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common in soil and aqueous environments, including wastewater (Yabuuchi and Kosako, 2005). They are also well known for their ability to degrade a wide variety of complex organic molecules. In the current work, Yin-Ru Chiang and his colleagues Yi-Lung Chen, Chang-Ping Yu, and others at the Academica Sinica and National Taiwan Normal University then identified 4-hydroxyestrone and pyridinestrone acid as likely intermediates in estrogen degradation by strain KC8. From these intermediates, it was possible to predict the types of enzyme likely to be present in the pathway. When the genome of stain KC8 was sequenced, two genes clusters were found to be differentially expressed following growth on estrogen. An enzyme, 4-hydroxyestrone 4,5dioxygenase, encoded by one of the clusters was also purified from estrogen-grown cells of strain KC8, and the gene identified by mass spectroscopy. Cloning and biochemical characterization of the recombinant enzyme further confirmed its role in estrogen degradation. These studies allowed the authors to propose the 4,5-seco pathway of aerobic estrogen degradation.

Chen et al. (2017) also addressed the issue of whether or not the estrogen degradation pathway found in Sphingomonas strain KC8 was widely distributed. They searched genome databases and found evidence for it in a variety of alphaproteobacteria and gammaproteobacteria. Moreover, following estrogen additions to river water and activated sludge, pyridinestrone acid was readily detected. These experiments suggest that this pathway is widely distributed in free-living bacteria. An exciting aspect of this work is that it is only the beginning. A number of other questions are immediately obvious. What are the roles of the other genes in the estrogen degradation clusters? What is the complete pathway for estrogen mineralization? Are there other pathways of estrogen degradation? The current studies provide the means to answer these more general questions. On the applied side, now that some of the genes of estrogen degradation are known, it is possible to use molecular ecological techniques to follow their distribution in the environment. If they correlate with estro-

gen-degrading activity, a new tool will be available to evaluate the impact of estrogen pollution and predict its recalcitrance.

REFERENCES Chen, T.S., Chen, T.C., Yeh, K.J.C., Chao, H.R., Liaw, E.T., Hsieh, C.Y., Chen, K.C., Hsieh, L.T., and Yeh, Y.L. (2010). Sci. Total Environ. 408, 3223–3230. Chen, Y.-L., Yu, C.-P., Lee, T.-H., Goh, K.-S., Chu, K.-H., Wang, P.-H., Ismail, W., Shih, C.-J., and Chiang, Y.-R. (2017). Cell Chem. Biol. 24, this issue, 712–724. Palme, R., Fischer, P., Schildorfer, H., and Ismail, M.N. (1996). Anim. Reprod. Sci. 43, 43–63. Roh, H., and Chu, K.H. (2010). Environ. Sci. Technol. 44, 4943–4950. Wise, A., O’Brien, K., and Woodruff, T. (2011). Environ. Sci. Technol. 45, 51–60. Yabuuchi, E., and Kosako, Y. (2005). In Bergey’s Manual of Systematic Bacteriology, Vol. 2C, D.J. Brenner, N.R. Krieg, and J.T. Staley, eds. (Springer), pp. 234–258. Young, W.F., Whitehouse, P., Johnston, I., and Sorokin, N. (2002). Proposed Predicted-No-EffectConcentrations (PNECs) for Natural and Synthetic Steroid Oestrogens in Surface Waters (England and Wales Environmental Agency Bristol).

Turning-ON Proteasomes Jan Henrik Krahn,1 Farnusch Kaschani,1 and Markus Kaiser1,*

€tsstraße 2, 45117 Essen, Germany Biology, University of Duisburg-Essen, ZMB, Faculty of Biology, Universita *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2017.06.005

1Chemical

While proteasome inhibitors are now well-established research tools and chemotherapeutics, proteasome activators are much less explored. In this issue of Cell Chemical Biology, in a study from the groups of Berkers and Ovaa (Leestemaker et al., 2017), a chemical screen was used to identify a p38 MAPK inhibitor as a proteasome activator. This compound furthermore enhanced clearance of protein aggregates, thereby implicating alternative chemotherapeutic options for treating neurodegenerative diseases. The ubiquitin-proteasome system (UPS) represents the core machinery of regulated protein degradation and is involved in almost all cellular processes. Many small molecule modulators of various components of the UPS are available, and chemical modulation of the UPS has emerged as a promising strategy for developing novel

chemotherapies (Huang and Dixit, 2016; Nalepa et al., 2006); e.g., through inhibition of the proteasome as the proteolytic ‘‘heart’’ of the UPS. Indeed, by far most known UPS modulators act via inhibition of the UPS. In contrast, the alternative approach, namely, small molecule mediated UPS activation by direct proteasome

activation, has so far been much less explored (Lee et al., 2010), despite its potential as a new chemotherapeutic approach, especially in the treatment of various neurodegenerative disorders (Chondrogianni et al., 2015). The lack of a suitable screening approach to identify proteasome activators is one reason why

