Harnessing the Power of Proteolysis for Targeted Protein Inactivation
2020; Elsevier BV; Volume: 77; Issue: 3 Linguagem: Inglês
10.1016/j.molcel.2020.01.010
ISSN1097-4164
AutoresRati Verma, Dane Mohl, Raymond J. Deshaies,
Tópico(s)CAR-T cell therapy research
ResumoTwo decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients. Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients. The eukaryotic cellular milieu is in dynamic flux with biomolecules synthesized and degraded continuously. Proteins that are destined to be degraded are targeted to the proteasome by ubiquitin (Ub), which is covalently attached to acceptor lysines on the substrate by a sequential cascade of Ub-activating (E1), Ub-conjugating (E2), and Ub-ligase (E3) enzymes (Figure 1). Ub conjugation can also serve as a signal for the autophagic pathway whereby cytosolic contents destined for degradation are first sequestered in a compartment bounded by a double membrane, after which the outer membrane fuses with the lysosome to deliver the encapsulated vesicle into the lysosome for destruction. Recent reviews have been written on the Ub-proteasome system (UPS) (Bard et al., 2018Bard J.A.M. Goodall E.A. Greene E.R. Jonsson E. Dong K.C. Martin A. Structure and Function of the 26S Proteasome.Annu. Rev. Biochem. 2018; 87: 697-724Crossref PubMed Scopus (104) Google Scholar) and autophagy (Dikic and Elazar, 2018Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (428) Google Scholar) and thus we will not describe them in further detail here other than to point out that the UPS operates in both the nuclear and cytosolic compartments whereas autophagy operates in the cytosol and can degrade individual proteins, protein complexes, macromolecular aggregates, and even entire organelles. In addition, both systems involve recognition of specific signals on targets and thus can potentially be repurposed to effect the specific elimination of individual proteins (via the UPS) or assemblages (via autophagy) from cells. A wealth of early molecular studies on DNA tumor viruses indicated that they subvert cellular regulatory pathways by coding for proteins that bind to and inactivate key cellular factors like the tumor suppressor proteins p53 and Rb (Dybas et al., 2018Dybas J.M. Herrmann C. Weitzman M.D. Ubiquitination at the interface of tumor viruses and DNA damage responses.Curr. Opin. Virol. 2018; 32: 40-47Crossref PubMed Scopus (6) Google Scholar, Mahon et al., 2014Mahon C. Krogan N.J. Craik C.S. Pick E. Cullin E3 ligases and their rewiring by viral factors.Biomolecules. 2014; 4: 897-930Crossref PubMed Scopus (25) Google Scholar). In a landmark study from Peter Howley’s group, investigation of the E6 protein of human papillomaviruses HPV-16 and HPV-18 illuminated an unexpected twist on this theme: the E6 protein of these viruses deploys the cellular E6AP (E6-associated protein) Ub ligase to ubiquitinate p53, resulting in rapid p53 degradation by the proteasome (Scheffner et al., 1993Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53.Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1811) Google Scholar). Another early “lesson from nature” that illustrated the power of hijacking the UPS was courtesy of HIV-1 (human immunodeficiency virus type 1). The viral protein VPU is an integral membrane phosphoprotein that binds to newly synthesized host membrane proteins such as the HIV receptor CD4 and MHC class I molecules. Two conserved phosphoserine residues in the cytoplasmic domain of VPU recruit the F-box protein β-TRCP1 resulting in the formation of a ternary complex comprising CD4, VPU, and Ub ligase SCFβ-TRCP, which triggers ubiquitination and proteasomal degradation of CD4 (Margottin et al., 1998Margottin F. Bour S.P. Durand H. Selig L. Benichou S. Richard V. Thomas D. Strebel K. Benarous R. A novel human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif.Mol. Cell. 1998; 1: 565-574Abstract Full Text Full Text PDF PubMed Google Scholar). It is now appreciated that inducing degradation of cellular proteins is a common strategy employed by a broad range of viruses to subvert cellular regulatory mechanisms and antiviral defenses (Mahon et al., 2014Mahon C. Krogan N.J. Craik C.S. Pick E. Cullin E3 ligases and their rewiring by viral factors.Biomolecules. 