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Cancer Mutations in SPOP Put a Stop to Its Inter-compartmental Hops

2018; Elsevier BV; Volume: 72; Issue: 1 Linguagem: Inglês

10.1016/j.molcel.2018.09.025

1097-4164

Julia F. Riley, Thuy P. Dao, Carlos A. Castañeda,

Lipid metabolism and biosynthesis

In this issue of Molecular Cell, Bouchard et al., 2018Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. et al.Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72 (this issue): 19-36Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar identify liquid-liquid phase separation as a mechanism for substrate-triggered localization of SPOP and ubiquitination machinery to different nuclear bodies and describe how cancer mutations disrupt this process. In this issue of Molecular Cell, Bouchard et al., 2018Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. et al.Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72 (this issue): 19-36Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar identify liquid-liquid phase separation as a mechanism for substrate-triggered localization of SPOP and ubiquitination machinery to different nuclear bodies and describe how cancer mutations disrupt this process. In addition to well-characterized membrane-bound organelles, a diverse population of membraneless organelles also exist. The latter category encompasses cytoplasmic and nuclear bodies, which have been proposed to form via liquid-liquid phase separation (LLPS) of macromolecules. Although the functions and compositions of these biomolecular condensates are not fully known, it is well documented that disease mutations in their phase separating components promote their dysfunction by disrupting disassembly and/or driving liquid-to-solid phase transitions (Boeynaems et al., 2018Boeynaems S. Alberti S. Fawzi N.L. Mittag T. Polymenidou M. Rousseau F. Schymkowitz J. Shorter J. Wolozin B. Van Den Bosch L. et al.Protein phase separation: a new phase in cell biology.Trends Cell Biol. 2018; 28: 420-435Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). Interestingly, many of these systems are associated with neurological disorders such as amyotrophic lateral sclerosis (ALS) and Alzheimer's and Parkinson's diseases. Here, Mittag and coworkers present a new mechanism through which disruption of LLPS could lead to cancer (Bouchard et al., 2018Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. et al.Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72 (this issue): 19-36Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The authors show that SPOP (speckle-type POZ protein), a substrate adaptor protein for cullin3-RING ubiquitin E3 ligase (CRL3), functions by phase separating with ubiquitination substrates to form distinct membraneless organelles. Cancer mutations in SPOP, which are found in up to 11% of prostate cancers (Barbieri et al., 2012Barbieri C.E. Baca S.C. Lawrence M.S. Demichelis F. Blattner M. Theurillat J.-P. White T.A. Stojanov P. Van Allen E. Stransky N. et al.Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer.Nat. Genet. 2012; 44: 685-689Crossref PubMed Scopus (1122) Google Scholar), negatively regulate the LLPS process between SPOP and substrates and prevent ubiquitination of substrates, leading to upregulated substrate levels and disrupted proteostasis. Contrary to its namesake, SPOP is found in various nuclear bodies including speckles, PML bodies, and DNA-damage loci (Marzahn et al., 2016Marzahn M.R. Marada S. Lee J. Nourse A. Kenrick S. Zhao H. Ben-Nissan G. Kolaitis R.-M. Peters J.L. Pounds S. et al.Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.EMBO J. 2016; 35: 1254-1275Crossref PubMed Scopus (117) Google Scholar, Nagai et al., 1997Nagai Y. Kojima T. Muro Y. Hachiya T. Nishizawa Y. Wakabayashi T. Hagiwara M. Identification of a novel nuclear speckle-type protein, SPOP.FEBS Lett. 1997; 418: 23-26Crossref PubMed Scopus (99) Google Scholar). Although underlying mechanisms of SPOP's localization to these different bodies were not known, the presence of substrates could be a contributing factor, as demonstrated by the formation of distinct membraneless bodies containing SPOP and one of its substrates, Gli3 (Marzahn et al., 2016Marzahn M.R. Marada S. Lee J. Nourse A. Kenrick S. Zhao H. Ben-Nissan G. Kolaitis R.-M. Peters J.L. Pounds S. et al.Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.EMBO J. 2016; 35: 1254-1275Crossref PubMed Scopus (117) Google Scholar). Since the cellular signals for the relocalization of SPOP to different bodies are largely unknown, Bouchard et al., 2018Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. et al.Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72 (this issue): 19-36Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar cleverly used transient overexpression of SPOP and substrates to trigger such an event. The authors showed that when transiently co-expressed in HeLa or prostate cancer cell lines, both SPOP and substrate moved away from their respective compartments to newly formed SPOP:substrate compartments. Thorough characterizations of wild-type and mutant SPOP and substrates revealed that SPOP oligomerization and SPOP:substrate interactions together promote the LLPS process that drives the assembly of SPOP-containing nuclear bodies (Figure 1). Oligomerization is closely associated with the ability to form the multivalent interactions that drive biomolecular condensation into liquid-like compartments (Dao et al., 2018Dao T.P. Kolaitis R.-M. Kim H.J. O'Donovan K. Martyniak B. Colicino E. Hehnly H. Taylor J.P. Castañeda C.A. Ubiquitin modulates liquid-liquid phase separation of UBQLN2 via disruption of multivalent interactions.Mol. Cell. 2018; 69: 965-978.e6Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, Mitrea et al., 2018Mitrea D.M. Cika J.A. Stanley C.B. Nourse A. Onuchic P.L. Banerjee P.R. Phillips A.H. Park C.-G. Deniz A.A. Kriwacki R.W. Self-interaction of NPM1 modulates multiple mechanisms of liquid-liquid phase separation.Nat. Commun. 2018; 9: 842Crossref PubMed Scopus (176) Google Scholar). While SPOP's oligomerization is required for its localization into membraneless organelles, SPOP does not independently undergo LLPS (Marzahn et al., 2016Marzahn M.R. Marada S. Lee J. Nourse A. Kenrick S. Zhao H. Ben-Nissan G. Kolaitis R.-M. Peters J.L. Pounds S. et al.Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.EMBO J. 2016; 35: 1254-1275Crossref PubMed Scopus (117) Google Scholar). Rather, Bouchard et al., 2018Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. et al.Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72 (this issue): 19-36Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar show that LLPS occurs when SPOP interacts with substrates such as death-domain-associated protein (DAXX) and androgen receptor (AR). Their data suggest that SPOP's oligomerization primes SPOP to phase separate with substrate targets. Convincingly, they show that oligomerization-incompetent SPOP mutants fail to phase separate with substrate in vitro and fail to colocalize with substrate in cells (Figure 1). Weak, multivalent interactions between biomolecules are necessary for phase separation under physiologically relevant conditions (Mittag and Parker, 2018Mittag T. Parker R. Multiple modes of protein-protein interactions promote RNP granule assembly.J. Mol. Biol. 2018; (Published online August 9, 2018)https://doi.org/10.1016/j.jmb.2018.08.005Crossref PubMed Scopus (118) Google Scholar). Therefore, Bouchard et al., 2018Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. et al.Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72 (this issue): 19-36Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar hypothesized that SPOP:substrate multivalent interactions promoted LLPS and, correspondingly, the formation of distinct SPOP:substrate membraneless nuclear compartments. The authors demonstrated that DAXX and many other SPOP substrates contain multiple SPOP-binding (SB) motifs comprising short Ser/Thr polar sequences. Through meticulous biophysical characterization, they determined that the intermolecular interactions between SB-motif peptides and the MATH domain of SPOP were weak, on the order of 40–500 μM. Together, these collective weak interactions are likely the "stickers" that promote SPOP:substrate LLPS. For confirmation, the authors scrambled the sequence of SB-motifs and found that both SPOP:substrate binding and LLPS propensity were significantly reduced. Furthermore, transient expression studies of binding-incompetent SPOP or substrate mutants failed to produce SPOP:substrate nuclear bodies. Interestingly, overexpression of disease-linked mutant SPOP and substrate partially rescues the formation of SPOP:substrate compartments. These findings are consistent with a model whereby increasing protein concentrations recover the transient interactions necessary to promote SPOP:substrate LLPS. In other words, SPOP cancer mutations not only reduce SPOP:substrate binding, but also increase the protein concentration threshold required for SPOP:substrate LLPS. As SPOP is a substrate adaptor protein for the Cullin3 ubiquitin (Ub) ligase, the authors tested whether SPOP:substrate liquid bodies are active