The TTLL10 polyglycylase is stimulated by tubulin glutamylation and inhibited by polyglycylation
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eLife Assessment
In their study, Cummings et al. provide a valuable advance in understanding the hierarchical regulation of tubulin polyglycylation, demonstrating that TTLL8 initiates monoglycylation which is a prerequisite for TTLL10-mediated polyglycylation. The evidence supporting these mechanistic insights is solid, relying on a compelling combination of purified biochemical assays, mass spectrometry, and microscopy. The work is further valued for revealing an unexpected crosstalk between polyglycylation and polyglutamylation that ensures a balanced post-translational modification landscape for proper cilia function.
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Abstract
Abstract
Microtubules in cells have complex and developmentally stereotyped posttranslational modifications that support diverse processes such as cell division, ciliary growth and axonal specification. Glycylation, the addition of glycines, singly (monoglycylation) or in chains (polyglycylation), is primarily found on axonemal microtubules where it functions in cilia maintenance and motility. It is catalyzed by three enzymes in the tubulin tyrosine ligase-like family, TTLL3, 8 and 10. We show that TTLL8 monoglycylates both α- and β-tubulin, unlike TTLL3 which prefers β-tubulin. Microscopy and mass spectrometry show that TTLL10 requires monoglycylation for high affinity microtubule binding and elongates polyglycine chains only from pre-existing glycine branches. Surprisingly, tubulin polyglycylation inhibits TTLL10 recruitment to microtubules proportional with the number of posttranslationally added glycines, suggesting an autonomous mechanism for polyglycine chain length control. In contrast, tubulin glutamylation, which developmentally precedes polyglycylation in cilia, increases TTLL10 recruitment to microtubules, suggesting a mechanism for sequential deposition of tubulin modifications on axonemes. Our work sheds light on how the tubulin code is written by establishing the substrate preference and regulation of TTLL glycylases and provides a minimal system for generating differentially glycylated microtubules for in vitro analyses of the tubulin code.
Article activity feed
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eLife Assessment
In their study, Cummings et al. provide a valuable advance in understanding the hierarchical regulation of tubulin polyglycylation, demonstrating that TTLL8 initiates monoglycylation which is a prerequisite for TTLL10-mediated polyglycylation. The evidence supporting these mechanistic insights is solid, relying on a compelling combination of purified biochemical assays, mass spectrometry, and microscopy. The work is further valued for revealing an unexpected crosstalk between polyglycylation and polyglutamylation that ensures a balanced post-translational modification landscape for proper cilia function.
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Reviewer #1 (Public review):
Summary:
In their current study, Cummings et al have approached this fundamental biochemical problem using a combination of purified enzyme-substrate reactions, MS/MS and microscopy in vitro to provide key insights into the hierarchy of generating polyglycylation in cilia and flagella. They first establish that TTLL8 is a monoglycylase, with the potential to add multiple mono glycine residues on both α- and β-tubulin. They then go on to establish that the monoglycylation is essential for TTLL10 binding and catalytic activity, which progressively reduces as the level of polyglycylation increases. This provides an interesting mechanism of how level of polyglycylation is regulated in the absence of a deglycylase. Finally, the authors also establish that for efficient TTLL10 activity, it is not just …
Reviewer #1 (Public review):
Summary:
In their current study, Cummings et al have approached this fundamental biochemical problem using a combination of purified enzyme-substrate reactions, MS/MS and microscopy in vitro to provide key insights into the hierarchy of generating polyglycylation in cilia and flagella. They first establish that TTLL8 is a monoglycylase, with the potential to add multiple mono glycine residues on both α- and β-tubulin. They then go on to establish that the monoglycylation is essential for TTLL10 binding and catalytic activity, which progressively reduces as the level of polyglycylation increases. This provides an interesting mechanism of how level of polyglycylation is regulated in the absence of a deglycylase. Finally, the authors also establish that for efficient TTLL10 activity, it is not just monoglycylation, but also polyglutamylation that is necessary, giving a key insight into how both these modifications interact with each other to ensure there is a balanced level of PTMs on the axonemes for efficient cilia function.
Strengths:
The manuscript is well written, and experiments are succinctly planned and outlined. The experiments used provide the conclusions to what the authors were hypothesising and provide some new novel possible mechanistic insights into the whole process of regulation of tubulin glycylation in motile cilia.
