Interaction of the Doa4 Deubiquitinating Enzyme with the Yeast 26S Proteasome

The Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin–proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

Apg7p/Cvt2p: A Novel Protein-activating Enzyme Essential for Autophagy

In the yeast Saccharomyces cerevisiae, the Apg12p–Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc–tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p–Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p–Apg5p conjugation.

The Doa4 Deubiquitinating Enzyme Is Required for Ubiquitin Homeostasis in Yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Δ mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Δ cells. This loss of viability and several other defects of doa4Δ cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.

A Ubiquitin-Conjugating Enzyme Is Essential for Developmental Transitions in Dictyostelium

We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for ∼10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.

Degradation of Hepatic Stearyl CoA Δ9-Desaturase

Δ9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt–washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12 and oleoyl Δ9,10 location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 102–103 times more potent than the corresponding trifluoromethyl ketones.

Dominant negative inhibition by fragments of a monomeric enzyme

Dominant negative inhibition is most commonly seen when a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein so that assembly of a functional oligomer is impaired. By analogy, it should be possible to interfere with the functional assembly of a monomeric enzyme by interfering with the folding pathway. Experiments in vitro by others suggested that fragments of a monomeric enzyme might be exploited for this purpose. We report here dominant negative inhibition of bacterial cell growth by expression of fragments of a tRNA synthetase. Inhibition is fragment-specific, as not all fragments cause inhibition. An inhibitory fragment characterized in more detail forms a specific complex with the intact enzyme in vivo, leading to enzyme inactivation. This fragment also associated stoichiometrically with the full-length enzyme in vitro after denaturation and refolding, and the resulting complex was catalytically inactive. Inhibition therefore appears to arise from an interruption in the folding pathway of the wild-type enzyme, thus suggesting a new strategy to design dominant negative inhibitors of monomeric enzymes.

BAX-induced cell death may not require interleukin 1β-converting enzyme-like proteases

Expression of BAX, without another death stimulus, proved sufficient to induce a common pathway of apoptosis. This included the activation of interleukin 1β-converting enzyme (ICE)-like proteases with cleavage of the endogenous substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation inhibitor for the rho family), as well as the fluorogenic peptide acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) successfully blocked this protease activity and prevented FAS-induced death but not BAX-induced death. Blocking ICE-like protease activity prevented the cleavage of nuclear and cytosolic substrates and the DNA degradation that followed BAX induction. However, the fall in mitochondrial membrane potential, production of reactive oxygen species, cytoplasmic vacuolation, and plasma membrane permeability that are downstream of BAX still occurred. Thus, BAX-induced alterations in mitochondrial function and subsequent cell death do not apparently require the known ICE-like proteases.

Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression

The Saccharomyces cerevisiae Mod5 protein catalyzes isopentenylation of A to i6A on tRNAs in the nucleus, cytosol, and mitochondria. The substrate for Mod5p, dimethylallyl pyrophosphate, is also a substrate for Erg20p that catalyzes an essential step in sterol biosynthesis. Changing the distribution of Mod5p so that less Mod5p is present in the cytosol decreases i6A on cytosolic tRNAs and alters tRNA-mediated nonsense suppression. We devised a colony color/growth assay to assess tRNA-mediated nonsense suppression and used it to search for genes, which, when overexpressed, affect nonsense suppression. We identified SAL6, TEF4, and YDL219w, all of which likely affect nonsense suppression via alteration of the protein synthesis machinery. We also identified ARC1, whose product interacts with aminoacyl synthetases. Interestingly, we identified ERG20. Midwestern analysis showed that yeast cells overproducing Erg20p have reduced levels of i6A on tRNAs. Thus, Erg20p appears to affect nonsense suppression by competing with Mod5p for substrate. Identification of ERG20 reveals that yeast have a limited pool of dimethylallyl pyrophosphate. It also demonstrates that disrupting the balance between enzymes that use dimethylallyl pyrophosphate as substrate affects translation.

Immunophilins, Refsum disease, and lupus nephritis: The peroxisomal enzyme phytanoyl-COA α-hydroxylase is a new FKBP-associated protein

FKBP52 (FKBP59, FKBP4) is a “macro” immunophilin that, although sharing high structural and functional homologies in its amino-terminal domain with FKBP12 (FKBP1), does not have immunosuppressant activity when complexed with FK506, unlike FKBP12. To investigate the physiological function of FKBP52, we used the yeast two-hybrid system as an approach to find its potential protein partners and, from that, its cellular role. This methodology, which already has allowed us to find the FK506-binding protein (FKBP)-associated protein FAP48, also led to the detection of another FKBP-associated protein. Determination of the sequence of this protein permitted its identification as phytanoyl-CoA α-hydroxylase (PAHX), a peroxisomal enzyme that so far was unknown as an FKBP-associated protein. Inactivation of this enzyme is responsible for Refsum disease in humans. The protein also corresponds to the mouse protein LN1, which could be involved in the progress of lupus nephritis. We show here that PAHX has the physical capacity to interact with the FKBP12-like domain of FKBP52, but not with FKBP12, suggesting that it is a particular and specific target of FKBP52. Whereas the binding of calcineurin to FKBP12 is potentiated by FK506, the specific association of PAHX and FKBP52 is maintained in the presence of FK506. This observation suggests that PAHX is a serious candidate for studying the cellular signaling pathway(s) involving FKBP52 in the presence of immunosuppressant drugs.

Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3

Two important cytokines mediating inflammation are tumor necrosis factor α (TNFα) and IL-1β, both of which require conversion to soluble forms by converting enzymes. The importance of TNFα-converting enzyme and IL-1β-converting enzyme in the production of circulating TNFα and IL-1β in response to systemic challenges has been demonstrated by the use of specific converting enzyme inhibitors. Many inflammatory responses, however, are not systemic but instead are localized. In these situations release and/or activation of cytokines may be different from that seen in response to a systemic stimulus, particularly because associations of various cell populations in these foci allows for the exposure of procytokines to the proteolytic enzymes produced by activated neutrophils, neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G). To investigate the possibility of alternative processing of TNFα and/or IL-1β by neutrophil-derived proteinases, immunoreactive TNFα and IL-1β release from lipopolysaccharide-stimulated THP-1 cells was measured in the presence of activated human neutrophils. Under these conditions, TNFα and IL-1β release was augmented 2- to 5-fold. In the presence of a specific inhibitor of NE and PR3, enhanced release of both cytokines was largely abolished; however, in the presence of a NE and Cat G selective inhibitor, secretory leucocyte proteinase inhibitor, reduction of the enhanced release was minimal. This finding suggested that the augmented release was attributable to PR3 but not NE nor Cat G. Use of purified enzymes confirmed this conclusion. These results indicate that there may be alternative pathways for the production of these two proinflammatory cytokines, particularly in the context of local inflammatory processes.

Memory landscapes of single-enzyme molecules

Immobilized single horseradish peroxidase enzymes were observed by confocal fluorescence spectroscopy during catalysis of the oxidation reaction of the nonfluorescent dihydrorhodamine 6G substrate into the highly fluorescent product rhodamine 6G. By extracting only the non-Markovian behavior of the spectroscopic two-state process of enzyme-product complex formation and release, memory landscapes were generated for single-enzyme molecules. The memory landscapes can be used to discriminate between different origins of stretched exponential kinetics that are found in the first-order correlation analysis. Memory landscapes of single-enzyme data shows oscillations that are expected in a single-enzyme system that possesses a set of transient states. Alternative origins of the oscillations may not, however, be ruled out. The data and analysis indicate that substrate interaction with the enzyme selects a set of conformational substates for which the enzyme is active.