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Referee #2
Evidence, reproducibility and clarity
Summary: In this study, the authors investigate novel functions of the tubulin typrosine ligase-like protein 6 (TTLL6), which covalently adds glutamate residues to the C-terminus of a given protein. The authors have already published previously on this topic. In the current study, the role of TTLL6 in colon function and pathologies was investigated. The study consists of two major parts.
In the first part, a mouse model is used to show that TTLL6 is expressed at elevated levels and activity in epithelial cells of the colon. A database search indicated that TTL6 expression positively correlates with prognosis of patients with colorectal cancer. The authors generated a TTLL6 KO mouse and showed that induced tumor growth at 40 days was more positive for vimentin in the crypts of these mice, which should correlate with tumor aggressiveness. This difference was not observed anymore after 70 days. Morphological analyses of the crypts showed that in TTLL6 KO mice the crypts increased in length, a difference in proliferation markers, and a change of cell types in the crypts was observed.<br />
In the second part, the authors used a modification-specific antibody to immunoprecipitate (IP) proteins modified by TTLLs. To identify TTLL6-dependently modified target proteins, they compared these results with IPs from TTLL6 KO mice. A total of 43 proteins were identified this way. Because of their similarity to the tubuline tail sequence, two of the enriched proteins, PurA and PurB, were further analyzed. The authors provide evidence that PurA but not PurB is modified by TTLL6, which as a result changes its subcellular localization.
While the first part of this work provides convincing novel insights into TTLL6's function with potential pathological relevance, the second part raises some concerns. I would therefore tend to rate the quality of the first part significantly higher than the second part.
Major comments:
1) When considering the results of the induced colorectal cancer test, the only significant difference between WT and KO was the moderately higher expression of Vimentin (figure 5E-F). Since this is the main evidence for a pathological relevance of TTLL6 in cancer, it is important to understand how the quantification of Vimentin in the complex tissue shown in figure 5E was done. A detailed description of how these images were analysed and perhaps a table with raw data would be essential to convince the reader of the conclusions. In the currently presented form, I find the analyses not too convincing.
2) Figure 7A: It was somewhat surprising that two of the least significant (PurA is just below the cutoff) were used for further analyses. Although the authors explain that both proteins have strong sequence similarity to the know TTLL6 target, tubuline, the C-terminal, genomically encoded protein sequence of PurA and PurB already contain several glutamates. This raises the concern that the polyE antibody in the IP possibly detected the non-modified C-terminal tail of PurA and PurB and that both proteins may not be modified by TTLL6. Because of this and the lower significance than other candidates, the authors should consider focussing on other hits (OPTIONAL). Besides being much more significant, they lack an accumulation of glutamates in their C-terminus (at least the ones I looked at). Alternatively, the concern of having potentially IP-ed unmodified proteins should be addressed.
3) Figure 8A: this figure compares PurA with a modified PurA that lacks the C-terminal EEE stretch. The authors conclude that the subcellular localization is different between both and that the nuclear localization of WT PURA must be due to modification by the co-expressed TTLL6. There are two major concerns with this conclusion:
Firstly, the expression of PurA without TTLL6 co-expression is a missing essential control. This would show if PurA itself is already predominantly located in the nucleus regardless of potential modifications (PurA seems to have different nucleocytoplasmic localization in different cell types). Secondly, both depicted cells look very different. In PurA the nuclei are much smaller and the cytoplasm seems also much smaller than in the PurA DDD-expressing cells. Furthermore, IF staining without proper quantification of several cells seem less than ideal for such conclusions. In case, the authors want to convincingly validate this conclusion such a quantification with several cells would be required. OPTIONAL: an alternative approach would be a nucleo-cytoplasmic fractionation experiment followed by a western blot.
Figure 8B: it seems that the contrast between the images of the upper and lower panel is very different. For this reason, I find it difficult to follow the conclusions. However, even when ignoring this aspect, I have great problems coming to the same conclusions as the authors.
Minor comments:
1) In figure 3A it would help if the legend describes what exactly "Control (+ or -)" means.
2) In figure 3E-F, a label inside of the figure (what is the red bar, what the blue) would help the reader to faster grasp the subfigures.
3) Figure 7C-D: these experiments are based on strong overexpression of TTLLs, which might result in unphysiological modifications of PurA. I would suggest to include a note of caution in the discussion that this is a possibility.
4) In the discussion (page 9, last paragraph), it is stated: "Our findings suggest that the polyglutamylation of PurA is essential for maintaining colonic homeostasis". I do not understand this statement, as this study does not provide any evidence that modification of PurA does play a functional role in the colon (expression itself is not an evidence for function importance or even being "essential"). I recommend to remove this statement.
5) Not all abbreviations are introduced properly (like CRC).
Significance
In general, this study addresses a very interesting aspect - i.e. the covalent addition of multiple glutamate residues to the C-terminus of a target gene by the enzyme TTLL6. The authors convincingly show that this protein regulates the morphology and composition of crypts in subregions of the colon. This is certainly a new and important finding that expands our knowledge about the functional breadth of this class of enzymes.
If convincingly validated (see major concerns), also the pathological relevance of this enzyme for cancer progression would be of general interest. However, this statement has to be considered with a note of caution as this is not my area of expertise.
The validation of novel targets of TTLL6 after IP is - at this stage of the manuscript - not very convincing to me. In particular the claim that PurA does play a functional role in the TTLL6-dependent regulation (of crypts) is not justified by the data. However, given that the list of other candidates contains several important gene regulators, this work might have the potential to open up to open up the field for new research directions.
The reviewer's areas of expertise: cell biology, biochemistry, histology.