Weaknesses:
There were some weaknesses in the initial submission of the manuscript, but the authors have addressed these in their revised version either by giving clear explanations in the text or through additional experiments.
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Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
“In their current study, Cummings et al have approached this fundamental biochemical problem using a combination of purified enzyme-substrate reactions, MS/MS, and microscopy in vitro to provide key insights into the hierarchy of generating polyglycylation in cilia and flagella. They first establish that TTLL8 is a monoglycylase, with the potential to add multiple mono glycine residues on both α- and β-tubulin. They then go on to establish that monoglycylation is essential for TTLL10 binding and catalytic activity, which progressively reduces as the level of polyglycylation increases. This provides an interesting mechanism of how the level of polyglycylation is regulated in the absence of a deglycylase. Finally, the authors …
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
“In their current study, Cummings et al have approached this fundamental biochemical problem using a combination of purified enzyme-substrate reactions, MS/MS, and microscopy in vitro to provide key insights into the hierarchy of generating polyglycylation in cilia and flagella. They first establish that TTLL8 is a monoglycylase, with the potential to add multiple mono glycine residues on both α- and β-tubulin. They then go on to establish that monoglycylation is essential for TTLL10 binding and catalytic activity, which progressively reduces as the level of polyglycylation increases. This provides an interesting mechanism of how the level of polyglycylation is regulated in the absence of a deglycylase. Finally, the authors also establish that for efficient TTLL10 activity, it is not just monoglycylation, but also polyglutamylation that is necessary, giving a key insight into how both these modifications interact with each other to ensure there is a balanced level of PTMs on the axonemes for efficient cilia function.”
Strengths:
The manuscript is well-written, and experiments are succinctly planned and outlined. The experiments were used to provide the conclusions to what the authors were hypothesising and provide some new novel possible mechanistic insights into the whole process of regulation of tubulin glycylation in motile cilia.”
We thank the reviewer for their support of our study and recognition of its importance to understanding microtubule glycylation and its regulation.
“The initial part of the manuscript where the authors discuss about the requirement of monoglycylation by TTLL8 is not new. This was established back in 2009 when Rogowski et al (2009) showed that polyglycylation of tubulin by TTLL10 occurs only when co-expressed in cells with TTLL3 or TTLL8. So, this part of the study adds very little new information to what was known. “
Our study provides the first in vitro evidence with purified recombinant components that human TTLL8 is exclusively a monoglycylase (Figure 1) and that polyglycylation by TTLL10 requires previous priming with monoglycylation (Figure 2). Studies with purified recombinant components are the gold standard for establishing the activity of an enzyme as cellular work can be obfuscated by the activity of other regulators. We did cite in our original submission the work by Rogowski, Gaertig and Janke from 2009 (reference 15 in the original submission) as well as that Ikegami and Setou 2009 work (reference 26 in the original submission) that established that TTLL10 polygyclylase activity requires co-expression with TTLL8 in cells. Specifically, we stated in our original submission and in the revised manuscript:
“Cellular overexpression studies coupled with the use of antibodies that recognize mono- and polyglycylation indicate that TTLL8 is also a glycyl-initiase, while TTLL10 a glycyl-elongase (15, 26). However, direct biochemical evidence with purified enzymes for segregated initiation and elongation activity for glyclases is still lacking as does knowledge of their substrate specificity and regulation.”
In addition to citing the Setou study, we now cite again the Rogowski, Gaertig and Janke 2009 study later in the manuscript when the cellular data are mentioned again. Specifically, we state in the revised manuscript:
“This is consistent with cellular overexpression data which showed that polyglycylation signal was detected via antibody only in tubulin from cells that co-expressed TTLL8 and TTLL10, but not TTLL10 alone (15, 26).”
“The study also fails to discuss the involvement of the other monoglycylase, TTLL3 in the entire study, which is a weakness as in vivo, in cells, both the monoglycylases act in concert and so, may play a role in regulating the activity of TTLL10. “
We previously showed that purified recombinant TTLL3, like TTLL8, adds only monoglycines, with a preference for the b-tubulin tail (Garnham et al., PNAS 2017). Given that TTLL10 requires priming by monoglycylation, we expect that, similarly to TTLL8, TTLL3 will allow elongation of the initial monoglycyline chains by TTLL10.
