Cottle T*, Joh L*, Posner C, DeCosta A, Kardon J†. An adaptor for feedback regulation of heme biosynthesis by the mitochondrial protease CLPXP. BioRxiv 602318 https://doi.org/10.1101/2024.07.05.602318                 *equal contribution  †corresponding author

CLPXP degrades ALAS in response to heme, a negative feedback mechanism to prevent heme oversupply and porphyrin precursor accumulation. Here, we biochemically reconstitute this feedback mechanism and discover that it requires an additional, heme-sensitive adaptor protein, POLDIP2.  We additionally determine that degradation requires the C-terminal element of ALAS and propose that this serves as the site for initiation of unfolding, providing a bivalent signal for degradation that is perturbed in several forms of erythropoietic protoporphyria.

*Kardon, J. R., Moroco, J. A., Engen, J. R., *Baker, T. A. (2018) Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding eLife 2020; 9:e54387 *co-corresponding author

In this manuscript, we used mutagenesis, biochemical reconstitution, and functional assays in vivo to describe a multivalent recognition and grip site in ALA synthase. Using hydrogen-deuterium exchange (in collaboration with John Engen's group, NEU) we observed that mitochondrial ClpX uses this site to initiate a specific partial unfolding of ALA synthase that exposes the active site, thus helping ALA synthase to bind its PLP cofactor and become active.

Kardon, J. R., Yien, Y. Y., Huston, N. C., Branco, D. S., Hildick-Smith, G. J., Rhee, K. Y., Paw, B. H., Baker, T. A. (2015) Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis. Cell 161(4):858-67

This paper describes our discovery of the mitochondrial unfoldase ClpX as an activator of the initial enzyme in heme biosynthesis, ALA synthase. Using genetics and metabolite profiling, we found that ClpX promotes the first step in heme biosynthesis, the synthesis of aminolevulinic acid (ALA). We additionally found that this function of ClpX is important for sustaining the high heme production necessary for red blood cell development (in collaboration with the Paw group, HMS).  Through biochemical reconstitution, we determined that ClpX directly activates ALA synthase by facilitating ALA synthase-cofactor complex formation.  

Yien, Y. Y., Ducamp, S., van der Vorm, L. N., *Kardon, J. R.,Manceau, H., Kannengiesser, C., Bergonia, H. A., Kafina, M. D., Karim, Z., Gouya, L., Baker, T. A., Puy, H., Phillips, J. D., Nicolas, G. & Paw, B. H. (2017) Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria. PNAS114:E8045–E8052 *co-second authors

This highly collaborative study combined human genetics, cell model systems for erythropoiesis, and biochemistry to describe mitochondrial ClpX as a new causative allele in erythropoietic protoporphyria. Surprisingly, a heterozygous loss-of-function allele of ClpX results in hyperstimulated heme biosythesis due to reduced degradation of ALA synthase. This result corroborates another recent report of ClpX-dependent degradation of ALA synthase that is triggered by heme binding, suggesting a possible feedback mechanism that switches ClpX from activation to degradation of ALA synthase when heme levels are too high.