Velocardiofacial Syndrome: Enzyme Stabilization Therapy?
Nearly half the US population carries a polymorphism of the gene for catechol-O-methyltransferase (COMT) that results in substitution of methionine for valine at position 108 of the soluble form of the protein (s-COMT) and position 158 of the membrane-bound form (mb-COMT). The Met variant of s-COMT loses activity rapidly at 37o, while the Val variant is stable. Individuals who are homozygous for the 108/158Met allele are at increased risk for several neuropsychiatric conditions. The instability of the Met variant appears to be a particularly serious problem in patients with velocardiofacial syndrome, who are missing the COMT gene entirely on one copy of chromosome 22. We propose to investigate why the Met variant is unstable, and to explore ways of stabilizing it. To determine whether the thermal inactivation reflects global unfolding or more subtle conformational changes, we will correlate the enzyme activity of 108Met and 108Val s-COMT with a panel of spectroscopic properties that report on protein conformation as functions of temperature, time, pH, and the substrate S-adenosylmethionine. Regions of the protein that unfold first or that dominate the rate-determining step of unfolding are potential targets for enzyme-stabilizing small molecules. We will identify these regions by using mass spectroscopy to measure the rates of deuterium exchange of amide hydrogens, by molecular-dynamics simulations of the thermal unfolding pathway, and by exploiting fluorescence correlation spectroscopy to examine the dynamics of structural fluctuations near the active site. Similar studies will be conducted on mb-COMT, which probably is the physiologically important form of COMT in the brain. We will select the most promising potential drug-binding sites based on the spectroscopic studies, deuterium exchange maps, and molecular-dynamics simulations, and we will then carry out iterative cycles of computational docking protocols and experimental assays to discover and refine molecules that stabilize 108/158Met COMT. Although beyond the scope of this proposal, lead compounds obtained from these searches can be tested in an existing mouse model of COMT deficiency.

Velocardiofacial Syndrome - Biological Significance
Velocardiofacial syndrome (VCFS) is now recognized to be the most common contiguous-gene deletion syndrome in humans, having an estimated prevalence of 1:4000 births (A contiguous-gene deletion syndrome refers to the loss of several contiguous genes in a particular chromosome). The majority of individuals with VCFS are missing a common stretch of 3 Mb on the short arm of chromosome 22. In most cases the disorder is not inherited, but rather appears to arise de novo from an error in homologous recombination. The error probably is mediated by duplicated sequences ("low-copy repeats") that flank the deleted region..
VCFS has a highly variable clinical phenotype, including more than 180 physical finding. DiGeorge originally described a condition of immunological insufficiency associated with developmental problems of the heart, vascular system and thymus. Shprintzen et al. independently described a syndrome of anomalies of the soft palate (velum) and face in addition to the heart. Anatomic anomalies of the brain have been found, and mild neurological impairment of cognition, attention, concentration and social-emotional function is common. Many individuals with VCFS develop a characteristic spectrum of severe neuropsychiatric manifestations that include pronounced separation anxiety, night terrors, and wide mood swings in early childhood, obsessive-compulsive disorder in later childhood, and a combination of an "ultra-ultra-rapid-cycling" bipolar disorder with characteristic "affective storms," anxiety, perseverative thoughts and hallucinations in adolescence. Some investigators consider the most common psychiatric symptoms to be consistent with schizophrenia or schizoaffective disorder, while others have emphasized features characteristic of bipolar and attention-deficit disorders. The neuropsychiatric manifestations of velocardiofacial syndrome are remarkably resistant to most neuropharmacological treatments, and have a devastating impact on the quality of life for many patients.
The commonly deleted region of chromosome 22q11 spans at least 30 genes, one of which is the gene for catechol-O-methyl transferase (COMT). COMT inactivates the neurotransmitter dopamine by transferring a methyl group from S-adenosyl-methionine (SAM) to a phenolic group of the catechol ring:

It acts similarly on epinephrine and norepinephrine. COMT also participates in the catabolism of catecholestrogens and of catechol drugs used for treatment of Parkinson disease, hypertension and asthma. In the brain, a deficiency of COMT could result in higher concentrations of dopamine, which could plausibly contribute to the neuropsychiatric manifestations of velocardiofacial syndrome. Other genes in the deleted region have been implicated in developmental aspects of VCFS and could contribute to the neuropsychiatric symptoms, but none of the other genes that have been identified has as clear a connection to neurochemical function.
Human COMT occurs as both a soluble protein with 216 residues (s-COMT, 24.4 kDa) and a membrane-bound protein with an N-terminal extension of 50 residues (mb-COMT, 30.0 kDa). The two proteins are coded by a single gene that has two start sites for transcription, but little is known about the factors that determine the relative amounts of the two forms in a given tissue. The soluble form of the protein predominates in most tissues except for brain and adrenal medulla, where mb-COMT is the major species. In the brain, the enzyme is found mainly in postsynaptic neurons and in glial cells near synapses and capillary walls. The membrane-bound form seems likely to play the main role in dopamine catabolism, because its Km for dopamine is about ten-time smaller than the Km of the soluble form.
The crystal structure of rat s-COMT has been determined at a resolution of 2 Å. Human s-COMT has not been crystallized, but probably has a 3-dimensional structure similar to that of the rat enzyme because the amino acid sequences are about 80% identical. No structure is available for mb-COMT; however, a hydropathy plot indicates that its additional N-terminal domain probably has a single transmembrane -helix.
A common polymorphism in the COMT gene results in substitution of methionine for valine at amino acid 108 of the soluble protein and position 158 of the membrane-bound enzyme. Approximately 25% of the US population is homozygous for the 108/158Met allele. The Met variant has markedly lower thermostability at physiological temperatures, and individuals who are homozygous for this allele have a 3- to 4-fold lower level of enzyme activity in their erythrocytes. Purified 108Met human s-COMT was found to lose about 80% of its activity in 30 min at 37o, while the activity of the 108Val enzyme was essentially unchanged. In patients with velocardiofacial syndrome, having the 108/158Met allele on the non-deleted chromosome could compound the effects of loss of the gene from the other copy of chromosome 22. In line with this reasoning, hemizygosity for the 108/158Met variant is strongly associated with some of the most severe neuropsychiatric manifestations of VCFS. In the general population, homozygosity for 108/158Met is associated with increased risk for obsessive-compulsive disorder in males, "ultra-rapid cycling" bipolar disorder, late-onset alcoholism and depressive disorder, and with aggressive, homicidal and suicidal behavior in schizophrenics. Although the 108/158Met variant of COMT is clearly not the cause of schizophrenia or bipolar disorder it evidently can intensify these disorders. In a mouse model, disruption of the COMT gene leads to increased aggression and behavioral changes indicative of increased anxiety. The less stable form of COMT also has been associated with increased risk for breast cancer in humans, although other investigators have found no such association.

Above are cartoon representations of the crystal structure of rat s-COMT from two viewpoints. The side chain of residue 108 (leucine in the rat) is shown in red. SAM, dinitrocatechol (DNC, a tightly binding inhibitor) and Mg2+ are in orange, yellow and black, respectively. Shades of blue and green indicate secondary structural elements ( -helices, -sheets and loops). The coordinates are from Brookhaven file 1VID.PDB.