(1) From the mass spec data, it appears that the Xaenopus Laevis TTLL10 can add up to 18 residues. However, the numbers indicated in Figure 2E seem to suggest that it is a maximum of 23 residues only at a particular position. Does this mean that the 13-18 residues observed are a collection of multiple short-chain polyglycylations or are there positions that the authors observed where there were chains of longer than 3 glycine residues? This would be an interesting point to note as when it was discovered in Paramecium, the polyglycyl chains were reported to be up to 34 residues (Redeker et al., Science 1994). If the authors could test the TTLL10 from Paramecium to observe if this is a consistent phenomenon across evolution or is there a biologically significant difference that is being developed, would be interesting to know.”
Figure 2E shows a subset of the modified tails that we identified and where the position of the posttranslationally added glycine can be mapped to a specific position, or range of positions. Additional species exist. We note that the mass spectra in Figure 2B are intact LC/MS, while those in Figure 2E are MS/MS. The ionization of tubulin tail peptides with larger number of glycines is not as efficient as for shorter glycine chains, reducing the sensitivity of detection of species that have higher number of glycines. This is not as pronounced when the mass spectra are obtained from the intact protein (Figure 2B). In summary, our data supports the fact that TTLL10 elongates polyglycine chains at multiple positions in the tubulin tail (shown in Figure 2E), however, we cannot ascertain the maximum polyglycine chain length, only the total number of glycyines added.
Testing the enzyme from Paramecium is an interesting proposal but outside the scope of this manuscript.
(2) While it is interesting to know that the TTLL10 binds to TTLL8-modified tubulin with a much higher affinity than unmodified tubulin, in vivo, the microtubules will be a mixture of both TTLL3- and TTLL8-modified tubulin. It would be good to see the binding of the enzyme to a tubulin that is modified by both TTLL3 and TTLL8 if the two have a greater influence on TTLL10 binding.”
Our previous work showed that purified recombinant TTLL3 has purely monoglycylase activity, with a preference for b-tubulin (Garnham et al., PNAS 2017). The sites of monoglycylation by TTLL3 overlap with those introduced by TTLL8 on b-tubulin (the difference being mainly that TTLL3 is more selective towards b-tubulin and thus has lower activity on a-tubulin). TTLL8 introduces additional monoGlys on the a-tubulin tail. Therefore, it is unlikely that TTLL10 will have a different response to microtubules that carry similar numbers of Gly residues, regardless of whether introduced by TTLL8 or TTLL3 and 8. Our data show that TTLL10 binding increases with Gly number, but that the gains in affinity plateau as the density of glycine residues on the tails increases above a certain threshold, likely because one TTLL10 molecule recognizes one monoGly branch, and steric hindrance on the tubulin tail prevents further recruitment of additional TTLL10 molecules.
(3) The authors have always increased the number of monoglycines in beta-tubulin more than in alpha-tubulin. Is there a rationale for this? Since TTLL8 is known to predominantly modify alphatubulin (Rogowski et al., 2009; Gadadhar et al., 2017) why did the authors not check for the increased binding of the TTLL10 on dimers where the number of monoglycines on alpha-tubulin is higher than 1.1? Especially when they themselves observe in their mass spec that even on alphatubulin there are 1, 2, and 3 glycines added. I would like to see what happens if the ratio is high alpha-G + low beta-G”
As our spectra in Figure 1 show, we find that TTLL8 is able to modify robustly in vitro both a- and b-tubulin but that it shows a slight preference for b-tubulin (Figure 1B). The work from the Janke group that the reviewer is referring to (Rogowski et al., 2009 and Gadahar et al., 2017) did not use recombinant, purified enzymes and unmodified microtubules as substrates and used axonemal tubulin (which carries many modifications), and so it is possible that the a-tubulin preference observed in that system when TTLL8 is overexpressed, is likely to other factors that do not reflect the biochemical property of the enzyme alone (for example, it could be because btubulin site are not available because they are already glutamylated). As can be seen from Figure 3D, the gain in affinity when increasing the number of glycines from one glycine is small, compared to the initial monoglycine added to the a- and the b-tubulin tail, likely reflecting that one tail cannot bind more than one TTLL10 at one time because of steric hindrance. Moreover, it is important here to note that glutamylation and glycylases compete for the same sites on the tubulin tails, as we have for example shown for TTLL3 and TTLL7 (Garnham et al., 2017), therefore the activity of these enzymes in vivo or with non-naïve substrates are context dependent and influences also what sites are available for TTLL10 to modify. In conclusion, by using recombinant enzymes and naïve tubulin we gain insight into the intrinsic property of these enzymes and therefore provide a framework for the interpretation of in vitro and in vivo observations.
