Targeted protein degradation using deGradFP in Trypanosoma brucei [version 2; peer review: 4 approved]

Targeted protein degradation is an invaluable tool in studying the function of proteins. Such a tool was not available in Trypanosoma brucei , an evolutionarily divergent eukaryote that causes human African trypanosomiasis. Here, we have adapted deGradFP (degrade green fluorescent protein [GFP]), a protein degradation system based on the SCF E3 ubiquitin ligase complex and anti-GFP nanobody, in T. brucei . As a proof of principle, we targeted a kinetoplastid kinetochore protein (KKT3) that constitutively localizes at kinetochores in the nucleus. Induction of deGradFP in a cell line that had both alleles of KKT3 tagged with yellow fluorescent protein (YFP) caused a more severe growth defect than RNAi in procyclic (insect form) cells. deGradFP also worked on a cytoplasmic protein (COPII subunit, SEC31). Given the ease in making GFP fusion cell lines in T. brucei , deGradFP can serve as a powerful tool to rapidly deplete proteins of interest, especially those with low turnover rates. Approaches for conditional expression of specific mRNAs and/or proteins have greatly facilitated experimental analyses of protein function in cells and organisms, particularly where protein depletion results in loss-of-fitness. One such approach involves targeted protein degradation, which was not previously reported for the African trypanosome, Trypanosoma brucei, an otherwise experimentally tractable parasite that causes often lethal diseases in humans and animals. The current study presents compelling data reporting adaptation of the deGradFP system for insect-form T. brucei; proof-of-principle is demonstrated by targeting a nuclear protein and a cytoplasmic protein. As the authors point out, the deGradFP approach relies upon the GFP-tagged protein retaining function and the VhhGFP4 nanobody being able to access the tag (meaning that the tag and the nanobody must be present in the same cellular compartment). This approach, as also noted, could be particularly powerful and effective for linking primary loss-of-function phenotypes to proteins with normally slow turnover, since protein degradation using deGradFP is rapid (detectable after 3 h), while knockdown by the widely used RNA interference approach can be relatively slow. This work reports the implementation of a degron approach for protein depletion in T. brucei . While T. brucei benefits from RNA interference, degron approaches that target the protein pool directly are likely to be valuable tools for rapid, selective, and more complete protein depletion. Ishii and Akiyoshi have identified a domain of a trypanosomatid-specific SCF E3 ubiquitin ligase complex (FBP75) that can target proteins for degradation, which is an important advance. They have fused this degron domain to a single-chain antibody that recognizes GFP, which when expressed using a tetracycline-inducible system targets FP-tagged proteins in cells, likely leading to their ubiquitination and subsequent degradation via the proteasome. Expression of the FBP75-Vhh fusion on its own does not appear to be toxic, which is essential for this method. The authors have shown degron-tag specific depletion of two proteins- the kinetochore protein KKT3, which is nuclear, and Sec31, which is a component of the COPII complex in the cytosol. They argue that the depletion of these proteins is more rapid than RNAi and provides more acute growth defects. Overall, this work provides the exciting prospect of a functional degron system for use in trypanosomatids, which could be extremely useful as a complement to RNAi in T. brucei and allow the conditional depletion of essential proteins in Leishmania and T. cruzi , which is a pressing issue. I have one significant concern that could be addressed by citing specific previous results: A deGradFP system that allows inducible, targeted protein degradation in T. brucei is described. As a proof-of-concept, the system is successfully used to target the degradation of one nuclear protein and one cytoplasmic protein. This is the first targeted protein degradation method reported in T. brucei , expanding the current molecular toolkits available in this important model organism. Rapid protein degradation makes it very useful for protein functional studies. As the method relies on the physical recruitment of the ubiquitin ligase complex to the targeted protein through YFP binding and KKT3 is present in a protein complex, it may be worth addressing whether other components in the KKT complex could also be degraded through this process. Such information may help to understand any potential “off-target” effects of this method. Another minor suggestion is to make the error bars larger in all the growth curves. They are barely visible in the current figures. the of


