The COP9 signalosome mediates the Spt23 regulated fatty acid desaturation and ergosterol biosynthesis.

The COP9 signalosome (CSN) is a conserved eukaryotic complex, essential for vitality in all multicellular organisms and critical for the turnover of key cellular proteins through catalytic and non-catalytic activities. Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of the CSN complex, since it includes a conserved enzymatic core but lacks non-catalytic activities, probably explaining its non-essentiality for life. A previous transcriptomic analysis of an S. cerevisiae strain deleted in the CSN5/RRI1 gene, encoding to the CSN catalytic subunit, revealed a downregulation of genes involved in lipid metabolism. We now show that the S. cerevisiae CSN holocomplex is essential for cellular lipid homeostasis. Defects in CSN assembly or activity lead to decreased quantities of ergosterol and unsaturated fatty acids (UFA); vacuole defects; diminished lipid droplets (LDs) size; and to accumulation of endoplasmic reticulum (ER) stress. The molecular mechanism behind these findings depends on CSN involvement in upregulating mRNA expression of SPT23. Spt23 is a novel activator of lipid desaturation and ergosterol biosynthesis. Our data reveal for the first time a functional link between the CSN holocomplex and Spt23. Moreover, CSN-dependent upregulation of SPT23 transcription is necessary for the fine-tuning of lipid homeostasis and for cellular health.

Statins interfere with the attachment of S. cerevisiae mtDNA to the inner mitochondrial membrane.

The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used Saccharomyces cerevisiae as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in S.cerevisiae by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins.

Targeted degradation of abscisic acid receptors is mediated by the ubiquitin ligase substrate adaptor DDA1 in Arabidopsis.

CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)-related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDB1-ASSOCIATED1 (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABA-mediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability.

PCI complexes: Beyond the proteasome, CSN, and eIF3 Troika.

The bipartite PCI domain serves as the principal scaffold for proteasome lid, CSN, and eIF3, complexes that influence protein life span. PCI domains are also found in newly identified complexes directing nucleic acid regulation. The breadth of functions associated with the extended PCI family is a factor of shared subunits, among them a common factor Sem1/DSS1 that facilitates complex assembly.

Yeast as a tool to select inhibitors of the cullin deneddylating enzyme Csn5.

The CSN complex plays a key role in various cellular pathways: through a metalloprotease activity of its Csn5 deneddylating enzyme, it regulates the activity of Cullin-RING ligases (CRLs). Indeed, Csn5 has been found amplified in many tumors, but, due to its pleiotropic effects, it is difficult to dissect its function and the involvement in cancer progression. Moreover, while growing evidences point to the neddylation function as a good target for drug development; specific inhibitors have not yet been developed for the CSN. Here, we propose the yeast Saccharomyces cerevisiae as a model system to screen libraries of small molecules as inhibitors of cullins deneddylation, taking advantage of the unique feature of this organism to survive without a functional CSN5 gene and to accumulate a fully neddylated cullin substrate. By combining molecular modeling and simple genetic tools, we were able to identify two small molecular fragments as selective inhibitors of Csn5 deneddylation function.

COP9 signalosome subunit Csn8 is involved in maintaining proper duration of the G1 phase.

The COP9 signalosome (CSN) is a conserved protein complex known to be involved in developmental processes of eukaryotic organisms. Genetic disruption of a CSN gene causes arrest during early embryonic development in mice. The Csn8 subunit is the smallest and the least conserved subunit, being absent from the CSN complex of several fungal species. Nevertheless, Csn8 is an integral component of the CSN complex in higher eukaryotes, where it is essential for life. By characterizing the mouse embryonic fibroblasts (MEFs) that express Csn8 at a low level, we found that Csn8 plays an important role in maintaining the proper duration of the G1 phase of the cell cycle. A decreased level of Csn8, either in Csn8 hypomorphic MEFs or following siRNA-mediated knockdown in HeLa cells, accelerated cell growth rate. Csn8 hypomorphic MEFs exhibited a shortened G1 duration and affected expression of G1 regulators. In contrast to Csn8, down-regulation of Csn5 impaired cell proliferation. Csn5 proteins were found both as a component of the CSN complex and outside of CSN (Csn5-f), and the amount of Csn5-f relative to CSN was increased in the Csn8 hypomorphic cells. We conclude that CSN harbors both positive and negative regulators of the cell cycle and therefore is poised to influence the fate of a cell at the crossroad of cell division, differentiation, and senescence.

Moonlighting and pleiotropy within two regulators of the degradation machinery: the proteasome lid and the CSN.

