Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs
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Evaluation Summary:
This is a very elegant study that through cross-species analysis describes the evolution of ubiquitin ligase adaptor protein LZTR1-mediated degradation of RAS-related GTPase RIT1 as a principal regulatory mechanism for RIT1 function and its role in Noonan syndrome, a prominent subgroup of RASopathy disorders. Extensive genetic experiments in Drosophila and mouse suggest that important pathological phenotypes observed in LZTR1-linked RASopathy models are mediated by its ubiquitination target RIT1 and less by the canonical RAS isoforms. While the work further supports the connection between LZTR1 mediated RIT1 level modulation, it does not fully rule out the significance of canonical RAS isoforms in LZTR1-associated RASopathies in humans.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. Here, we have analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, we show that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1 . Overall, our results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.
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Evaluation Summary:
This is a very elegant study that through cross-species analysis describes the evolution of ubiquitin ligase adaptor protein LZTR1-mediated degradation of RAS-related GTPase RIT1 as a principal regulatory mechanism for RIT1 function and its role in Noonan syndrome, a prominent subgroup of RASopathy disorders. Extensive genetic experiments in Drosophila and mouse suggest that important pathological phenotypes observed in LZTR1-linked RASopathy models are mediated by its ubiquitination target RIT1 and less by the canonical RAS isoforms. While the work further supports the connection between LZTR1 mediated RIT1 level modulation, it does not fully rule out the significance of canonical RAS isoforms in LZTR1-associated RASopathies in humans.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
This is a very elegant study that through cross-species analysis describes the evolution of ubiquitin ligase adaptor protein LZTR1-mediated degradation of RAS-related GTPase RIT1 as a principal regulatory mechanism for RIT1 function and its role in Noonan syndrome, a prominent subgroup of RASopathy disorders. Extensive genetic experiments in Drosophila and mouse suggest that important pathological phenotypes observed in LZTR1-linked RASopathy models are mediated by its ubiquitination target RIT1 and less by the canonical RAS isoforms. While the work further supports the connection between LZTR1 mediated RIT1 level modulation, it does not fully rule out the significance of canonical RAS isoforms in LZTR1-associated RASopathies in humans.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This manuscript seeks to clarify which Ras GTPases are regulated by the Cullin adaptor protein LZTR1. Prior work from other groups identified Ras proteins (H/K/M/N-Ras) as substrates of Lztr1, whereas prior work from the authors has identified RIT1 as the major substrate. Here the authors take an evolutionary genetic approach by comparing the function of Lztr1 in Drosophila and mice. They find that Lztr1 appears to have co-evolved closely with RIT1, but not other Ras family members. This is supported by sequence conservation through evolution, and by an apparently higher binding affinity of Lztr1 to Rit1 than to other Ras GTPases. The authors go on to show that inactivation of Lztr1 in Drosophila and mice results in increased RIT1 protein abundance, but has a minimal effect on other Ras family members. …
Reviewer #1 (Public Review):
This manuscript seeks to clarify which Ras GTPases are regulated by the Cullin adaptor protein LZTR1. Prior work from other groups identified Ras proteins (H/K/M/N-Ras) as substrates of Lztr1, whereas prior work from the authors has identified RIT1 as the major substrate. Here the authors take an evolutionary genetic approach by comparing the function of Lztr1 in Drosophila and mice. They find that Lztr1 appears to have co-evolved closely with RIT1, but not other Ras family members. This is supported by sequence conservation through evolution, and by an apparently higher binding affinity of Lztr1 to Rit1 than to other Ras GTPases. The authors go on to show that inactivation of Lztr1 in Drosophila and mice results in increased RIT1 protein abundance, but has a minimal effect on other Ras family members. Together, these data support the author's conclusion that Rit1 is the primary substrate of Lztr1.
A strength of this work is the genetic epistasis experiment in mice, where the authors show that deletion of Rit1 partially rescues the embryonic lethality of Lztr1 mutant mice. Another strength is the very compelling effect on Rit1 upregulation observed across multiple species and experiments upon Lztr1 inactivation.
