Mitochondrial+Trifunctional+Protein+Deficiency

=**What is Mitochondrial Trifunctional Protein (MTP)**?=

1. The key role of MTP in mitochondrial β-oxidation
The mitochondrial β-oxidation of long-chained fatty acid is a complex catabolic pathway that provides the primary source of energy for skeletal muscle, the heart, and other high energy-requiring tissues. When a fatty acid reaches the mitochondrial matrix, it undergoes a sequential two-carbon cleavage that yields two products (Figure 1):


 * Fatty acyl-Coenzymes A (**acyl-CoA**), which are further reduced in length by two carbons;
 * Acetyl-coenzyme A molecules (**acetyl-CoA**), which feed directly into the Krebs cycle to generate energy.



Mitochondrial trifunctional protein (also called trifunctional protein) is a multienzyme complex found in the inner mitochondrial membrane that operates right after acyl-CoA dehydrogenase to carry out the last three enzymatic activities involved in β-oxidation. As illustrated in Figure 1, MTP transforms 2,3 Enoyl Co-A into both acetyl-CoA and acyl-CoA through the successive activity of the following enzymes (Ach et al, 2006):


 * 2,3-enoyl-CoA hydratase (**LCEH**), which adds a water molecule across the double bond;
 * 3-Hydroxyacyl-CoA dehydrogenase (**LCHAD**), which carries out an oxidation;
 * 3-ketoacyl-CoA thiolase (**LKAT**), which cleaves off acetyl-CoA units and thus shortens the fatty acyl-CoA.

Figure 1 also shows the importance of the electron transport chain. Indeed, LCHAD catalyzes a reaction that requires the electron carrier NAD+ to compensate for the two extra electrons produced after oxidizing 3 Hydroxyacyl-CoA. By contributing to the proton gradient in the mitochondrial inner membrane, this process indirectly promotes ATP generation.

2. The relationship between the role and structure of MTP


MTP is a hetero-octamer composed of four α-subunits and four β- subunits (Spiekerkoetter et al, 2003), which are 20 and 16 exons long respectively as shown in Figure 2 (Ibdah, 2006). Exons, or coding sequences, are nucleotide sequences in DNA that encode the final mRNA molecules and define a protein’s amino acid sequence.

The presence of both subunits is essential to the proper functioning of MTP since α-subunits harbor the LCEH and LCHAD domains, and β- subunits harbor the LKAT domain. Besides, their association directly affects the stabilization of the MTP enzyme.

3. Functional understanding of MTP deficiency
General MTP deficiency is one of the two human defects in the MTP complex. It is defined as the reduced activity of all three enzymes and the loss of both α- and β- subunits, which are encoded by separate genes located in the same region of the chromosome 2p23 (Spiekerkoetter et al, 2003). Figure 3 illustrates the level at which MTP deficiency occurs in the biochemical pathway.



=How do genetic mutations cause MTP deficiency?=

The discoveries related to this recessive inherited disease are relatively recent:


 * 1992: Description of the first patient with MTP deficiency evidence
 * 1995: Discovery of the first disorder-causing mutation in the α-subunit (Spiekerkoetter et al, 2003)
 * 1996: First demonstration of the disease-causing mutations in the β-subunit (Ushikubo et al, 1996)

Although both a mutant β-subunit and an abnormal α-subunit can originate MTP deficiency, more mutations have been identified on the α subunit than on the β subunit (Matern, 1999). Besides, according to Spiekerkoetter et al., mutations carried on either subunit are molecularly heterogeneous and can occur at various regions of the protein.

1. Mutations in the alpha subunit
Among the mutations in the α-subunit, the most common discovered are the two that affect the exon 3 donor splice site of the trifunctional protein (Figure 2). These two different mutations on two different alleles produce an incorrect mRNA splicing and a universal exon 3 skipping (71 bp). This event generates very premature stop codons in the misspliced, but stable, mature mRNA: none of the normal 727 amino acids of the subunit are generated due to the frameshift.