The distinction between pleiotrotic and moonlighting roles of proteins is challenging; however, this distinction may be clearer when it comes to multiprotein complexes. Two examples are the proteasome lid and the COP9 signalosome (CSN), which are twin enzymes with 1:1 paralogy between subunits. In each complex, one out of eight subunits harbours a JAMM/MPN⁺ metalloprotease motif. This motif contributes the canonical activity of each complex: hydrolysis of covalently attached ubiquitin by Rpn11 in the proteasome lid and hydrolysis of ubiquitin-related 1 (Rub1/Nedd8) from Cullins by Csn5 in the CSN. In both complexes, executing this activity suggests pleiotropic effects and requires an assembled full complex. However, beyond canonical functions, both Rpn11 and Csn5 are involved in additional unique, complex-independent functions, herein referred to as moonlighting activities.

The Ubiquitin-like Proteins of Saccharomyces cerevisiae.

In this review, we present a comprehensive list of the ubiquitin-like modifiers (Ubls) of Saccharomyces cerevisiae, a common model organism used to study fundamental cellular processes that are conserved in complex multicellular organisms, such as humans. Ubls are a family of proteins that share structural relationships with ubiquitin, and which modify target proteins and lipids. These modifiers are processed, activated and conjugated to substrates by cognate enzymatic cascades. The attachment of substrates to Ubls alters the various properties of these substrates, such as function, interaction with the environment or turnover, and accordingly regulate key cellular processes, including DNA damage, cell cycle progression, metabolism, stress response, cellular differentiation, and protein homeostasis. Thus, it is not surprising that Ubls serve as tools to study the underlying mechanism involved in cellular health. We summarize current knowledge on the activity and mechanism of action of the S. cerevisiae Rub1, Smt3, Atg8, Atg12, Urm1 and Hub1 modifiers, all of which are highly conserved in organisms from yeast to humans.

Strategies for Monitoring "Ubiquitin C-Terminal Hydrolase 1" (Yuh1) Activity.

The family of ubiquitin C-terminal hydrolases (UCHs(releases ε-linked amide bonds positioned at the C-terminus of ubiquitin. UCHL3 is a highly conserved and dual functional member of this family, recognizing C-terminal extensions of two paralogous modifiers: ubiquitin and NEDD8. The Saccharomyces cerevisiae orthologue of UCHL3, namely, Yuh1, is the only UCH family member in this organism. Like UCHL3, Yuh1 recognizes ubiquitin as well as Rub1, the direct orthologue of NEDD8 in S. cerevisiae. We describe here a method for examining the activity of bacteria and yeast expressed Yuh1 by monitoring the C-terminal trimming of UBB + 1 and Rub1 + 1 through immunoblotting and the increased AMC fluorescence readout detected through a plate reader.

Fluorescence Detection of Increased Reactive Oxygen Species Levels in Saccharomyces cerevisiae at the Diauxic Shift.

The budding yeast Saccharomyces cerevisiae is a facultative organism that is able to utilize both anaerobic and aerobic metabolism, depending on the composition of carbon source in the growth medium. When glucose is abundant, yeast catabolizes it to ethanol and other by-products by anaerobic fermentation through the glycolysis pathway. Following glucose exhaustion, cells switch to oxygenic respiration (a.k.a. "diauxic shift"), which allows catabolizing ethanol and the other carbon compounds via the TCA cycle and oxidative phosphorylation in the mitochondria. The diauxic shift is accompanied by elevated reactive oxygen species (ROS) levels and is characterized by activation of ROS defense mechanisms. Traditional measurement of the diauxic shift is done through measuring optical density of cultures grown in a batch at intermediate time points and generating a typical growth curve or by estimating the reduction of glucose and accumulation of ethanol in growth media over time. In this manuscript, we describe a method for determining changes in ROS levels upon yeast growth, using carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA). H2-DCFDA is a widely used fluorescent dye for measuring intracellular ROS levels. H2-DCFDA enables a direct measurement of ROS in yeast cells at intermediate time points. The outcome of H2-DCFDA fluorescent readout measurements correlates with the growth curve information, hence providing a clear understanding of the diauxic shift.

Saccharomyces cerevisiae as a Toolkit for COP9 Signalosome Research.