Introduction
Kinetoplastids are a group of unicellular flagellated eukaryotes found in diverse environmental conditions (d' Avila-Levy et al., 2015). They belong to the phylum Euglenozoa (Discoba/Excavata) and are evolutionarily distant from commonly studied model eukaryotes such as yeasts, worms, flies, and humans (Opisthokonta) (Cavalier-Smith, 2010; Keeling & Burki, 2019). Understanding their biology could therefore provide insights into the extent of conservation/divergence among eukaryotes and lead to a deeper understanding of biological systems and evolution of eukaryotes. Importantly, three neglected tropical diseases are caused by parasitic kinetoplastids: African trypanosomiasis, Chagas disease, and leishmaniasis (Horn, 2022; Rao et al., 2019). Human African trypanosomiasis (sleeping sickness) is caused by Trypanosoma brucei, which also causes the cattle disease, nagana, that leads to weight loss and anemia in livestock and imposes a huge burden on economic development in affected regions. Understanding the biology of kinetoplastids could facilitate the design of new drugs against kinetoplastid parasites.
Inducible depletion of a target protein is an essential tool in biology (Prozzillo et al., 2020). In Trypanosoma brucei, this can be achieved by RNAi (Alsford et al., 2011;Ngô et al., 1998) and Tet-off system (Merritt & Stuart, 2013) at the RNA level, as well as by conditional knockout at the gene level using Cre-LoxP (Kim et al., 2013). Although powerful in many cases, these approaches are not efficient in reducing the level of proteins that have slow turnover rates. Targeted  . However, such tools were not available in Trypanosoma brucei, to our knowledge.
In this study, we have adapted the deGradFP (degrade green fluorescent protein) system which was originally established in Drosophila melanogaster (Caussinus et al., 2011). It relies on the expression of VhhGFP4 fused with a truncated F-box protein. VhhGFP4 is an anti-GFP nanobody that recognizes GFP and some derivatives such as yellow fluorescent proteins (YFP) and Venus (Saerens et al., 2005), while an F-box protein is a substrate-recognition subunit of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex that catalyzes the ubiquitylation of target proteins (Petroski & Deshaies, 2005). In deGradFP, a substrate-recognition domain of an F-box protein is replaced by VhhGFP4 so that GFP-fusion proteins are ubiquitylated by the SCF complex, leading to their degradation via the 26S proteasome pathway ( Here, we show that deGradFP successfully depletes a kinetochore protein and a COPII subunit in the procyclic form of T. brucei cells.

Results
To establish a targeted protein degradation method in T. brucei, we chose a protein that has a slow turnover rate. KKT3 is a kinetochore protein that constitutively localizes at kinetochores and does not show any obvious fluctuation in its abundance during the cell cycle, implying that it is a stable protein (Akiyoshi & Gull, 2014). In fact, the half-life of KKT3 has been estimated to be much longer than transiently-localized kinetochore proteins (Tinti et al., 2019). To apply deGradFP in T. brucei, we made a construct that expresses an F-box domain fused with a nuclear localization signal (NLS) and the anti-GFP nanobody VhhGFP4 (Saerens et al., 2005) ( Figure 1A, B). The fusion protein was expressed from a derivative of pDEX777 that integrates at the 177 bp repeats on minichromosomes and allows doxycycline-inducible expression ( Figure 1C), the construct containing Tb927.5.700 caused growth defects, implying that expression of Tb927.5.700 1-200 -NLS-VhhGFP4 led to degradation of KKT3-YFP. In contrast, expression of CFB1C 1-200 -NLS-VhhGFP4 did not cause strong growth defects. This means that expression of VhhGFP4 (at least when fused to the F-box of CFB1C) was not sufficient to disrupt the function of KKT3-YFP. We therefore used the F-box from Tb927.5.700 (named FBP75 herein for F-box protein 75 kDa) for subsequent experiments. Besides the construct with an NLS to target nuclear proteins (pBA2675: NLS-deGradFP), we also made another one without an NLS to target cytoplasmic proteins (pBA2705: deGradFP). Induction of these deGradFP systems in wild-type procyclic cells with 1 µg/mL doxycycline did not cause any growth defect ( Figure 1D), showing that expression of deGradFP itself does not cause growth defects.
We next used a cell line in which both alleles of KKT3 were C-terminally tagged with YFP using a PCR-based method in one transfection step (Dean et al., 2015). We found that induction of NLS-deGradFP in this cell line caused more severe growth defects than RNAi (Figure 2A and B) (Marcianò et al., 2021). We did not observe severe growth defects with deGradFP without NLS in KKT3-YFP cells, showing the importance of NLS to target nuclear proteins in our system ( Figure 2C). Microscopy analysis confirmed that NLS-deGradFP caused more significant depletion of KKT3 at six hours than RNAi ( Figure 2D and E). The fact that