ubiquitination centers. They elegantly constructed an in vitro ubiquitination phase-separating system containing Ub-charged E2, neddylated cullin E3 ligase, SPOP, and DAXX substrate. Using fluorescently labeled Ub, they demonstrated that Ub colocalized inside SPOP membraneless bodies only in the presence of functional ubiquitinating enzymes, and that DAXX was ubiquitinated. Ubiquitination occurred in either filamentous or liquid assemblies of SPOP/DAXX. Surprisingly, ubiquitination kinetics appeared to be the same in the presence or absence of mesoscale assemblies in vitro. However, it is important to note that the dynamics in SPOP/DAXX bodies are significantly reduced compared to the dilute phase, as shown by fluorescence recovery after photobleaching (FRAP) experiments. This decrease in dynamics in mesoscale assemblies might offset the increase in local protein concentration, accounting for lack of improvement in enzymatic activity. Bouchard et al., 2018Bouchard J.J. Otero J.H. Scott D.C. Szulc E. Martin E.W. Sabri N. Granata D. Marzahn M.R. Lindorff-Larsen K. Salvatella X. et al.Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments.Mol. Cell. 2018; 72 (this issue): 19-36Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar provide convincing evidence that phase-separated droplets are not just sequestration centers, but active compartments. Their research provokes a myriad of questions. Can different SPOP:substrate membraneless compartments exist concurrently in cell nuclei? What are the cellular signals (including post-translational modifications) that promote endogenous SPOP recruitment to these different substrate bodies? Once substrates are ubiquitinated in SPOP phase-separated bodies, how are they degraded? Is proteasome machinery recruited into these SPOP bodies, as recently demonstrated in stress granules (Turakhiya et al., 2018Turakhiya A. Meyer S.R. Marincola G. Böhm S. Vanselow J.T. Schlosser A. Hofmann K. Buchberger A. ZFAND1 recruits p97 and the 26S proteasome to promote the clearance of arsenite-induced stress granules.Mol. Cell. 2018; 70: 906-919.e7Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar)? Are ubiquitinated substrates able to leave on their own, or are they directed out of membraneless bodies to proteasome or degradation machinery elsewhere? This work is both highly relevant to the pathology of SPOP-related diseases and far-reaching in implications. Currently, the membraneless organelles predominantly associated with protein quality control are stress granules. The recognition that ubiquitination occurs inside SPOP mesoscale assemblies, the formation of which might be vital to the correct localization of some E3 ubiquitin ligases to their substrates, is an intriguing twist in the current narrative. SPOP:substrate LLPS-directed ubiquitination is an interesting new example of protein quality control of phase-separated proteins. This work provides a possible mechanism for the recruitment of ubiquitination machinery to other membraneless compartments, including stress granules, in which ubiquitin-like post-translational modifications (e.g., SUMOylation and NEDDylation) have been shown to occur. Interestingly, disruption to phase separating components of stress granules could also be disease-causing, possibly leading to protein-containing ubiquitinated inclusions characteristic of neurological disorders such as ALS. Recently, Schuster et al., 2018Schuster B.S. Reed E.H. Parthasarathy R. Jahnke C.N. Caldwell R.M. Bermudez J.G. Ramage H. Good M.C. Hammer D.A. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles.Nat. Commun. 2018; 9: 2985Crossref PubMed Scopus (177) Google Scholar show that LLPS systems can be robustly engineered to recruit and release cargo. Membraneless organelles can be programmed to compartmentalize proteins, much in the same way SPOP is compartmentalized to substrate-dependent membraneless bodies. These works provide a potential foundation for developing therapeutic avenues for SPOP-mediated cancers once we understand the molecular determinants and cellular signals for localization to different membraneless organelles. Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated CompartmentsBouchard et al.Molecular CellSeptember 20, 2018In BriefMutations in the tumor suppressor SPOP are known to cause solid tumors. Bouchard and Otero et al. show that SPOP phase separates with substrates in vitro; the same interactions mediate co-localization in membraneless organelles in cells. SPOP cancer mutations disrupt liquid-liquid phase separation, which correlates with loss of function. Full-Text PDF Open Archive