THE IMPACT OF NEW DISCOVERIES ON UREA CYCLE DISORDERS
Rubio V
Instituto de Biomedicina de Valencia (IBV-CSIC) and group 739 of CIBERER-ISCIII, Valencia,
Spain
Background.The mutational database for the classical urea cycle disorder carbamoyl
phosphate synthetase (CPSI) deficiency has recently been expanded very importantly. The
disease-causing nature of the many missense mutations reported in patients with CPSI
deficiency must be ascertained in most cases. Although a bacterial CPS, from Eschericha coli
(EcCPS),was produced recombinantly and used for site-directed mutagenesis studies,and had
its crystal structure determined, this enzyme differs importantly from CPSIin amino acid
sequence and in some keyfeatures, including the nitrogenous substrate used (glutamine,
whereas CPSI uses exclusively ammonia with high affinity) and in the lack of effect of the
essential activator of CPSI, N-acetyl-L-glutamate (NAG), rendering EcCPS a poor model for
human CPSI. Thus, a recombinant source of abundant genuine CPSIis needed for site-directed
mutagenesis and as the base for pursuing structural studies with CPSI. This need is even more
acute now that postranslational modifications of CPSI including lysine acylations have been
reported to occur and to influence strongly CPSI activity,having been linked to control by
deacylation by the anti-aging mitochondrial protein sirtuin 5.
An inborn defect in the bifunctional enzyme Δ1-pyrroline-5-carboxylate synthetase(P5CS) was
reported by others to cause clinical hyperammonemia. This enzyme catalyzes the initial two
steps of the de novo biosynthesis of ornithine, and, therefore, its deficiency could be linked to
a lack of ornithine supply to the urea cycle and therefore to urea cycle dysfunction under
conditions of limited arginine (as a precursor of ornithine) arginine supply. These observations
were based on the knowledge of only two families with mutations in this gene, one clearly
exhibiting clinical hyperammonemia and another one in which no patent hyperammonemia
was reported. Now a crop of new families with this deficiency has been reported.
Objectives. 1) To discuss new advances on thein vitro production of human CPSI, highlighting
the potential of these advances: a) for clarifying the disease-causing nature of CPS1 missense
mutations; b) for probing the effects of postranslational modifications of CPSI; c) for advancing
towards the determination of the structure of CPSI; and d) for investigating the mechanisms of
secondary hyperammonemias due to poor CPSI function.
2) To discuss recently reported cases carrying clinical mutations in the ALDH18A1gene, which
encodes P5CS, and, in particular, to discuss the evidence that mutations affecting this
bifunctional protein can result in either recessive or dominant inheritance of the associated
clinical syndrome.
Materials and methods.For CPSI, experimental study based on the utilization of baculovirus
expression of CPSI and illustration of ongoing work of the laboratory using this system. For
P5CS, analysis of the recently reported cases.
Results and Discussion. Human CPSI is produced now in the author's laboratory in a
baculovirus/insect cell expression system. Using the pure protein generated in this way it has
been possibleto clarify key features of this enzyme, including its domain composition, the
possibility to substitute partially the activator NAG by cryoprotectants such as glycerol, and the
protective effects of NAG and of its drug analogue N-carbamoyl-L-glutamate (NCG) against
CPSI degradation or inactivation. The latter findings raise the possibility of using NCG as a
chemical chaperone for therapy of CPSI deficiency.Such therapy is much needed particularly if
the results of a pilot study of our laboratory with a few CPS1 missense mutations reported in
patients with CPSI deficiency could be generalized to most CPSI mutations. This pilot study
revealed that the majority of the eight mutations tested decreased CPSI stability. Indeed, the
way is now open to analyze experimentally the impact of many clinical mutations onCPSI
activity and stability, andwe are presently carrying out collaborative work with clinical
laboratoriesfor the experimental study of clinical mutations affecting mutationally highly
eloquent regions of the CPSIprotein . The study of the effects of acylations affecting given
lysine residues can also be attempted using our expression system , although this must be
done indirectly, by using acylation-mimicking mutations of these lysines. The fact that we have
an abundant source of purified human CPSIhas allowed us to put to test whether metabolites
accumulating in organic acidemias or whether potential acylations of CPSI can collaborate to
cause the hyperammonemia that is observed in organic acidemiasand in fatty acid oxidation
defects. Furthermore, our laboratory has been successful in crystallizing human CPSIas a first
step towards determination of the CPSI structure and the structural understanding of the
effects of CPSI mutations and postranslationalmodifications, and to clarify why is CPSI inactive
in the absence of NAG and how can NAG or cryoprotectants activate the enzyme.
Concerning P5CS clinical mutations, all the reported cases will be reviewed,with the goal to
understand why some have a more pronounced "metabolic" presentation related to impaired
ornithine synthesis, whereas in other families connective tissue defects possibly related to
impaired proline synthesis appear to play the central role, without clear evidence for
hyperammonemia. We will also reflect on the possible mechanisms accounting for the recent
report of P5CS deficiency due to a dominant mutation, presenting novel alleys for approaching
experimentally the examination of this question.
Supported by grants Prometeo 2009/051 from the Valencian Government, BFU2011-30407
from the Science Department of the Spanish Government, and from the Fundación Alicia
Koplowitz.