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

The rejuvenating power of the Buena Vista Social Club

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes

In the land of the rising sun with the COP9 signalosome and related Zomes. Symposium on the COP9 signalosome, Proteasome and eIF3

International audienc

Duplication and familial promiscuity within the proteasome lid and COP9 signalosome kin complexes

Two paralogous complexes, the proteasome lid and the COP9 signalosome (CSN), have diverged from a common ancestor; yet fulfill distinctive roles within the ubiquitin-proteasome sphere. The CSN regulates the largest family of E3 ubiquitin ligases, called CRLs (Cullin-RING ubiquitin Ligases), while the lid is a subcomplex of the 26 S proteasome, a proteolytic machinery responsible for the degradation of ubiquitinated proteins. Remarkably, in many organisms, several subunits of both complexes are duplicated, a circumstance that can hypothetically increase the number of different complexes that can be formed. Duplication, however, is not the only complexity trait within the lid and the CSN, because many of their subunits are not fully committed only to one of the two complexes, but they are able to associate with both. Indeed, their corresponding mutants have features that can be due to the absence of more than one complex. This could be simply explained by the subunits being able to carry an identical function within more than one paralogdus complex or by the subunits having a certain level of promiscuity, i.e. being able to carry more than one function, depending on the complex they are associating with. Recent data show that both options are possible and, although their functional relevance still needs to be fully uncovered, evidence is accumulating, which indicates a promiscuous trading of paralogous subunits, and suggests that this may occur transiently, and/or in response to particular environmental conditions. (C) 2013 Elsevier Ireland Ltd. All rights reserved

Cullin E3 Ligases and Their Rewiring by Viral Factors

The ability of viruses to subvert host pathways is central in disease pathogenesis. Over the past decade, a critical role for the Ubiquitin Proteasome System (UPS) in counteracting host immune factors during viral infection has emerged. This counteraction is commonly achieved by the expression of viral proteins capable of sequestering host ubiquitin E3 ligases and their regulators. In particular, many viruses hijack members of the Cullin-RING E3 Ligase (CRL) family. Viruses interact in many ways with CRLs in order to impact their ligase activity; one key recurring interaction involves re-directing CRL complexes to degrade host targets that are otherwise not degraded within host cells. Removal of host immune factors by this mechanism creates a more amenable cellular environment for viral propagation. To date, a small number of target host factors have been identified, many of which are degraded via a CRL-proteasome pathway. Substantial effort within the field is ongoing to uncover the identities of further host proteins targeted in this fashion and the underlying mechanisms driving their turnover by the UPS. Elucidation of these targets and mechanisms will provide appealing anti-viral therapeutic opportunities. This review is focused on the many methods used by viruses to perturb host CRLs, focusing on substrate sequestration and viral regulation of E3 activity

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

Centrosomal Chk2 in DNA damage responses and cell cycle progession

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators

S.cerevisiae as a tool to select inhibitors of the deneddylating activity of the COP9 signalome Angela Cirigliano, Sergio Menta, Mattia Mori, Valerio Licursi, Svetlana Danovska, Valentina Vapore, Giovanna Serino, Elah Pick, Bruno Botta, Rodolfo Negri, and Teresa Rinaldi

The COP9 signalosome (CSN) protein complex plays a key role in regulating cullin-RING ligases and is a central mediator of cellular functions essential for cancer progression. The CSN5/Jab1 gene, encoding the catalytic subunit of the complex, has been found amplified in many tumors; however, due to its pleiotropic effects, it has been difficult to dissect Csn5 function and its involvement in cancer progression. Moreover, while a growing body of evidence point to the neddylation pathway as a good target for drug development, specific inhibitors have not yet been developed for the Csn5 enzyme. Deneddylation by the CSN is conserved in the budding yeast Saccharomyces cerevisiae and, in contrast to human or plants, lack of Csn5 does not compromise viability of budding yeast. We have recently performed a transcriptomic and proteomic analysis of a csn5 strain to assess its function in budding yeast, and we have shown that Csn5 is involved in the modulation of the genes controlling amino acid and lipid metabolism and in particular of the ergosterol biosynthesis; this observation correlates with lower ergosterol level in csn5 cells (Licursi et al., (2014). FEBS Journal, 281(1), 175-190) In the study shown here, we have used budding yeast as a model to identify novel inhibitors of Csn5 deneddylating activity. We present our preliminary results obtained using a combined approach of molecular modelling and simple genetic tools to identify small molecules as selective inhibitors of Csn5 deneddylating functio