The absence of the α-subunit alters the interaction with the LKAT-containing β subunit and interferes with the stabilization of the mitochondrial trifunctional protein. Activities of all three enzymes are therefore lost.

Another mutation in exon 9 has also been reported by Ibdah (2006). This mutation affects both the heterooctamer complex formation and the subunit interactions since exon 9 is responsible for encoding a “linker domain” region (Figure 2) that plays an important role in such situations.

2. Mutations in the beta subunit
Although no identical mutations on the β subunit gene have been reported (Orii et al, 1997), three point mutations were identified as disorder causing mutations (Spiekerkoetter et al, 2003). Among the abnormalities are:


 * A-788-to-G substitution;
 * G-182-to-A substitution;
 * G-740-to-A substitutions.

All three missenses seem to account for destabilizing the trifunctional protein complex, resulting in the rapid degradation of both subunits (Ushikubo et al, 1996). Although missense mutations (typical for β subunit) generally present milder phenotypes than early termination or frameshift mutations (typical of α subunit), they alter MTP stability and structure enough to significantly reduce activities of both subunits: thiolase protein expression is extremely reduced (Spiekerkoetter et al, 2003).

Another relevant point is that Spiekerkoetter et al. (2003) noted that most of β-subunit mutated patients are compound heterozygotes, meaning that the individuals have two different, harmful, mutant alleles at the same loci.

3. Resulting phenotypes
According to Spiekerkoetter et al. (2003), patients with either α- or β-subunit mutations express heterogeneous but similar phenotypes. Among such are sudden unexplained infant death, Reye-like syndrome, cardiomyopathy, and skeletal myopathy (Brackett et al, 1995).

In β subunit mutations, the mild myopathic phenotype that results from MTP disease is the most widespread phenotype. In the heterozygous case, the β-oxidation is functional enough to ensure long-term survival. This observation logically correlates with the abundance of missense mutations. On the other hand, homozygous missense mutation often results in the lethal phenotype (Spiekerkoetter et al, 2003).

=References=

Ach T, Kolling G, Rohrschneider K, Richter C, Haas D, Schmidt-Bacher A. Ocular signs of a mitochondrial trifunctional protein defect. //Der Ophtalmologe: Zeitschrift der Deutschen Opthalmologischen Gesellschaft// 2012; **109**: 277-282

Brackett JC, Sims HF, Rinaldo P, Shapiro S, Powell CK, Bennett MJ, Strauss AW. Two alpha subunit donor splice site mutations cause human trifunctional protein deficiency. //The Journal of clinical investigation// 1995; **95**: 2076-2082

Ibdah JA. Acute fatty liver of pregnancy: An update on pathogenesis and clinical implications. //World Journal of Gastroenterology// 2006; **12**: 7397-7404

Matern D. Diagnosis of mitochondrial trifunctional protein deficiency in a blood spot from the newborn screening card by tandem mass spectrometry and DNA analysis. //Pediatric research// 1999; **46**: 45

Orii KE, Aoyama T, Wakui K, Fukushima Y, Miyajima H, Yamaguchi S, Orii T, Kondo N, Hashimoto T. Genomic and mutational analysis of the mitochondrial trifunctional protein beta-subunit (HADHB) gene in patients with trifunctional protein deficiency. //Human molecular genetics// 1997; **6**: 1215-1224

Spiekerkoetter U, Sun B, Khuchua Z, Bennett MJ, Strauss AW. Molecular and phenotypic heterogeneity in mitochondrial trifunctional protein deficiency due to beta-subunit mutations. //Human Mutations// 2003; **21**: 598-607

 Ushikubo S, Aoyama T, Kamijo T, Wanders RJ, Rinaldo P, Vockley J, Hashimoto T. Molecular characterization of mitochondrial trifunctional protein deficiency: formation of the enzyme complex is important for stabilization of both alpha- and beta-subunits. //American journal of human genetics// 1996; **58**:979-988.