HAX1 drives assembly and activation of the mitochondrial intermembrane space chaperone CLPB
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eLife Assessment
This in vitro study provides important evidence that the mitochondrial intermembrane space protein HAX1 functions as a direct activating cofactor of the AAA+ chaperone CLPB, resolving a longstanding question arising from the strong genetic, proteomic, and disease associations between these proteins. The convincing work advances our understanding of mitochondrial proteostasis by demonstrating that HAX1 promotes CLPB oligomerization and will potentially even enable the generation of small molecules that mimic the function of HAX1 in CLPB activation. The study will be of general interest to biochemists working on chaperones and/or mitochondrial biology.
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Abstract
The multicellular metazoan lineage acquired a novel chaperone in the mitochondrial intermembrane space, the AAA+ disaggregase and refoldase CLPB. Although it is not known how they function together, CLPB and the intrinsically disordered IMS protein HAX1 interact and share disease and cellular phenotypes; loss of function in either gene causes severe congenital neutropenia as well as neuropathology and causes many proteins in the IMS and its bounding membranes to become insoluble. Through biochemical reconstitution, we here find that HAX1 is a direct stimulatory cofactor of CLPB. HAX1 promotes oligomerization of CLPB into an active disaggregase and stimulates the ATPase and refoldase activities of the oligomeric complex. A short peptide within HAX1 is necessary for direct interaction with the ankyrin domain of CLPB, but stimulation of CLPB activity requires additional elements of HAX1. Characterization of CLPB and CLPB-HAX1 oligomers indicates that HAX1 shifts the predominant oligomeric state of CLPB from a dodecamer to an apparent hexamer elaborated with HAX1, suggesting that this smaller oligomer is important during the cycle of CLPB function with clients.
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eLife Assessment
This in vitro study provides important evidence that the mitochondrial intermembrane space protein HAX1 functions as a direct activating cofactor of the AAA+ chaperone CLPB, resolving a longstanding question arising from the strong genetic, proteomic, and disease associations between these proteins. The convincing work advances our understanding of mitochondrial proteostasis by demonstrating that HAX1 promotes CLPB oligomerization and will potentially even enable the generation of small molecules that mimic the function of HAX1 in CLPB activation. The study will be of general interest to biochemists working on chaperones and/or mitochondrial biology.
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Reviewer #1 (Public review):
The mitochondrial intermembrane space (IMS) is a compartment under constant proteostasis stress. During the development of multicellularity, the IMS acquired the AAA+ ATPase CLPB, a disaggregase whose absence from cells results in aggregate formation in the IMS and whose mutation in humans leads to rare but severe human diseases.
The precise molecular function of CLPB in the human IMS remains unresolved and is addressed in this study, in particular the crosstalk with the IMS protein HAX1, which is a prominent interaction partner of CLPB.
The authors find HAX1 to serve as an activating cofactor of CLPB as a disaggregase and refoldase in a purified system. This is well in line with the similar phenotypes of HAX1 and CLPB loss, and is an exciting finding as it not only assigns a direct role for HAX1 in the IMS …
Reviewer #1 (Public review):
The mitochondrial intermembrane space (IMS) is a compartment under constant proteostasis stress. During the development of multicellularity, the IMS acquired the AAA+ ATPase CLPB, a disaggregase whose absence from cells results in aggregate formation in the IMS and whose mutation in humans leads to rare but severe human diseases.
The precise molecular function of CLPB in the human IMS remains unresolved and is addressed in this study, in particular the crosstalk with the IMS protein HAX1, which is a prominent interaction partner of CLPB.
The authors find HAX1 to serve as an activating cofactor of CLPB as a disaggregase and refoldase in a purified system. This is well in line with the similar phenotypes of HAX1 and CLPB loss, and is an exciting finding as it not only assigns a direct role for HAX1 in the IMS but also yields the potential for CLPB activity regulation by regulating amounts of HAX1. The experimental support for this finding is strong and acquired by a combination of different and carefully executed in vitro enzyme activity assays.
One potential weakness of this study is that it does not consider recently identified players in the CLPB-HAX1 axis, FAM136A (now called TIMCC) and MIA40, that might additionally modulate/regulate CLPB activity. This might constitute an exciting route for future research.
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Reviewer #2 (Public review):
The authors sought to determine the mechanistic relationship between the mitochondrial intermembrane space proteins CLPB and HAX1, whose genetic, proteomic, and disease associations have long suggested a functional partnership. Using biochemical reconstitution with purified proteins, they provide evidence that HAX1, an intrinsically disordered protein, acts as a direct activating cofactor of CLPB, stimulating its oligomerisation, ATPase activity, disaggregase activity, and refoldase activity. They further identify a region of HAX1 required for CLPB interaction and propose a model in which HAX1 promotes formation of a distinct active CLPB assembly.
A major strength of the work is the comprehensive biochemical approach. The authors combine activity assays, mutational analysis, interaction studies, and …
Reviewer #2 (Public review):
The authors sought to determine the mechanistic relationship between the mitochondrial intermembrane space proteins CLPB and HAX1, whose genetic, proteomic, and disease associations have long suggested a functional partnership. Using biochemical reconstitution with purified proteins, they provide evidence that HAX1, an intrinsically disordered protein, acts as a direct activating cofactor of CLPB, stimulating its oligomerisation, ATPase activity, disaggregase activity, and refoldase activity. They further identify a region of HAX1 required for CLPB interaction and propose a model in which HAX1 promotes formation of a distinct active CLPB assembly.
A major strength of the work is the comprehensive biochemical approach. The authors combine activity assays, mutational analysis, interaction studies, and oligomerisation measurements to address the central question from multiple angles. The data provide convincing evidence that HAX1 is not simply a client of CLPB but instead functions as a positive regulator.
The principal weakness concerns the mechanistic model of oligomer remodelling. While the SEC and stoichiometric analyses are consistent with the formation of a smaller HAX1-bound CLPB complex, the proposed transition from a dodecameric to a hexameric assembly is inferred rather than directly demonstrated. Additional structural or biophysical evidence would strengthen this aspect of the study.
Overall, the authors largely achieve their aims. The evidence strongly supports the conclusion that HAX1 is a direct stimulatory cofactor of CLPB and provides an important mechanistic framework linking two proteins associated with overlapping mitochondrial and hematological disease phenotypes. Some aspects of the proposed oligomeric remodeling mechanism should be interpreted more cautiously, as they remain supported primarily by indirect evidence.
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