2014/06/16

Tuning intracellular homeostasis of human uroporphyrinogen III synthase by engineering a mutational hotspot found in congenital erythropoietic porphyria

Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. CEP presents with a plethora of different symptoms, with patients suffering from skin defects produced by the massive accumulation of porphyrins in the body. The most widespread mechanism for the disease is the enzyme destabilization after inheriting the mutation. For example, C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases.

In a collaborative effort within the Metabolism & Disease Program, the groups of Juan Manuel Falcón and Oscar Millet have studied the structural basis by which certain mutations located at this hotspot lead to UROIIIS destabilization. The work, published in Human Molecular Genetics, provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.

First, a thorough in vitro characterization unravelled a strong interdependency between the volume of the side chain at position 73 and the half-life time of the protein's folded conformation. Interestingly, when such experiment was expanded to the cellular environment, there is a good agreement between the in vitro half-life of the mutated protein and their expression levels in eukaryotic cell lines. This result suggests that C73 is an important position to preserve the molecular architecture and, remarkably, phylogenetic analysis shows that different species only use "stabilizing" residues for this position. Molecular modelling was used to rationalize the results, revealing a coupling between the mutation and the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability.
A putative restitution of the catalytic activity by means of rescue mutations was also investigated. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. The goal now is to produce a similar effect using chemical compounds acting as molecular chaperones.

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