Amendments from Version 1
In the revised manuscript, we added new results showing that CFB1C's F-box does not cause growth defects in KKT3-YFP cells, that expression of deGradFP that lacks an NLS does not cause growth defects in KKT3-YFP cells, and that NLS-deGradFP did not work on YFP-KKT18. We also made changes to respond to referee comments. induction of NLS-deGradFP in wild-type cells did not cause any growth defect ( Figure 1D) means that the observed growth defect was due to specific degradation of YFP-tagged KKT3. In the deGradFP system, YFP-tagged target proteins ubiquitylated by the SCF ubiquitin ligase complex are degraded by the proteasome system (Caussinus & Affolter, 2016). Consistent with this possibility, addition of the proteasome inhibitor MG132 prevented degradation of KKT3-YFP ( Figure 2F), suggesting that our deGradFP system in T. brucei relies on the proteasome-dependent protein degradation pathway as expected. We note that NLS-deGradFP did not work well for another kinetochore protein KKT18 ( Figure 2G). In contrast, RNAi-mediated depletion of KKT18 caused growth defects and reduction of protein.
We next targeted a cytoplasmic protein SEC31 using a deGradFP construct that lacks an NLS. SEC31 is a subunit of COPII and localizes at the endoplasmic reticulum (ER) exit site (Hu et al., 2016). Both alleles of SEC31 were C-terminally tagged in a CRISPR cell line (Beneke et al., 2017). Induction of deGradFP caused a strong growth defect ( Figure 3A and B), which is apparently more severe than RNAi-mediated depletion of SEC31 reported in a previous study (Hu et al., 2016). These results therefore show that deGradFP can efficiently deplete both nuclear and cytoplasmic proteins in T. brucei.

Discussion
In T. brucei, it is easy to tag genes at the endogenous locus using plasmid-or PCR-based methods (Beneke et al., 2017;  , we have shown that deGradFP can induce targeted protein degradation in T. brucei. The depletion kinetics is faster than the RNAi-mediated depletion method, at least for KKT3. Our results therefore show that deGradFP can be a powerful tool in characterizing depletion phenotypes in T. brucei. It is, however, important to note that deGradFP has some limitations. For example, it has been suggested that deGradFP does not work if GFP is not accessible (Caussinus et al., 2011; Caussinus & Affolter, 2016). It is also essential that target proteins have lysines that can be ubiquitylated by deGradFP. Furthermore, it is critical that GFP-fusion proteins retain enough functionality to support cell growth because the deGradFP system utilizes the VhhGFP4 nanobody that recognizes GFP or its derivatives. If necessary, this system could be modified to use nanobodies against other epitope tags or even the protein of interest itself to induce degradation of the target (Aguilar et al., 2019).
The function of the F-box protein used in this study (FBP75) remains unknown. We also do not know which SKP1 or cullin proteins interact with FBP75 and whether those proteins are expressed in other life stages. It therefore remains unknown whether FBP75-based deGradFP works in bloodstream form cells. If it does not work, other F-box proteins could be utilized to deplete proteins of interest in bloodstream form cells (Benz & Clayton, 2007;Rojas et al., 2017). In any case, it is our hope that deGradFP will prove to be a useful protein degradation tool to facilitate studies of Trypanosoma brucei.