The conclusions of the paper are mostly well supported by the data presented. An exception to this would be the signaling data in Figure 4g, in which the difference betweeen Lztr1/Rit1 double KO and Lztr1 single KO cells is minimal at best. Quantitative assessment of these signaling differences might help strengthen this weak point. The paper would benefit from more quantitative analyses in other areas, such as the description of fly and mouse phenotypes in Figure 2 and 3. In addition, some of the conclusions overlap with results in Castel et al., Science 2019, so the findings are not totally novel, though this reviewer does not find that to be a concern. Finally, inclusion of human data would further increase the impact of the work.
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Reviewer #2 (Public Review):
LZTR1 mediated degradation modulates the signaling output of several GTPases and has important implications for health and disease as loss of function mutations have been implicated in the genetic disorders collectively referred to RASopathies. This study employs a series of elegant and rigorous approaches, combining phylogenetic analysis and cross-species biochemical and functional analysis to define that RIT1 is the preferential GTPase binding partner of Lztr1, that this preferential interaction and mode of regulation of RIT1 emerged early in evolution, and it is the major determinant of the pathobiological function of Lztr1. The authors also systematically characterize the biological consequences of heterozygous and homozygous deletion of Lztr1. Whereas previous studies had suggested that in the context …
Reviewer #2 (Public Review):
LZTR1 mediated degradation modulates the signaling output of several GTPases and has important implications for health and disease as loss of function mutations have been implicated in the genetic disorders collectively referred to RASopathies. This study employs a series of elegant and rigorous approaches, combining phylogenetic analysis and cross-species biochemical and functional analysis to define that RIT1 is the preferential GTPase binding partner of Lztr1, that this preferential interaction and mode of regulation of RIT1 emerged early in evolution, and it is the major determinant of the pathobiological function of Lztr1. The authors also systematically characterize the biological consequences of heterozygous and homozygous deletion of Lztr1. Whereas previous studies had suggested that in the context of Noonan Syndrome, a major subgroup of RASopathies, Lztr1 is happloinsufficient, the authors data strongly indicates that Lztr1 is, instead, happlosufficient. Consisted with previous studies, homozygous deletion of Lztr1 is embryonic lethal. Extending these observations, the authors link lethality to developmental vascular and cardiac defects that are prominent characteristics of Noonan Syndrome, and also demonstrate that mechanistically, the embryonic lethal phenotype is driven by Lztr1 mediated regulation of Rit1. The study has several strengths, in particular the cross-species study of this interaction and evolution of functional output in organismal development and disease states, as well as rigorous and carefully executes experimental approaches. This study is likely to be of broad interest to the RasGTPase field and those studying Rasopathies.
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Reviewer #3 (Public Review):
The paper of Cuevas-Navarro et al. strengthens the mechanistic and physiological link between RIT1 and LZTR1.
They first performed an in silico analysis of the similarity of the RIT1, KRAS and LZTR1 protein sequences from a number of model organisms ranging from yeast to human. While KRAS is highly conserved, both RIT1 and LZTR1 show a higher divergence in simpler organisms. The co-evolution of RIT1 and LZTR1 is further supported by qualitative interaction data (pull-downs) showing that between four species the interaction preference of LZTR1-orthologues for RIT1-, as compared to KRAS-orthologues, is conserved.
Next, they studied the effect of LZTR1 loss of function using two knockouts in Drosophila finding that RIT1-orthologue levels are much more increased than RAS-orthologue levels relative to the …
Reviewer #3 (Public Review):
The paper of Cuevas-Navarro et al. strengthens the mechanistic and physiological link between RIT1 and LZTR1.
They first performed an in silico analysis of the similarity of the RIT1, KRAS and LZTR1 protein sequences from a number of model organisms ranging from yeast to human. While KRAS is highly conserved, both RIT1 and LZTR1 show a higher divergence in simpler organisms. The co-evolution of RIT1 and LZTR1 is further supported by qualitative interaction data (pull-downs) showing that between four species the interaction preference of LZTR1-orthologues for RIT1-, as compared to KRAS-orthologues, is conserved.