(4) I wonder why the authors did not use the human TTLL10 to test if this also shows similar binding to the glycylated tubulin despite the fact that it is enzymatically inactive. If it does, then it would be interesting to see the kinetics of binding of this enzyme to see if the fall off of the enzyme from the tubulin is solely driven by the level of polyglycylation only, or if it has any other mechanism involved as well.”
Work with human recombinant TTLL10, a TTLL10 homolog which was proposed to be inactive, will be an interesting future direction but outside the scope of this manuscript. We did note in our previous manuscript (Garnham et al., 2017, Figure S5) that the residues which are mutated in the human enzyme compared to other mammals are on the dorsal face of the enzyme, far away from the active site, raising an interesting question of how they inactivate the enzyme. We need however to emphasize that our work clearly shows that it is polyglycylation on the microtubules that reduces binding of TTL10 to microtubules because experiments done in the absence of glycylating activity i.e. with enzyme that was incubated with microtubules that were pre-modified with polyglycline chains, but in the absence of glycyine substrate (precluding any glycylation activity during the binding assay) show that the binding decreases monotonically with the number of polyglycines on the microtubule (Figures 4A, B).
(5) In Figure 5, the authors use monoglycylated tubulin that is either glutamylated or not to show that the activity of TTLL10 is enhanced by the extent of polyglutamylation present on the tubulin. However, there is no evidence of the enzyme binding to microtubules that are only glutamylated. It would be good to test this to determine if the binding is also dependent on both monoglycylation and glutamylation or is it only the enzyme activity.
Figure 5E shows that TTLL10 binding increases with monoglycylation alone, and that glutamylation is additive and Figures 4A, B show that it is not the enzyme activity that affects the binding, but the glycylation state of the microtubule. We did not determine binding to microtubules that were only glutamylated, because TTLL10 would not be able to elongate polyglycine chains on those microtubules, even if it bound.
(6) The level of polyglycylation used in Figure 5 is quite low. It would be good to see how the length of the polyglycine chain impacts TTLL10 activity in the presence of polyglutamylation, and whether this has any cooperative effect leading to longer chain polyglycylation than what is seen with only monoglycylated tubulin.
We expect longer chain polyglycylation to have an inhibitory effect as we show in Figure 4.
“(7) In the overall study, the authors fail to discuss whether the activity of both the glycylases at different sites on tubulin is sequential, or modifications at different residues happen all at once. If the authors were to do a sequential time course of the modification followed by MS/MS analysis, they could get some indications about this.”
As the data in Figure 3D shows, the effect of adding more monoGly site on a tubulin tail has a muted effect on binding, indicating that the additional mono-Gly branches do not lead to more TTLL10 recruitment because of steric hindrance i.e. multiple TTLL10 enzymes cannot be accommodated on the same tail at the same time efficiently. This is consistent with the overall dimensions of the enzyme and the positions of its active site, which were modeled initially in our previous publication (Garnham et al., PNAS 2017). The site of TTL10 action is pre-determined by the position of the mono-Gly branch introduced by TTLL3 or TTLL8. The length of the tubulin tail and the proximity of mono-Gly sites to each other precludes TTLL10 acting at multiple positions at once on the same tail.
“(8) Do the modifications have any cooperative effect with respect to the sites of modification? Does modifying a particular site enhance the kinetics of modification of the other sites? Can the authors test this?”
This would be an interesting line of future investigations.
“Minor points:
(1’) The authors opine that the level of polyglycylation is regulated by the decreased binding of the TTLL10 to the polyglycylated tubulin. While this is an interesting argument, which could be a possibility based on the data they present, it would still not answer if this is a mechanism followed by TTLL10 of all species or not. If they could test the efficacy of TTLL10 from another species, to see the binding efficiency of that enzyme, it could potentially strengthen their argument of this possible mechanism.”
