A Liquid-to-Solid Phase Transition Enhances the Catalytic Activity of SARM1
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Curated by eLife
Evaluation Summary:
This manuscript describes an interesting regulatory mechanism that activates SARM1, an enzyme that degrade NAD+ and promote axon degeneration. Previous structural and biochemical studies mostly focus on how SARM1 is auto-inhibited at basal conditions and this manuscript provides evidences supporting that phase transition could promote its activity, thus providing new understanding about its regulatory mechanism. The finding also enables in vitro assays to be carried out more easily and thus could facilitate the development of small molecule modulators of SARM1 for therapeutics purposes.
(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 #2 agreed to share their names with the authors.)
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Abstract
Sterile alpha and toll/interleukin receptor (TIR) motif–containing protein 1 (SARM1) is a neuronally expressed NAD + glycohydrolase whose activity is increased in response to various stressors. The consequent depletion of NAD + triggers axonal degeneration (i.e., Wallerian degeneration), which is a characteristic feature of neurological diseases, including peripheral neuropathies and traumatic brain injury. Notably, SARM1 knockout mice show minimal degeneration in models of peripheral neuropathy and traumatic brain injury, making SARM1 a promising therapeutic target. However, the development of SARM1 inhibitors has been challenging as the purified enzyme is largely inactive. Herein, we report that SARM1 activity is increased ∼2000–fold by a liquid-to-solid phase transition. These findings provide critical insights into SARM1 biochemistry with important implications for the situation in vivo . Moreover, they will facilitate the discovery of novel SARM1–targeted therapeutics.
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Reviewer #3 (Public Review):
Advances in understanding the biochemical and cellular mechanism of neuronal damage are investigated here and are to be appreciated. The strength of this work on SARM1 is its success in establishing that a concentration-dependent phase change activates the enzyme to degrade NAD, an essential component of neuronal integrity. Cellular significance is demonstrated in C. elegans neuronal damage triggered by citrate. Weaknesses are that high citrate is required for SARM1 effects but low citrate is used in the C. elegans model without establishing concentration dependence in the C. elegans system. The progression on neuronal damage from enzyme activation to neuronal damage in C. elegans is missing the quantitation of NAD change. A strength of the work is to provide a solid stepping-stone to permit the next steps …
Reviewer #3 (Public Review):
Advances in understanding the biochemical and cellular mechanism of neuronal damage are investigated here and are to be appreciated. The strength of this work on SARM1 is its success in establishing that a concentration-dependent phase change activates the enzyme to degrade NAD, an essential component of neuronal integrity. Cellular significance is demonstrated in C. elegans neuronal damage triggered by citrate. Weaknesses are that high citrate is required for SARM1 effects but low citrate is used in the C. elegans model without establishing concentration dependence in the C. elegans system. The progression on neuronal damage from enzyme activation to neuronal damage in C. elegans is missing the quantitation of NAD change. A strength of the work is to provide a solid stepping-stone to permit the next steps in cementing the biochemical pathways of initiating cellular damage to neurons.
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Reviewer #2 (Public Review):
The latest manuscript of Loring and coworkers solves a number of important problems of SARM1 structure and function at once, namely why the purified enzyme has little activity, what size is the active multimer, whether it produces cADPR on the way to ADPR, and how this enzyme may overcome autoinhibition by NAD+ in vivo. In work that is technically sound, the authors describe a phase transition that can be induced by macroviscogens and by citrate in which we are able to see cryoEM images of activated multimers and the induction of SARM1 activity in worms by citrate. Working with concentrated enzyme, the authors are further able to characterize SARM1 activity in detail and clearly show which cations are most inhibitory and that ADPR and not cADPR is the primary product of the reaction.
There is clearly a lot …
Reviewer #2 (Public Review):
The latest manuscript of Loring and coworkers solves a number of important problems of SARM1 structure and function at once, namely why the purified enzyme has little activity, what size is the active multimer, whether it produces cADPR on the way to ADPR, and how this enzyme may overcome autoinhibition by NAD+ in vivo. In work that is technically sound, the authors describe a phase transition that can be induced by macroviscogens and by citrate in which we are able to see cryoEM images of activated multimers and the induction of SARM1 activity in worms by citrate. Working with concentrated enzyme, the authors are further able to characterize SARM1 activity in detail and clearly show which cations are most inhibitory and that ADPR and not cADPR is the primary product of the reaction.