2014; 4: 897-930Crossref PubMed Scopus (25) Google Scholar). It is estimated that no fewer than two dozen viruses deploy one or more proteins that serve as a molecular bridge to link a cellular protein target to a Ub ligase. HIV-1 is perhaps the most opportunistic, employing four different proteins (VPU, VPR, VPX, and VIF) to target host proteins for elimination. Apart from SCFβ-TRCP (a member of the CRL1 cullin-RING ligase family), VPR and VPX hijack CRL4 and VIF repurposes CRL5 to degrade antiviral proteins (Mahon et al., 2014Mahon C. Krogan N.J. Craik C.S. Pick E. Cullin E3 ligases and their rewiring by viral factors.Biomolecules. 2014; 4: 897-930Crossref PubMed Scopus (25) Google Scholar, Yu et al., 2003Yu X. Yu Y. Liu B. Luo K. Kong W. Mao P. Yu X.F. Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex.Science. 2003; 302: 1056-1060Crossref PubMed Scopus (926) Google Scholar). It is particularly instructive that viruses, which are under intense evolutionary pressure and must make do with compact genomes, have repeatedly selected for targeted degradation as a means to circumvent cellular defenses. Auxin (indole-3-acetic acid or IAA) is a plant hormone that regulates plant development by promoting degradation of the Aux/IAA family of transcriptional repressors via the Ub ligase SCFTIR1. Auxin binds directly to the F-box substrate receptor TIR1 (Dharmasiri et al., 2005Dharmasiri N. Dharmasiri S. Estelle M. The F-box protein TIR1 is an auxin receptor.Nature. 2005; 435: 441-445Crossref PubMed Scopus (1343) Google Scholar, Kepinski and Leyser, 2005Kepinski S. Leyser O. The Arabidopsis F-box protein TIR1 is an auxin receptor.Nature. 2005; 435: 446-451Crossref PubMed Scopus (1147) Google Scholar) and stabilizes association of Aux/IAA substrates—which share a homologous “auxin-inducible degron” (AID)—with SCFTIR1, resulting in ubiquitination and degradation of the Aux/IAA proteins (Figure 2). X-ray crystallography of a TIR1-auxin-AID ternary complex revealed that auxin occupies a cavity in the Aux/IAA binding site of TIR1, and thereby creates a platform that makes contact with and stabilizes binding of the AID (Tan et al., 2007Tan X. Calderon-Villalobos L.I. Sharon M. Zheng C. Robinson C.V. Estelle M. Zheng N. Mechanism of auxin perception by the TIR1 ubiquitin ligase.Nature. 2007; 446: 640-645Crossref PubMed Scopus (911) Google Scholar). Even more remarkable is that the plant hormone jasmonate works by an identical mechanism through the F-box substrate receptor COI1 (Thines et al., 2007Thines B. Katsir L. Melotto M. Niu Y. Mandaokar A. Liu G. Nomura K. He S.Y. Howe G.A. Browse J. JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.Nature. 2007; 448: 661-665Crossref PubMed Scopus (1342) Google Scholar), and strigolactones, salicylic acid, and cytokinins also manipulate protein degradation in different ways (Larrieu and Vernoux, 2015Larrieu A. Vernoux T. Comparison of plant hormone signalling systems.Essays Biochem. 2015; 58: 165-181Crossref PubMed Google Scholar). Together with the data on viruses summarized above, these examples illustrate the extraordinary potential of using small molecules and engineered proteins to induce targeted degradation of proteins in either the therapeutic or research space. The examples provided by viral hijacking of the UPS inspired an effort to develop bi-specific small molecules that could mimic the action of E6 and VPU and induce proximity of a specific cellular “protein of interest” (POI) with a Ub ligase. At the time, the limiting factor was the paucity of portable, well-defined ligands for Ub ligases. As proof of concept, a ten amino acid phosphopeptide degron from IkBα that binds the β-TRCP substrate receptor subunit of Ub ligase SCFβ-TRCP (Winston et al., 1999Winston J.T. Strack P. Beer-Romero P. Chu C.Y. Elledge S.J. Harper J.W. The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro.Genes Dev. 1999; 13: 270-283Crossref PubMed Google Scholar, Yaron et al., 1998Yaron A. Hatzubai A. Davis M. Lavon I. Amit S. Manning A.M. Andersen J.S. Mann M. Mercurio F. Ben-Neriah Y. 