Advances in Targeted Degradation of Endogenous Proteins

Cellular and Molecular Life Sciences https://doi.org/10.1007/s00018-019-03112-6 Cellular andMolecular Life Sciences REVIEW Advances in targeted degradation of endogenous proteins Sascha Röth1 · Luke J. Fulcher1 · Gopal P. Sapkota1 Received: 11 January 2019 / Revised: 23 March 2019 / Accepted: 16 April 2019 © The Author(s) 2019 Abstract Protein silencing is often employed as a means to aid investigations in protein function and is increasingly desired as a therapeutic approach. Several types of protein silencing methodologies have been developed, including targeting the encoding genes, transcripts, the process of translation or the protein directly. Despite these advances, most silencing systems sufer from limitations. Silencing protein expression through genetic ablation, for example by CRISPR/Cas9 genome editing, is irreversible, time consuming and not always feasible. Similarly, RNA interference approaches warrant prolonged treatments, can lead to incomplete protein depletion and are often associated with of-target efects. Targeted proteolysis has the potential to overcome some of these limitations. The feld of targeted proteolysis has witnessed the emergence of many methodologies aimed at targeting specifc proteins for degradation in a spatio-temporal manner. In this review, we provide an appraisal of the diferent targeted proteolytic systems and discuss their applications in understanding protein function, as well as their potential in therapeutics. Keywords Nanobody · Monobody · Ubiquitin · Proteasome · Auxin · Afnity-directed protein missile · VHL · CRBN · AiD · HALO · FKBP12 · Thalidomide · Proteolysis targeting chimera · PROTAC Introduction against viral infections, which acts to cleave foreign DNA or RNA from invading pathogens, thereby limiting infection Understanding the inherent function of a protein is a cor- [1]. Back in 2012, this system was adapted as a powerful nerstone in life sciences. Many biochemical and biophysical genome editing tool [2] which could be utilized to either properties of proteins can be ascertained in vitro; however, knockout a target gene, or introduce new DNA sequences a key drawback of these approaches is the absence of the into the genome in eukaryotic cells (Fig. 1, top). While cellular context. In cellulo research on the function of a pro- CRISPR/Cas9 has since become an easy, cost-effective tein of interest (POI) often relies on its deletion or depletion method for genome editing, it cannot be used for all targets, from cells to screen for changes in, for example, phenotype, as single cell clones of essential gene knockouts will not signalling or gene expression patterns. Two current rou- survive the clonal selection process. Additionally, CRISPR/ tine approaches used to achieve POI deletion or depletion Cas9 genome editing usually does not interfere with tran- are genome editing, for example by using CRISPR/Cas9- scription, but relies on the deletion of the start codon or mediated gene knockout, and RNA interference (RNAi), introduction of nonsense mutations. However, mutagenesis respectively. by CRISPR can result in exon skipping, generating a difer- With recent advances in CRISPR/Cas9 genome editing, ent start codon, resulting in truncations of the protein still targeted knockout of genes in cells, tissues and whole organ- being translated [3]. Even in the case of a true gene knock- isms has been widely utilized to assess protein function. out, however, the time required for a single cell clone to CRISPR/Cas9 is a bacterial and archaeal defence mechanism propagate might be sufcient for cells to establish genetic compensation, thereby altering expression patterns of related proteins to make up for the loss of one protein [4]. Because * Gopal P. Sapkota gene knockouts are irreversible, the homeostatic changes in [email protected] knockout cell clones might be very diferent from parental 1 MRC Protein Phosphorylation and Ubiquitylation Unit, cells. Therefore, any interpretation of changes due to the School of Life Sciences, University of Dundee, Dow Street, loss of a POI relies on the ability to rescue phenotypes by Dundee DD1 5EH, UK Vol.:(0123456789)1 3 S. Röth et al. PROCON - Irreversible CRISPR/Cas9 DNA - Complete KO cells through all stages Tr - Can keep and use cells - Not feasible for anscriptio over time essential genes or certain - Can target isoforms based cell systems on splicing pattern - Compensation - Truncation possible n - Can target essential RNAi RNA gene products Tr - Can target gene families anslation based on sequence similarities relies on protein turnover - Can target isoforms - Prolonged treatments based on unique required sequences - Can be time consuming Targeted Protein - Independent of to develop, based on Proteolysis regulation on