The COP9 signalosome (CSN) is a highly conserved eukaryotic multi-subunit enzyme, regulating cullin RING ligase activities and accordingly, substrate ubiquitination and degradation. We showed that the CSN complex of Saccharomyces cerevisiae that is deviated in subunit composition and in sequence homology harbors a highly conserved cullin deneddylase enzymatic core complex. We took advantage of the non-essentiality of the S. cerevisiae CSN-NEDD8/Rub1 axis, together with the enzyme-substrate cross-species activity, to develop a sensitive fluorescence readout assay, suitable for biochemical assessment of cullin deneddylation by CSNs from various origins. We also demonstrated that the yeast catalytic subunit, CSN5/Jab1, is targeted by an inhibitor that was selected for the human orthologue. Treatment of yeast by the inhibitor led to the accumulation of neddylated cullins and the formation of reactive oxygen species. Overall, our data revealed S. cerevisiae as a general platform that can be used for studies of CSN deneddylation and for testing the efficacy of selected CSN inhibitors.

Mammalian DET1 regulates Cul4A activity and forms stable complexes with E2 ubiquitin-conjugating enzymes.

DET1 (de-etiolated 1) is an essential negative regulator of plant light responses, and it is a component of the Arabidopsis thaliana CDD complex containing DDB1 and COP10 ubiquitin E2 variant. Human DET1 has recently been isolated as one of the DDB1- and Cul4A-associated factors, along with an array of WD40-containing substrate receptors of the Cul4A-DDB1 ubiquitin ligase. However, DET1 differs from conventional substrate receptors of cullin E3 ligases in both biochemical behavior and activity. Here we report that mammalian DET1 forms stable DDD-E2 complexes, consisting of DDB1, DDA1 (DET1, DDB1 associated 1), and a member of the UBE2E group of canonical ubiquitin-conjugating enzymes. DDD-E2 complexes interact with multiple ubiquitin E3 ligases. We show that the E2 component cannot maintain the ubiquitin thioester linkage once bound to the DDD core, rendering mammalian DDD-E2 equivalent to the Arabidopsis CDD complex. While free UBE2E-3 is active and able to enhance UbcH5/Cul4A activity, the DDD core specifically inhibits Cul4A-dependent polyubiquitin chain assembly in vitro. Overexpression of DET1 inhibits UV-induced CDT1 degradation in cultured cells. These findings demonstrate that the conserved DET1 complex modulates Cul4A functions by a novel mechanism.

Phosphatidylinositol-3-kinase as a therapeutic target in melanoma.

PURPOSE: Phosphatidylinositol-3 kinases (PI3K) are critical for malignant cellular processes including growth, proliferation, and survival, and are targets of drugs in clinical development. We assessed expression of PI3K in melanomas and nevi, and studied associations between PI3K pathway members and in vitro response to a PI3K inhibitor, LY294002. EXPERIMENTAL DESIGN: Using Automated Quantitative Analysis, we quantified expression of p85 and p110alpha subunits in 540 nevi and 523 melanomas. We determined the IC(50) for LY294002 for 11 melanoma cell lines and, using reverse phase protein arrays, assessed the association between levels of PI3K pathway members and sensitivity to LY294002. RESULTS: p85 and p110alpha tend to be coexpressed (P < 0.0001); expression was higher in melanomas than nevi (P < 0.0001) for both subunits, and higher in metastatic than primary melanomas for p85 (P < 0.0001). Although phospho-Akt (pAkt) levels decreased in all cell lines treated with LY294002, sensitivity was variable. We found no association by t tests between baseline p85, p110alpha, and pAkt levels and sensitivity to LY294002, whereas pS6 Ser(235) and Ser(240) were lower in the more resistant cell lines (P = 0.01 and P = 0.004, respectively). CONCLUSIONS: Expression of p85 and p110alpha subunits is up-regulated in melanoma, indicating that PI3K is a good drug target. Pretreatment pS6 levels correlated with sensitivity to the PI3K inhibitor, LY294002, whereas PI3K and pAkt did not, suggesting that full activation of the PI3K pathway is needed for sensitivity to PI3K inhibition. pS6 should be evaluated as a predictor of response in melanoma patients treated with PI3K inhibitors, as these drugs enter clinical trials.

The necessity of NEDD8/Rub1 for vitality and its association with mitochondria-derived oxidative stress.

Access of molecular oxygen to the respiratory electron transport chain at the mitochondria costs in the generation of reactive oxygen-derived species (ROS). ROS induces progressive damage to macromolecules in all living cells, hence, rapid defense mechanisms to maintain cellular redox homeostasis are vital. NEDD8/Rub1 is a highly conserved ubiquitin-like modifier that has recently been identified as a key regulator of cellular redox homeostasis. In this review, I will present NEDD8/Rub1, its modification cascade of enzymes, substrates and hydrolases. After introduction, I will show that the NEDD8/Rub1 pathway is linked with mitochondria physiology, namely, oxidative stress. In the rest of the review, I will approach the Ascomycota phylum of the kingdom fungi instrumentally, to present existing links between NEDD8/Rub1 vitality and the aerobic lifestyle of model species belonging to three subphyla: Saccharomycotina (S. cerevisiae and C. albicans), Pezizomycotina (A. nidulans and N. crassa), and Taphrinomycotina (S. pombe).

