-RanBP2 binding affected SUMO-2 but not SUMO-1 conjugation to Sp100 and to a newly identified RanBP2 substrate, PML. RanBP2 contains a binding site specific for SUMO-1 but not SUMO-2, indicating that a Ubc9-SUMO-1 thioester could be recruited to RanBP2 via SUMO-1 in the absence of strong binding between Ubc9 and RanBP2. Thus we show that E2-E3 interactions are not conserved across the ubiquitin-like protein superfamily and identify a RanBP2-dependent mechanism for SUMO paralog-specific conjugation.     

A novel mechanism by which thiazolidinediones facilitate the proteasomal degradation of cyclin D1 in cancer cells

This study identifies a novel mechanism by which thiazolidinediones mediate cyclin D1 repression in prostate cancer cells. Based on the finding that the thiazolidinedione family of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists mediated PPARgamma-independent cyclin D1 degradation, we developed a novel PPARgamma-inactive troglitazone derivative, STG28, with high potency in cyclin D1 ablation. STG28-mediated cyclin D1 degradation was preceded by Thr-286 phosphorylation and nuclear export, which however, were independent of glycogen synthase kinase 3beta. Mutational analysis further confirmed the pivotal role of Thr-286 phosphorylation in STG28-induced nuclear export and proteolysis. Of several kinases examined, inhibition of IkappaB kinase alpha blocked STG28-mediated cytoplasmic sequestration and degradation of cyclin D1. Pulldown of ectopically expressed Cul1, the scaffold protein of the Skp-Cullin-F-box E3 ligase, in STG28-treated cells revealed an increased association of cyclin D1 with beta-TrCP, whereas no specific binding was noted with other F-box proteins examined, including Skp2, Fbw7, Fbx4, and Fbxw8. This finding represents the first evidence that cyclin D1 is targeted by beta-TrCP. Moreover, beta-TrCP expression was up-regulated in response to STG28, and ectopic expression and small interfering RNA-mediated knock-down of beta-TrCP enhanced and protected against STG28-facilitated cyclin D1 degradation, respectively. Because cyclin D1 lacks the DSG destruction motif, mutational and modeling analyses indicate that cyclin D1 was targeted by beta-TrCP through an unconventional recognition site, (279)EEVDLACpT(286), reminiscent to that of Wee1. Moreover, we obtained evidence that this beta-TrCP-dependent degradation takes part in controlling cyclin D1 turnover when cancer cells undergo glucose starvation, which endows physiological relevance to this novel mechanism.