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The same approach was employed to target the estrogen (ER) and androgen (AR) receptors for degradation (Sakamoto et al., 2003Sakamoto K.M. Kim K.B. Verma R. Ransick A. Stein B. Crews C.M. Deshaies R.J. Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation.Mol. Cell. Proteomics. 2003; 2: 1350-1358Crossref PubMed Scopus (170) Google Scholar). Shortly after the original Sakamoto et al., 2001Sakamoto K.M. Kim K.B. Kumagai A. Mercurio F. Crews C.M. Deshaies R.J. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation.Proc. Natl. Acad. Sci. USA. 2001; 98: 8554-8559Crossref PubMed Scopus (478) Google Scholar report was published a patent was issued that proposes the use of peptides that bind the Ub ligase UBR1 linked to different ligands to trigger ubiquitination and degradation of target proteins. However, no data were shown to illustrate the effectiveness of this approach (US Patent No. US6306663B1). Because the first PROTACs employed a phosphopeptide ligand for β-TRCP, they did not penetrate cells. An opportunity to engineer cell-permeable PROTACs presented itself with the discovery of a seven residue hydroxyproline-containing peptide from HIF-1α that specifically binds the substrate receptor subunit VHL (Von Hippel-Lindau) of ubiquitin ligase CRL2VHL (Hon et al., 2002Hon W.C. Wilson M.I. Harlos K. Claridge T.D. Schofield C.J. Pugh C.W. Maxwell P.H. Ratcliffe P.J. Stuart D.I. Jones E.Y. Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL.Nature. 2002; 417: 975-978Crossref PubMed Scopus (482) Google Scholar, Min et al., 2002Min J.H. Yang H. Ivan M. Gertler F. Kaelin Jr., W.G. Pavletich N.P. Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling.Science. 2002; 296: 1886-1889Crossref PubMed Scopus (515) Google Scholar). This peptide was used to assemble PROTACs that induce degradation of AR and FKBP12 (FK506-binding protein) in cells (Schneekloth et al., 2004Schneekloth Jr., J.S. Fonseca F.N. Koldobskiy M. Mandal A. Deshaies R. Sakamoto K. Crews C.M. Chemical genetic control of protein levels: selective in vivo targeted degradation.J. Am. Chem. Soc. 2004; 126: 3748-3754Crossref PubMed Scopus (206) Google Scholar). Although PROTACs based on the VHL peptide ligand work in cells, they suffer from relatively low potency, most likely due to poor bioavailability. This spurred engineering of the first “all small molecule” PROTAC based on Nutlin-3a, a ligand of the Ub ligase MDM2 that competes for binding of the MDM2 substrate p53. Nutlin-3 conjugated to the AR antagonist hydroxyflutamide specified AR degradation in a proteasome-dependent manner (Schneekloth et al., 2008Schneekloth A.R. Pucheault M. Tae H.S. Crews C.M. Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics.Bioorg. Med. Chem. Lett. 2008; 18: 5904-5908Crossref PubMed Scopus (176) Google Scholar). Another approach to the development of small molecule degraders is based on a ligand for the Ub ligase IAP (Inhibitor of Apoptosis) (Fulda and Vucic, 2012Fulda S. Vucic D. Targeting IAP proteins for therapeutic intervention in cancer.Nat. Rev. Drug Discov. 2012; 11: 109-124Crossref PubMed Scopus (523) Google Scholar). Bestatin methyl ester is linked to ligands that bind a POI to create bifunctional SNIPERS (specific and nongenetic IAP-dependent protein erasers) that direct degradation of targets such as CRABPI and CRABPII (Itoh et al., 2012Itoh Y. Ishikawa M. Kitaguchi R. Okuhira K. Naito M. Hashimoto Y. Double protein knockdown of cIAP1 and CRABP-II using a hybrid molecule consisting of ATRA and IAPs antagonist.Bioorg. Med. Chem. 