The differences between the properties of TTLL10 from different organisms will be an interesting focus of future investigations, but outside the scope of this present study. However, we would like to point out that the level of sequence conservation between TTLL10 makes it unlikely that other TTLL10 do not follow a similar mechanism, albeit with possible differences in the extent of the response. We also note that we have shown that polyglycylation also inhibits binding to the microtubule of the severing enzyme katanin (Szczesna et al., Dev. Cell 2022). Therefore, these studies suggests that polyglycylation might be a more general mechanism for reducing microtubule binding affinity since glycylation reduces the negative charge on the tubulin tails, which frequently interact with positively charged domains or interfaces in microtubule associated proteins.
“(2) The authors indicate that glycylases act on pre-glutamylated microtubules. However, in their assays, they use unmodified tubulin, which I would presume is also not glutamylated. If this is the case, how can they justify that the enzymes prefer pre-glutamylated microtubules? This is a bit unclear. Do they mean that their tubulin is already pre-glutamylated? Have they tested this?”
The statement regarding the action of these enzymes on glutamylated microtubules refer to the in vivo situation where polyglycylated microtubules appear in cilia biogenesis after the microtubules in the axoneme are already glutamylated. In vitro, by using microtubules that are only monoglycylated and microtubules that are both glutamylated and monoglycylated, we show that glutamylation further increases recruitment of TTLL10 to microtubules that are monoglycyated. Therefore, glutamylated microtubules will be polyglycylated preferentially over those that are not glutamylated.
We state: “Axonemal microtubules are abundantly glutamylated. Glutamylation appears during cilia development first, followed by glycylation (12, 13), indicating that in this scenario glycylases act on pre-glutamylated microtubule substrates.”
“(3) In continuation with the previous point, an immunoblot of their purified tubulin showing no reactivity to anti-glycylation or anti-glutamylation antibodies, which upon treatment with TTLL8 reacts to the anti-glycylation antibody would be confirmatory evidence to show that the isolated tubulin was indeed unmodified.”
We have now included a Western blot of our TOG-purified tubulin as Figure S3 in our revised manuscript. This shows a faint signal with the pep-G1 antibody and a very strong signal after TTLL8 treatment. We are not sure whether the low signal with the pep-G1 antibody for the unmodified tubulin is due to low bona fide monoglycylation-specific signal or a low affinity nonspecific interaction of this antibody (raised against mono-glycylated tubulin tail peptides) with the unmodified tubulin. We note that this signal is clearly visible only when loading at least 0.2 micrograms of the purified tubulin. At this loading level the signal for the glycylated species is saturated. It is also important to note that we have not detected glycylated species in this tubulin either by LC-MS or MS/MS. Therefore, our data strongly indicate that the tubulin purified from tsA201 cells is not glycylated or has at most extremely low levels of glycylation. Importantly, this potential trace level of monoglycylated tubulin does not affect any of the conclusions in this study. The Western blot also shows no detectable signal with the polyglycyation antibody in the unmodified tubulin and a very strong, saturated signal after the tubulin was treated with both TTLL8 and TTLL10. We also added an additional Figure S8 that shows that the tSA201 tubulin does not give a detectable signal for glutamylation. Please see also Figure 3 from Vemu et al., Methods Enzymology 2017 where we also published a Western blot from our TOG-purified tubulin using anti-glutamylation antibodies.
“(4) In their study, the authors have used polyglycylation of up to 10-13 residues. This brings me to my first point that in the case of Paramecium, the number was identified to be up to 34, which would mean that this enzyme has higher binding or catalytic activity. I would like to know the authors' perspective on this, as to what could potentially determine the difference in the activities of TTLL10 across species.”
The Xenopus TTLL10 enzyme can add more glycines than the 10-13 range that we show here if the enzyme is incubated for longer periods. The fact that glycine numbers as high as 34 were detected in Paramecium does not necessarily mean that the Paramecium enzyme is more active since there is no equivalent data to compare it with from Xenopus. The only way to address potential species differences in enzyme specific activity is to purify enzymes from different species and compare their activity side-by-side.
(5) How was the completion of the reaction of monoglycylation and polyglycylation determined? If the enzymes were left for more than 20 minutes, did TTLL8/ TTLL10 add more glycines? What is the reason for using less tubulin (1:20 enzyme:tubulin molar ratio) for monoglycylation by TTLL8, and more tubulin (1:50 enzyme:tubulin molar ratio) for polyglycylation by TTLL10?