There is clearly a lot of regulation in the system with NAD+ inhibiting and NMN activating this enzyme and NMNAT, which controls conversion of NMN to NAD+ being localized to the outer Golgi membrane. Golgi and mitochondria are both moved along axons in processes that are totally dependent on cellular energetics. Given the broad contributions that are made by this work, I would not mind if the authors considered whether citrate, either from stressed mitochondria or from inhibition of the cytosolic enzyme ATP-citrate lyase, might be produced at high enough concentration to push SARM1 into the phase transition described herein.
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Reviewer #1 (Public Review):
SARM1 is an enzyme that is present in neurons and degrade NAD+. Previous studies have shown that disrupting SARM1 inhibits axon degeneration and thus it could be a target for treating neurodegenerative diseases. NAD+ is also an important metabolite that is required for many biological pathways. Thus, SARM1 activity must be carefully regulated. Recent studies have provided structural and biochemical insights about how SARM1 activity is auto-inhibited in basal states. The manuscript by Dr. Thompson and coworkers provide a nice new model regarding how SARM1 could be potentially activated. They provide strong in vitro data to support that phase transition, promoted by PEG molecules and citrate, could dramatically increase the activity of SARM1 TIR domain (which is the catalytic domain) in vitro. The authors also …
Reviewer #1 (Public Review):
SARM1 is an enzyme that is present in neurons and degrade NAD+. Previous studies have shown that disrupting SARM1 inhibits axon degeneration and thus it could be a target for treating neurodegenerative diseases. NAD+ is also an important metabolite that is required for many biological pathways. Thus, SARM1 activity must be carefully regulated. Recent studies have provided structural and biochemical insights about how SARM1 activity is auto-inhibited in basal states. The manuscript by Dr. Thompson and coworkers provide a nice new model regarding how SARM1 could be potentially activated. They provide strong in vitro data to support that phase transition, promoted by PEG molecules and citrate, could dramatically increase the activity of SARM1 TIR domain (which is the catalytic domain) in vitro. The authors also showed that in the worm, C. elegans, citrate promotes SARM1 puncta formation and axon degeneration, which is consistent with the in vitro data. They also generated multiple mutants of SARM1 TIR domain and showed many of the mutants have decreased phase transition and decreased activity in vitro. One of mutant, G601P, also showed decreased puncta formation when expressed in HEK 293T cells as SARM1 SAM-TIR domains E462A mutant (a catalytic mutant so that expression will not cause toxicity) fused with GFP.
The manuscript has many strengths, including the strong and very careful in vitro characterization of the purified SARM1 TIR domain, which provide a lot of useful information regarding the kinetic parameters, substrate specificity, and inhibition profiles. The worm data with citrate is consistent with the in vitro data, which is also a strength.
The impact of the finding lies in two aspects. First, it provides a new understanding about how SARM1 activity might be regulated in vivo by phase transition. This is especially true given most studies so far focuses on how it is inhibited at basal conditions. It also adds another example to the list of enzymes that are regulated by phase separation. Second, the finding that PEG and citrate strongly activate SARM1 in vitro also provides a much improved assay for the development of small molecule modulators of SARM1 for potential therapeutic applications.
There are two minor weaknesses associated with the studies of the manuscript. One is that all the in vitro studies used just the TIR domain of SARM1, not the full length SARM1. Another minor weakness is associated with the data in Figure 5. Most of the mutants have dramatically lower catalytic activities (>100-fold), but the precipitate formation is only modestly affected (2-fold). Although this does not affect the overall conclusion of the manuscript, it prevents the mutants from being more useful for mechanistic dissection.
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Evaluation Summary:
This manuscript describes an interesting regulatory mechanism that activates SARM1, an enzyme that degrade NAD+ and promote axon degeneration. Previous structural and biochemical studies mostly focus on how SARM1 is auto-inhibited at basal conditions and this manuscript provides evidences supporting that phase transition could promote its activity, thus providing new understanding about its regulatory mechanism. The finding also enables in vitro assays to be carried out more easily and thus could facilitate the development of small molecule modulators of SARM1 for therapeutics purposes.
(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 #2 agreed to share their …
Evaluation Summary:
This manuscript describes an interesting regulatory mechanism that activates SARM1, an enzyme that degrade NAD+ and promote axon degeneration. Previous structural and biochemical studies mostly focus on how SARM1 is auto-inhibited at basal conditions and this manuscript provides evidences supporting that phase transition could promote its activity, thus providing new understanding about its regulatory mechanism. The finding also enables in vitro assays to be carried out more easily and thus could facilitate the development of small molecule modulators of SARM1 for therapeutics purposes.
(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 #2 agreed to share their names with the authors.)
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