Trypanosome cells
All cell lines used in this study were derived from the TREU 927 procyclic form cells and are listed in Table 2 To make the homozygous KKT3-YFP cell line, two YFP-tagging cassettes were amplified from pPOTv7 (YFP, Hyg) or pPOTv7 (YFP, G418) (Dean et al., 2015) by PCR using BA1821/ BA1822 (Table 3). 25 µL of 2x PrimeSTAR MAX (Takara),

David Horn
The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, UK Approaches for conditional expression of specific mRNAs and/or proteins have greatly facilitated experimental analyses of protein function in cells and organisms, particularly where protein depletion results in loss-of-fitness. One such approach involves targeted protein degradation, which was not previously reported for the African trypanosome, Trypanosoma brucei, an otherwise experimentally tractable parasite that causes often lethal diseases in humans and animals.
The current study presents compelling data reporting adaptation of the deGradFP system for insect-form T. brucei; proof-of-principle is demonstrated by targeting a nuclear protein and a cytoplasmic protein. As the authors point out, the deGradFP approach relies upon the GFP-tagged protein retaining function and the VhhGFP4 nanobody being able to access the tag (meaning that the tag and the nanobody must be present in the same cellular compartment). This approach, as also noted, could be particularly powerful and effective for linking primary loss-of-function phenotypes to proteins with normally slow turnover, since protein degradation using deGradFP is rapid (detectable after 3 h), while knockdown by the widely used RNA interference approach can be relatively slow.
I suggest some minor/simple edits to improve clarity: Fig. 1C, 2A, 2B, 3: The error bars are unclear. Suggest increasing the line weight. 1. First paragraph of results: Replace "177 bp locus" with "177 bp repeats on minichromosomes".

3.
Some further suggestions that the authors may want to consider: It's likely that the two example proteins are degraded in a proteasome-dependent manner, 1.
but this could be tested using a proteasome inhibitor such as bortezomib.
First paragraph of results: The authors could add some detail explaining how Tb927.5.700 was selected/prioritised as the source of the F-box to be fused to the nanobody.

2.
Discussion: deGradFP may also effectively target 'nuclear' proteins, either before they reach the nucleus or by acting directly in the nucleus. Indeed, it remains unclear at this point whether the centrally located La NLS effectively directs NLS-deGradFP to the nucleus. Also, some proteins that function in both compartments may be effectively targeted by deGradFP.

3.
Consider depositing key plasmids with a plasmid repository such as Addgene. 4.

Are sufficient details provided to allow replication of the method development and its use by others? Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes Competing Interests: No competing interests were disclosed.
Response: We have increased the line weight for these errors bars as suggested. Fig. 1 legend: Replace "dashed dashed" with "dashed lines".

© 2022 de Graffenried C.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Christopher L. de Graffenried
Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, USA This work reports the implementation of a degron approach for protein depletion in T. brucei. While T. brucei benefits from RNA interference, degron approaches that target the protein pool directly are likely to be valuable tools for rapid, selective, and more complete protein depletion. Ishii and Akiyoshi have identified a domain of a trypanosomatid-specific SCF E3 ubiquitin ligase complex (FBP75) that can target proteins for degradation, which is an important advance. They have fused this degron domain to a single-chain antibody that recognizes GFP, which when expressed using a tetracycline-inducible system targets FP-tagged proteins in cells, likely leading to their ubiquitination and subsequent degradation via the proteasome. Expression of the FBP75-Vhh fusion on its own does not appear to be toxic, which is essential for this method. The authors have shown degron-tag specific depletion of two proteins-the kinetochore protein KKT3, which is nuclear, and Sec31, which is a component of the COPII complex in the cytosol. They argue that the depletion of these proteins is more rapid than RNAi and provides more acute growth defects.
Overall, this work provides the exciting prospect of a functional degron system for use in trypanosomatids, which could be extremely useful as a complement to RNAi in T. brucei and allow the conditional depletion of essential proteins in Leishmania and T. cruzi, which is a pressing issue. I have one significant concern that could be addressed by citing specific previous results: As the authors mention, the FBP75 domain has just been identified as a potential degron and we do not currently know which Skp1 or cullins the domain interacts with -this specific pathway in T. brucei is complex. Can the authors show that the Vhh antibody on its own is not sufficient to disrupt the function of or degrade the YFP-tagged proteins? It appears that the original Cassinus paper has this data ( Figure 2B). Considering that a new degron candidate is being employed here, the evidence that the Vhh on its own does not cause degradation in other systems should be specifically mentioned.