DNA/RNA - Can target essential - Limited availability of proteins POI-targeting nanobod- - Tractable based on ies or small molecules system being used - Limited number of Fig. 1 Comparison of protein silencing tools and their advantages transcripts. This in turn efectively blocks translation of the protein. (PRO) and disadvantages (CON). Top: CRISPR/Cas9 genome editing Bottom: targeted proteolysis: While both transcription and translation results in mutations of target gene sequence, typically through dele- remain unafected, through mechanisms described for the diferent tion of the start ATG codon, and introduction of early stop codons proteolytic methods, the target protein is directly marked for protea- or frame shifts. While transcripts are still produced, translation is somal degradation blocked. Middle: RNAi approaches result in cleavage of the mRNA restoring the POI expression. This approach is not always the turnover of the target protein for efcient reduction in feasible and often relies on the use of artifcial and constitu- target protein levels. For silencing of most POIs, RNAi is tive promoters to drive protein expression. usually efective only after 48–72 h of treatment. However, RNAi-mediated depletion of a target POI transcript for those proteins that have very slow turnover rates that sur- is a potential way to circumvent some of the problems of pass this time frame [7], RNAi approaches will not yield the CRISPR/Cas9 genome editing highlighted above, as this desired knockdown of target transcripts. Another common technique is performed in a much shorter time frame than issue associated with RNAi appears to be of-target silenc- that required for a genetic knockout. In metazoan cells, RNAi ing of transcripts based on partial sequence complementa- approaches include short interfering RNAs (siRNAs), micro- rity [8], despite great eforts going into the optimization of RNAs (miRNAs) and short hairpin RNAs (shRNA) [5]. In siRNA sequence design or delivery to minimize potential all of these cases, the RNAi machinery triggers the activa- of-target efects [9]. tion of the RNAi-induced silencing complex (RISC) and, An alternative approach, circumventing the challenges of depending on sequence complementarity, results in either protein silencing through genomic alterations or transcript degradation of the target RNA or inhibition of its transla- inhibition, is the direct targeting of proteins for destruction (Fig. 1, middle) [6], leading to post-transcriptional gene tion. Targeted proteolysis is desirable both in therapeutic silencing. While these methods enable the opportunity to applications and in basic research. To date, such proteolytic target transcripts of essential genes, they ultimately rely on approaches have relied on the cells’ built-in protein recycling 1 3 Advances in targeted degradation of endogenous proteins centre, known as the ubiquitin–proteasome system (UPS). In of transcriptional repressor proteins are rapidly degraded short, proteins are marked for destruction through the cova- through a specifc form of the SCF (SKP–cullin–F-Box)- lent attachment of the small protein ubiquitin onto primarily RING E3 Ligase system [24, 25]. Auxin hormones bind to lysine residues on the target protein [10, 11]. The frst step the F-box transport inhibitor response 1 (TIR1) protein and, of the reaction is mediated by the E1 ubiquitin-activating in doing so, promote the association between the SCF-TIR1 enzyme that activates the ubiquitin monomer. Subsequently, E3 ligase complex with AUX/IAA transcriptional repres- the activated ubiquitin is passed on to the E2 ubiquitin-con- sors, ultimately leading to the ubiquitination and degrada- jugating enzyme, before ultimately being attached to the tion of the AUX/IAA transcriptional repressors [24, 25]. target protein through the action of an E3 ubiquitin ligase. Whilst other eukaryotes lack this auxin-inducible response, The ubiquitin monomers can be further ubiquitinated to form the SCF degradation pathway is conserved, thereby raising chains, with distinct chain types linked to specifc biologi- the tantalizing possibility that the auxin-inducible degrada- cal processes [12–14]. For example, and of relevance here, tion pathway could be transferred into other eukaryotic cells. if ubiquitin molecules added to the target protein form a The Kanemaki lab demonstrated this possibility with rapid, lysine 48-linked chain, the ubiquitinated protein is marked reversible degradation of essential POIs in cell lines derived for proteasomal degradation [12, 13]. The proteasome itself from various organisms including chicken, mouse, yeast, and is a cylindrical multimeric protein complex composed of humans [24]. They achieved this through fusion of an IAA17 a 20S core particle

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