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Figure 1. The Experimental Workflow for Identifying p38 MAPK Pathway Inhibition as a Proteasome Activating Mechanism (A) Chemical structure of the proteasome-specific activity-based probe (ABP) used in the highthroughput assay. The reactive vinyl sulfone moiety binding covalently to the active site is highlighted in yellow, and the fluorophore for detecting labeled proteasomes is highlighted in magenta. (B) Mechanistic representation of proteasome labeling by the ABP that is, among other factors, dependent on the opening of the proteasome gate for gaining access to the active site. (C) Overview of the FACS-based high-throughput screen for proteasome activators. Labeling of active proteasomes by the proteasome-specific probe leads to a fluorescence signal that can be read out and quantified by FACS or, as an alternative analysis method, SDS-PAGE. This workflow led to identification of 11 small molecule proteasome activators, among them the p38 MAPK inhibitor PD169316. (D) Overview of the performed systematic small molecule or siRNA modulations of the p38 MAPK pathway. Inhibition of the pathway leads to amplification of proteasomal activity while its activation leads to reduced proteasomal activity.

such promising compounds are currently not forthcoming. The persistent challenge to elucidate such direct proteasome activators has now been taken up by the groups of Celia R. Berkers and Huib Ovaa (Leestemaker et al., 2017). To this end, they first established a novel FACS-based highthroughput screen for directly measuring proteasome activity in living cells. This assay was based on a known proteasome-directed activity-based probe (ABP) that binds to the proteasomal active sites in an activity-dependent manner (Figure 1A) (Verdoes et al., 2006). Cellular proteasome activity is regulated by diverse factors; for example by regulating spatial access to the proteolytic sites via a gating mechanism (Figure 1B) (Finley et al., 2016). The ABP is tagged with a fluorophore so that labeling of active proteasomes results in a fluorescence signal. Its intensity thereby correlates with proteasome activity and can be directly quantified in living cells via FACS. A cellular pre-incubation with chemical modulators before application of the proteasome ABP will thus allow a technically simple cell-based screening for chemical proteasome activators and inhibitors. By screening a library of 2750 compounds, the authors were able to identify overall 11 small molecules with diverse chemical structures that amplified proteasomal activity (Figure 1C). Of these, one compound, PD169316, was particularly interesting because it is 654 Cell Chemical Biology 24, June 22, 2017

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a known active site p38 MAPK inhibitor, thereby indicating a regulatory link between the p38 MAPK signaling pathway and the UPS. Accordingly, the authors focused their studies on this compound and the corresponding signal transduction cascade. To confirm that the observed proteasome activation indeed stemmed from p38 MAPK inhibition, the proteasome activation assay was repeated with another set of commercially available p38 MAPK inhibitors, among them the highly p38a-selective compound skepinone-L, as well as via siRNA experiments versus all four p38 MAPK isoforms. Interestingly, in both experiment series, inhibition or downregulation of p38a MAPK resulted in the highest proteasome activation. To better understand which component of the p38 MAPK pathway besides p38a MAPK contributes to proteasome activation, the authors performed an siRNA screen for depleting all major kinases of this signaling pathway. They found the upstream components ASK1 and MKK6 as well as the downstream kinase MK2 as proteasome activity modulating factors. This indicates that proteasome activity is at least partly modulated by the kinase MK2 and application of the pharmacological MK2 inhibitor III indeed led to proteasome activation (Figure 1D). The authors therefore next asked how the p38 MAPK pathway affects proteasome activity. This could for example be achieved by post-translational modification, in particular phosphorylation, of the proteasome. To test whether p38 MAPK inhibition-mediated proteasome activation is persistent, the authors used a tagged proteasome species that allowed isolation of proteasomes after p38 MAPK pathway inhibition. Indeed, they found that isolated proteasomes retained their higher proteolytic activity resulting from p38 MAPK inhibition, indicating that the observed activation is caused by direct proteasome modulation such as post-translational modifications. However, all further experiments to better understand the molecular basis of this effect did not reveal a clear mechanism. For example, phosphoproteomics analyses of the proteasome after MAPK pathway inhibition showed no significant changes in the phosphosite signature. All other tested known proteasome activity influ-