1.
Smaller comments not necessary to address in this manuscript but would be important for testing the mechanism and generality of the approach: Does the expression of the Vhh-FBP75 fusion lead to the appearance of higher MW bands corresponding to ubiquitinated versions of the YFP-tagged proteins? 1.
Does blocking proteasomal activity via MG132 limit the loss of YFP signal? Are the proteins mislocalized, even if they are still present in the cell? 2.
It seems possible that degron-mediated depletion of proteins could provide different phenotypes compared to RNAi. Since the method could actively target proteins that are already part of stable protein complexes, such as the KKTs, ubiquitination of these proteins could lead to the direct disruption of complexes. That might provide different phenotypes compared to RNAi. So, speed of depletion is just one aspect of the potential benefits of the degron. Have the authors observed any differences in how the phenotypes manifest? 3.
The authors do not provide a side-by-side comparison of the Sec31 RNAi and Sec31 degron. The paper they cite for the Sec31 RNAi shows a slower growth defect than the degron, but it should be noted that at longer time points the RNAi is showing a very lethal phenotype while the degron is showing what appears to be a rebound in the population. This could be due to a loss of expression of the Vhh-degron, which can occur with tetracycline-inducible systems. This might be a persistent issue with this implementation of the degron approach.
4. Figure 1 legend: "Green dashed dashed" -Remove one of the "dashed." 5. To our knowledge, the CRISPR/Cas9 approach used/described in Beneke et al., 2017 is not a conditional KO. It is described as such in the introduction.

Is the description of the method technically sound? Yes
Are sufficient details provided to allow replication of the method development and its use by others? Yes If any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes 1. As the authors mention, the FBP75 domain has just been identified as a potential degron and we do not currently know which Skp1 or cullins the domain interacts with -this specific pathway in T. brucei is complex. Can the authors show that the Vhh antibody on its own is not sufficient to disrupt the function of or degrade the YFP-tagged proteins? It appears that the original Cassinus paper has this data ( Figure 2B). Considering that a new degron candidate is being employed here, the evidence that the Vhh on its own does not cause degradation in other systems should be specifically mentioned.
Response: Expression of the F-box of Tb927.1.4580 (CFB1C) fused with VhhGFP4 did not cause any significant growth defect ( Figure 1C) or reduction of KKT3-YFP signals (not shown). We therefore think that expression of VhhGFP4 itself is not sufficient to disrupt the function of KKT3-YFP or degrade KKT3-YFP. We added the following sentence in the Results section "This means that expression of VhhGFP4 (at least when fused to the F-box of CFB1C) was not sufficient to disrupt the function of KKT3-YFP".
Smaller comments not necessary to address in this manuscript but would be important for testing the mechanism and generality of the approach: 1. Does the expression of the Vhh-FBP75 fusion lead to the appearance of higher MW bands corresponding to ubiquitinated versions of the YFP-tagged proteins?
We did not observe additional bands that may correspond to ubiquitylated species at least for KKT3-YFP.
2. Does blocking proteasomal activity via MG132 limit the loss of YFP signal? Are the proteins mislocalized, even if they are still present in the cell?
We found that MG132 inhibited the degradation of KKT3-YFP by NLS-deGradFP. This result confirmed that KKT3 was degraded in a proteasome-dependent manner as expected. Interestingly, we still observed kinetochore-like dots, suggesting that ubiquitylated KKT3-YFP stays at kinetochores.
3. It seems possible that degron-mediated depletion of proteins could provide different phenotypes compared to RNAi. Since the method could actively target proteins that are already part of stable protein complexes, such as the KKTs, ubiquitination of these proteins could lead to the direct disruption of complexes. That might provide different phenotypes compared to RNAi. So, speed of depletion is just one aspect of the potential benefits of the degron. Have the authors observed any differences in how the phenotypes manifest?
We have not thoroughly examined the phenotype of KKT3 depletion using NLS-deGradFP. We agree that it will be interesting to compare the phenotype of this targeted protein degradation system to that of RNAi.