Yes, if the enzymes were incubated longer, they added more glycines. The extent of glycylation was determined from the LC-MS and the incubation time was varied to obtain samples with fewer or more glycines. The lower ratio used for TTLL10 is because of the higher specific activity of that enzyme compared to TTLL8.
(6) Figure S2 A, b2 ion is not indicated in the peptide sequence, while it is shown in the m/z graph.
We thank the reviewer for the careful reading. We have corrected this in our MS/MS spectrum.
Reviewer #2 (Public review):
“In their manuscript, Cummings et al. focus on the enzymatic activities of TTLL3, TTLL8, and TTLL10, which catalyze the glycylation of tubulin, a crucial posttranslational modification for cilia maintenance and motility. The experiments are beautifully performed, with meticulous attention to detail and the inclusion of appropriate controls, ensuring the reliability of the findings. The authors utilized in vitro reconstitution to demonstrate that TTLL8 functions exclusively as a glycyl initiase, adding monoglycines at multiple positions on both α- and β-tubulin tails. In contrast, TTLL10 acts solely as a tubulin glycyl elongase, extending existing glycine chains. A notable finding is the differential substrate recognition between TTLL glycylases and TTLL glutamylases, highlighting a broader substrate promiscuity in glycylases compared to the more selective glutamylases. This observation aligns with the greater diversification observed among glutamylases. The study reveals a hierarchical mechanism of enzyme recruitment to microtubules, where TTLL10 binding necessitates prior monoglycylation by TTLL8. This binding is progressively inhibited by increasing polyglycine chain length, suggesting a self-regulatory mechanism for polyglycine chain length control. Furthermore, TTLL10 recruitment is enhanced by TTLL6mediated polyglutamylation, illustrating a complex interplay between different tubulin modifications. In addition, they uncover that polyglutamylation stimulates TTLL10 recruitment without necessarily increasing glycylation on the same tubulin dimer, due to the potential for TTLLs to interact with neighboring tubulin dimers. This mechanism could lead to an enrichment of glycylation on the same microtubule, contributing to the complexity of the tubulin code. The article also addresses a significant challenge in the field: the difficulty of generating microtubules with controlled posttranslational modifications for in vitro studies. By identifying the specific modification sites and the interplay between TTLL activities, the authors provide a valuable tool for creating differentially glycylated microtubules. This advancement will facilitate further studies on the effects of glycylation on microtubule-associated proteins and the broader implications of the tubulin code. In summary, this study substantially contributes to our knowledge of posttranslational enzymes and their regulation, offering new insights into the biochemical mechanisms underlying microtubule modifications. The rigorous experimental approach and the novel findings presented make this a pivotal addition to the field of cellular and molecular biology.”
We thank the reviewer for their support of our work.
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eLife assessment
In their manuscript, Cummings et al. use in vitro reconstitution to examine the differential activities of tubulin polyglycylases, providing valuable insights into the enzymatic regulation of microtubule glycylation and its mechanistic role in maintaining cilia function and microtubule dynamics. The convincing evidence, supported by well-designed experiments and appropriate controls, significantly advances our understanding of the tubulin code and its biochemical mechanisms.
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Reviewer #1 (Public Review):
Summary:
In their current study, Cummings et al have approached this fundamental biochemical problem using a combination of purified enzyme-substrate reactions, MS/MS, and microscopy in vitro to provide key insights into the hierarchy of generating polyglycylation in cilia and flagella. They first establish that TTLL8 is a monoglycylase, with the potential to add multiple mono glycine residues on both α- and β-tubulin. They then go on to establish that monoglycylation is essential for TTLL10 binding and catalytic activity, which progressively reduces as the level of polyglycylation increases. This provides an interesting mechanism of how the level of polyglycylation is regulated in the absence of a deglycylase. Finally, the authors also establish that for efficient TTLL10 activity, it is not just …
Reviewer #1 (Public Review):
Summary:
In their current study, Cummings et al have approached this fundamental biochemical problem using a combination of purified enzyme-substrate reactions, MS/MS, and microscopy in vitro to provide key insights into the hierarchy of generating polyglycylation in cilia and flagella. They first establish that TTLL8 is a monoglycylase, with the potential to add multiple mono glycine residues on both α- and β-tubulin. They then go on to establish that monoglycylation is essential for TTLL10 binding and catalytic activity, which progressively reduces as the level of polyglycylation increases. This provides an interesting mechanism of how the level of polyglycylation is regulated in the absence of a deglycylase. Finally, the authors also establish that for efficient TTLL10 activity, it is not just monoglycylation, but also polyglutamylation that is necessary, giving a key insight into how both these modifications interact with each other to ensure there is a balanced level of PTMs on the axonemes for efficient cilia function.