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Further derivatization of the IAP ligand module yielded SNIPER(ER)-110, which recruits XIAP and prompts degradation of both cIAP and ERα (Ohoka et al., 2018Ohoka N. Morita Y. Nagai K. Shimokawa K. Ujikawa O. Fujimori I. Ito M. Hayase Y. Okuhira K. Shibata N. et al.Derivatization of inhibitor of apoptosis protein (IAP) ligands yields improved inducers of estrogen receptor α degradation.J. Biol. Chem. 2018; 293: 6776-6790Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). By 2010 PROTACs able to induce degradation of multiple substrates had been described, yet the approach remained challenged by the lack of high-affinity Ub ligase ligands as well as a compelling proof of concept that PROTACs had the potential to become actual drugs. This began to change with the important discovery that cereblon (CRBN), a substrate receptor of Ub ligase CRL4, is the primary target of the multiple myeloma drug thalidomide (Ito et al., 2010Ito T. Ando H. Suzuki T. Ogura T. Hotta K. 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The finding that lenalidomide recruits IKZF proteins and CK1α to CRBN (Figure 4) was a puzzle because no obvious degron sequences are shared between these proteins. Crystallographic studies of CRBN bound to CK1α in the presence of lenalidomide shed considerable light on how IMiDs mediate ternary complex formation (Petzold et al., 2016Petzold G. Fischer E.S. Thomä N.H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase.Nature. 2016; 532: 127-130Crossref PubMed Scopus (158) Google Scholar). As was shown previously for the mechanism of signaling by auxin, IMiDs sit at the Ub ligase-neosubstrate interface and stabilize the complex. The glutarimide ring of lenalidomide nestles in the “tri-Trp” pocket of CRBN whereas the phthalimide ring remains solvent exposed, allowing contact with a β-hairpin loop containing a critical glycine (amino acids 35–41) in CK1α. Residues within the same CK1α loop also contact CRBN. The complex is thus formed by a balance of lenalidomide-protein and protein-protein contacts at the interface. Importantly, the atoms in the β-hairpin that contact CRBN are in the main chain and not the side chains. This explains how lenalidomide stabilizes complex formation with diverse neosubstrates, because similar β-hairpin loops are present in zinc finger proteins including IKZF1/3 and other IMiD-dependent CRL4CRBN neosubstrates including ZFP91 and SALL4 (An et al., 2017An J. Ponthier C.M. Sack R. Seebacher J. Stadler M.B. Donovan K.A. Fischer E.S. pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4CRBN ubiquitin ligase.Nat. Commun. 2017; 8: 15398Crossref PubMed Scopus (47) Google Scholar, Donovan et al., 2018Donovan K.A. An J. Nowak R.P. Yuan J.C. Fink E.C. Berry B.C. Ebert B.L. Fischer E.S. 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Interestingly, a β-hairpin with a key glycine residue promotes CRBN binding and degradation of the translation termination factor GSPT1 by the glutarimide analog CC-885 (Matyskiela et al., 2016Matyskiela M.E. Lu G. Ito T. Pagarigan B. Lu C.C. Miller K. Fang W. Wang N.Y. Nguyen D. Houston J. et al.A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase.Nature. 2016; 535: 252-257Crossref PubMed Scopus (133) Google Scholar). Since CRBN is widely expressed and GSPT1 is essential, its degradation represents a potential liability for CRBN-based degraders. However, structure-activity analyses aided by chemoproteomics are allowing for the design of more specific IMiD analogs that no longer degrade GSPT1 (Hansen et al., 2018Hansen J.D. Condroski K. Correa M. Muller G. Man H.W. Ruchelman A. Zhang W. Vocanson F. Crea T. 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Phthalimide conjugation as a strategy for in vivo target protein degradation.Science. 2015; 348: 1376-1381Crossref PubMed Scopus (546) Google Scholar, Zengerle et al., 2015Zengerle M. Chan K.H. Ciulli A. Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4.ACS Chem. Biol. 2015; 10: 1770-1777Crossref PubMed Scopus (302) Google Scholar), a set of potent PROTACs based on VHL-1 and IMiDs was described. These papers established that PROTACs:(1)Work on multiple different proteins. Targets as varied as FKBP12, the BET family of epigenetic readers BRD2-4, the steroid receptor ERRα, and protein kinase RIPK2 could be efficiently degraded (see Table 1 for a more complete list of proteins targeted by PROTACs thus far).Table 1Compilation of Cellular Proteins Targeted by Non-peptidic PROTACsTarget(s)Liga
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