encing factors, such as proteasome assembly or differential recruitment of proteasome-associated factors, were also uninfluenced by p38 MAPK inhibitor treatments. In the last part of their study, the authors then turned their attention to the biomedical applicability of proteasome activation via p38 MAPK pathway inhibition. To this end, they first determined the maximal level of proteasome activation that can be achieved by impairing this pathway. High activation levels up to 4-fold were reached, particularly at low basal proteasome activity levels, as, for example, observed in neurodegenerative disease conditions. This amplification of proteolytic activity, however, did not result in a massive increase in proteasomal protein turnover, but only in changes in levels of selected proteins in accordance with the known finding that under normal cellular conditions, ubiquitination and not degradation represents the rate-limiting step in the proteolysis of most proteins. For proteins with artificially triggered ubiquitination, as, for example, through the use of a PROTAC approach that forces ubiquitination of target proteins (Lai and Crews, 2017), p38 MAPK inhibition increased protein degradation rates. Finally, the authors were also able to show that p38 MAPK pathway inhibition increased clearance of toxic protein assemblies via a cell-based a-synuclein aggregation model. In such conditions, proteasome activity is usually very low, and these experiments therefore strongly indicate that proteasome activation is not resulting in massive, potentially harmful cellular changes under normal but only under non-homeostatic conditions. Proteasome activation might thus represent a feasible alternative chemotherapeutic strategy to combat neurodegenerative diseases. Altogether, the study by Berkers and Ovaa represents an impressive advance in the development of chemical tools and the biomedical exploitation and our functional understanding of the ubiquitin-proteasome system (Leestemaker et al., 2017). First, they were able to develop a systematic high-throughput screening approach for identifying direct small molecule proteasome activators that despite a limited set of only 2750 compounds, delivered many starting points for chemical probe development.

Clearly, a repetition of the screening campaign with a larger small molecule library has the potential to identify further, chemically distinct proteasome activators that might then be further developed into tool compounds or even drugs. Such compounds, with in addition a subsequent target identification approach, may thus reveal many novel and scientifically rewarding insights into the cellular regulation of proteasome activity. In fact, the focused study on the identified p38 MAPK signaling pathway inhibitor already revealed that the p38 MAPK downstream kinase MK2 is involved in proteasome activity regulation, which was unknown before this study. Moreover, the authors were able to show the chemotherapeutic potential of small molecule-triggered proteasome activation. Although further studies in more complex systems are undoubtedly still required, the present work thus opens new avenues for chemotherapeutic inventions, in particular for neurodegenerative disease treatments. It is therefore reasonable to assume that the present study has only been the first step in the field of proteasome activation and will foster future research into this direction.

REFERENCES Chondrogianni, N., Voutetakis, K., Kapetanou, M., Delitsikou, V., Papaevgeniou, N., Sakellari, M., Lefaki, M., Filippopoulou, K., and Gonos, E.S. (2015). Ageing Res. Rev. 23 (Pt A), 37–55. Finley, D., Chen, X., and Walters, K.J. (2016). Trends Biochem. Sci. 41, 77–93. Huang, X., and Dixit, V.M. (2016). Cell Res. 26, 484–498. Lai, A.C., and Crews, C.M. (2017). Nat. Rev. Drug Discov. 16, 101–114. Lee, B.H., Lee, M.J., Park, S., Oh, D.C., Elsasser, S., Chen, P.C., Gartner, C., Dimova, N., Hanna, J., Gygi, S.P., et al. (2010). Nature 467, 179–184. Leestemaker, Y., de Jong, A., Witting, K.F., Penning, R., Schuurman, K., Rodenko, B., Zaal, E.A., van de Kooij, B., Laufer, S., Heck, A.J.R., et al. (2017). Cell Chem. Biol. 24, this issue, 725–736. Nalepa, G., Rolfe, M., and Harper, J.W. (2006). Nat. Rev. Drug Discov. 5, 596–613. Verdoes, M., Florea, B.I., Menendez-Benito, V., Maynard, C.J., Witte, M.D., van der Linden, W.A., van den Nieuwendijk, A.M., Hofmann, T., Berkers, C.R., van Leeuwen, F.W., et al. (2006). Chem. Biol. 13, 1217–1226.

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