High HSP90 expression is associated with decreased survival in breast cancer.

The heat shock protein HSP90 chaperones proteins implicated in breast cancer progression, including Her2/neu. HSP90-targeting agents are in clinical trials for breast cancer. HSP90 expression is high in breast cancer cell lines, yet no large studies have been conducted on expression in human tumors and the association with clinical/pathologic variables. Tissue microarrays containing 10 cell lines and primary specimens from 655 patients with 10-year follow-up were assessed using our automated quantitative analysis (AQUA) method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured HSP90 expression within the mask using Cy5-conjugated antibodies. We similarly assessed estrogen receptor, progesterone receptor, and Her2/neu expression. HSP90 expression was more variable in human tumors than in cell lines (P < 0.0001). High HSP90 expression was associated with decreased survival (P = 0.0024). On multivariable analysis, high HSP90 expression remained an independent prognostic marker. High HSP90 expression was associated with high Her2/neu and estrogen receptor, large tumors, high nuclear grade, and lymph node involvement. Although HSP90 levels were high in all our cell lines, expression in tumors was more variable. High HSP90 expression in primary breast cancer defines a population of patients with decreased survival. Evaluation of HSP90 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.

The Proteasome Lid Triggers COP9 Signalosome Activity during the Transition of Saccharomyces cerevisiae Cells into Quiescence.

The class of Cullin-RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in Saccharomyces cerevisiae). Cullin modification is then reversed by hydrolytic action of the COP9 signalosome (CSN). As the NEDD8-Rub1 catalytic cycle is not essential for the viability of S. cerevisiae, this organism is a useful model system to study the alteration of Rub1-CRL conjugation patterns. In this study, we describe two distinct mutants of Rpn11, a proteasome-associated deubiquitinating enzyme, both of which exhibit a biochemical phenotype characterized by high accumulation of Rub1-modified Cdc53-Cullin1 (yCul1) upon entry into quiescence in S. cerevisiae. Further characterization revealed proteasome 19S-lid-associated deubiquitination activity that authorizes the hydrolysis of Rub1 from yCul1 by the CSN complex. Thus, our results suggest a negative feedback mechanism via proteasome capacity on upstream ubiquitinating enzymes.

Assessing expression of apoptotic markers using large cohort tissue microarrays.

Apoptotic markers include proteins from the intrinsic and extrinsic pathways. These cascades include both pro-apoptotic and anti-apoptotic elements. The expression levels of these elements can be assessed by immunohistochemistry (IHC) and can indicate general trends in prov-ersus anti-apoptotic tendencies of the cells. IHC is particularly useful when studying large cohorts of paraffin-embedded specimens. Advances in tissue microarray (TMA) technology have facilitated evaluation of large cohorts of specimens, as cores from hundreds of patients can be represented on a single glass slide and stained in a uniform fashion. In this chapter, we discuss construction and staining methods of TMAs and present examples of assessment of apoptotic marker expression in malignant and benign cells using a novel method of automated, quantitative analysis of in situ protein expression.

Immunodepletion and Immunopurification as Approaches for CSN Research.

The COP9 signalosome (CSN) is an evolutionary conserved complex that is found in all eukaryotes, and implicated in regulating the activity of Cullin-RING ubiquitin Ligases (CRLs). Activity of CRLs is highly regulated; complexes are active when the cullin subunit is covalently attached to the ubiquitin like modifier, Nedd8. Neddylation/deneddylation cycles are required for proper CRLs activity, and deneddylation is performed by the CSN complex.We describe here a method utilizing resin-coupled antibodies to deplete the CSN from human cell extracts, and to obtain endogenous CSN complexes by immunopurification. In the first step, the cross-linked primary antibodies recognize endogenous CSN complexes, and deplete them from cell extract as the extract passes through the immunoaffinity column. The resulting "CSN-depleted extract" (CDP) is rich in neddylated cullins that can be used as a substrate for cullin-deneddylation assay for CSN complexes purified from various eukaryotes. Consequently, regeneration of the column results in dissociation of a highly purified CSN complex, together with its associated proteins. Immunopurification of the CSN from various human tissues or experimental conditions is advantageous for the generation of numerous CSN-interaction maps.