Strengths:
The manuscript is well-written, and experiments are succinctly planned and outlined. The experiments were used to provide the conclusions to what the authors were hypothesising and provide some new novel possible mechanistic insights into the whole process of regulation of tubulin glycylation in motile cilia.
Weaknesses:
The initial part of the manuscript where the authors discuss about the requirement of monoglycylation by TTLL8 is not new. This was established back in 2009 when Rogowski et al (2009) showed that polyglycylation of tubulin by TTLL10 occurs only when co-expressed in cells with TTLL3 or TTLL8. So, this part of the study adds very little new information to what was known.
The study also fails to discuss the involvement of the other monoglycylase, TTLL3 in the entire study, which is a weakness as in vivo, in cells, both the monoglycylases act in concert and so, may play a role in regulating the activity of TTLL10.
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Reviewer #2 (Public Review):
In their manuscript, Cummings et al. focus on the enzymatic activities of TTLL3, TTLL8, and TTLL10, which catalyze the glycylation of tubulin, a crucial posttranslational modification for cilia maintenance and motility. The experiments are beautifully performed, with meticulous attention to detail and the inclusion of appropriate controls, ensuring the reliability of the findings. The authors utilized in vitro reconstitution to demonstrate that TTLL8 functions exclusively as a glycyl initiase, adding monoglycines at multiple positions on both α- and β-tubulin tails. In contrast, TTLL10 acts solely as a tubulin glycyl elongase, extending existing glycine chains. A notable finding is the differential substrate recognition between TTLL glycylases and TTLL glutamylases, highlighting a broader substrate …
Reviewer #2 (Public Review):
In their manuscript, Cummings et al. focus on the enzymatic activities of TTLL3, TTLL8, and TTLL10, which catalyze the glycylation of tubulin, a crucial posttranslational modification for cilia maintenance and motility. The experiments are beautifully performed, with meticulous attention to detail and the inclusion of appropriate controls, ensuring the reliability of the findings. The authors utilized in vitro reconstitution to demonstrate that TTLL8 functions exclusively as a glycyl initiase, adding monoglycines at multiple positions on both α- and β-tubulin tails. In contrast, TTLL10 acts solely as a tubulin glycyl elongase, extending existing glycine chains. A notable finding is the differential substrate recognition between TTLL glycylases and TTLL glutamylases, highlighting a broader substrate promiscuity in glycylases compared to the more selective glutamylases. This observation aligns with the greater diversification observed among glutamylases. The study reveals a hierarchical mechanism of enzyme recruitment to microtubules, where TTLL10 binding necessitates prior monoglycylation by TTLL8. This binding is progressively inhibited by increasing polyglycine chain length, suggesting a self-regulatory mechanism for polyglycine chain length control. Furthermore, TTLL10 recruitment is enhanced by TTLL6-mediated polyglutamylation, illustrating a complex interplay between different tubulin modifications. In addition, they uncover that polyglutamylation stimulates TTLL10 recruitment without necessarily increasing glycylation on the same tubulin dimer, due to the potential for TTLLs to interact with neighboring tubulin dimers. This mechanism could lead to an enrichment of glycylation on the same microtubule, contributing to the complexity of the tubulin code. The article also addresses a significant challenge in the field: the difficulty of generating microtubules with controlled posttranslational modifications for in vitro studies. By identifying the specific modification sites and the interplay between TTLL activities, the authors provide a valuable tool for creating differentially glycylated microtubules. This advancement will facilitate further studies on the effects of glycylation on microtubule-associated proteins and the broader implications of the tubulin code. In summary, this study substantially contributes to our knowledge of posttranslational enzymes and their regulation, offering new insights into the biochemical mechanisms underlying microtubule modifications. The rigorous experimental approach and the novel findings presented make this a pivotal addition to the field of cellular and molecular biology.
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