The authors do not provide a side-by-side comparison of the Sec31
RNAi and Sec31 degron. The paper they cite for the Sec31 RNAi shows a slower growth defect than the degron, but it should be noted that at longer time points the RNAi is showing a very lethal phenotype while the degron is showing what appears to be a rebound in the population. This could be due to a loss of expression of the Vhh-degron, which can occur with tetracycline-inducible systems. This might be a persistent issue with this implementation of the degron approach.
As shown by many researchers, this kind of rebound phenotype is frequently observed when essential genes are depleted by RNAi or other methods, likely due to loss of key components required for depletion (e.g. loss of deGradFP-containing minichromosomes due to chromosome mis-segregation). Given that we will focus on examining cells at early time points (e.g. between 6 hours and 24 hours, in the case of KKT3-YFP, before growth defects step in) to avoid secondary or indirect effects, we do not think this is a critical issue. Figure 1 legend: "Green dashed dashed" -Remove one of the "dashed."

5.
We fixed the typo.

Figures 1 -3: Error bars aren't clear. I would recommend making them more visible by using a thicker line size.
We made the error bars clearer. Figures 2 and 3: The authors could zoom in to a smaller area for the fluorescence images so that we can see the YFP image in more detail.

7.
We included zoomed in images as suggested.
8. To our knowledge, the CRISPR/Cas9 approach used/described in Beneke et al., 2017 is not a conditional KO. It is described as such in the introduction.
We removed the reference from that sentence. and secondary effects. The authors carefully highlight the possible drawbacks, especially not knowing whether it works in bloodstream forms, and target protein accessibility. It is likely that proteins that are in membrane-bound compartments (glycosome, mitochondrion, ER) will be less susceptible. In future, it would be interesting to know whether it was really necessary to express the nuclear-targeted version of the nanobody fusion in order to get degradation of the kinetochore protein, and what happens for proteins that shuttle between nucleus and cytoplasm. It's also possible that some "accessible" proteins will be less amenable than others, as happens with other degron systems -only testing will reveal this.
It would have been useful to supply annotated versions of the plasmid sequences and some maps. I'm sure researchers could work this out but it would save time to supply the details in advance. On the other hand it's really nice that the sequences are already supplied, and the colour-coding on the sequence Table is already very helpful.
I have a small quibble. The statement "kinetoplastids may represent one of the earliest-branching eukaryotes based on a number of unique molecular features" with the reference to Cavalier-Smith's paper in 2010 should probably be removed. Most of the arguments in that paper have since been invalidated. Cavalier-Smith's conclusion is based mainly on the absence of Tom40 and ORC complexes. Both Tom40 and ORC complexes are known to be present, although highly diverged from those of other model organisms. In contrast, for example, some Kinetoplastid protein sequences (e.g. Tom22) resemble plants more than yeast. Other remaining arguments for things that are missing or unusual are also out of date. Possibly more remarkable is the completely new kinetochore composition in trypanosomes, discovered by the current author. However, it depends on what you judge to be important -what about (for example) the novel proteins that replace histones in Dinoflagellates? Burki et al. (2019 1 ), based on seqeunces, put all of the Excavata and various other things as branching separately, but on the same "level" as separation of Amorphea from Plants and SAR, with no way of seeing what came first. Trying to pick out a few organisms as being earliest-branching based on particular features is simply too subjective.
Are the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes findings presented in the article? Yes