Next, they studied the effect of LZTR1 loss of function using two knockouts in Drosophila finding that RIT1-orthologue levels are much more increased than RAS-orthologue levels relative to the parental background. These data are supported by transgenic knock-in experiments with HA-tagged RAS- and RIT1-proteins that are again increased in the LZTR1 knockout background.
Subsequent mouse experiments produced results that conflict with previously published data by Steklov et al. The authors find here that the heterozygous deletion, does not produce a NS-like phenotype. They comment that also in human NS patients bi-allelic LZTR1 loss is found. To address this, they go on asking the important question, whether the lethal phenotype that is observed in LZTR1-/- could depend on the mouse strain background. Indeed, only in the mixed background (of C57BL/6N and 129sv mouse strains) did they obtain a few viable homozygous LZTR1-/- mice, which display typical NS-like phenotypes and show increased RIT1 levels in all major tissues. This ko phenotype is further examined in late embryos of these mice with particular attention to the cardiovascular system, finding that it is broadly perturbed (vascularization defects leading to bleeding and heart growth defects).
The authors then performed epistasis experiments, revealing that an additional RIT1 ko can rescue the LZTR1 ko lethality. Finally, they claim that serum stimulation of MEFs isolated from the double-ko animals results in a noticeable decrease in MAPK-levels, as compared to LZTR1-/-.
Strengths:
The evolutionary conservation of the stronger dependency between LZTR1 and RIT1 orthologs underscores that RIT1 is a major target of LZTR1. This is in line with the fact that RIT1 is constitutively GTP-bound and therefore regulated by proteolysis. The former conclusion is supported by qualitative interaction data (Fig. 1b), differential proteomics data in LZTR1 knockout and transgenic flies (Fig. 1c-e), mouse knockout (Fig. 3g) and epistasis experiments (Fig. 4).
The use of two genetic model organisms, laborious breeding schemes in mouse and the state-of-the-art quantification of major RASopathy phenotypes in mice are a major strength.An implicit conclusion that could be more elaborated is that genetic modifiers seem to regulate the strength of the LZTR1 and RIT1 dependency. These may explain the discrepancies in the heterozygous LZTR1 phenotypes of the mice generated here as compared to those by Steklov et al.
Such modifiers are suggested by both tissue-specific RIT1 levels (Fig. 3g), as well as mouse strain specific differences in the LZTR1-mutant phenotypes (Fig. 3e).Weaknesses:
While manuscript and data quality are overall very high, there are some important shortcomings.
Most importantly, the rescue of the lethal LZTR1-/- phenotype by the additional bi-allelic knockout of RIT1 (Fig. 4d), is only insufficiently documented and open to alternative interpretations. While a rescue of the heart phenotype (Fig. 4e) is qualitatively indicated, a full phenotypic quantification as done in Fig. 3 is missing, albeit the authors claim 'resulting DKO mice appeared normal, were fertile, and were absent of any detectable phenotype that resembled other NS mouse models, as assessed by size, heart weight, and cranial morphology'.
Furthermore, it should be noted by the authors that the rescue of the lethal phenotype and the limited assessment of phenotypes overall may not allow to detect more subtle RASopathy-like manifestations. This may be particularly relevant in the double-knockout animals, where the rescue from lethality may mostly relate to the rescue of the vascularization and heart defects.
Finally, some Western blotting data need more repeats and quantification for proper interpretation. Notably, data shown in Fig. 4g need to be substantiated and possibly reinterpreted. In all LZTR1 wt conditions, LZTR1 is induced by serum, at later times than MAPK-output (pMEK and pERK), while RIT1 levels remain constant over time. A dependency between LZTR1 and RIT1 cannot be recognized here, and a modulation of MAPK-output by RIT1 abundance is also not seen (LZTR1+/+ and LZTR1-/- samples). The levels of the canonical Ras proteins should be investigated and the suggested relation between RIT1 elevation and increased MAPK-output re-examined; this is even more relevant, as previous reports did not suggest Raf as effector of RIT1.
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