Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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$Heart failure with preserved ejection fraction - European Heart Journal$

1126 Table 4 Characteristics of the three patients carrying dilated cardiomyopathy-associated TXNRD2 mutations Patient TXNRD2 mutation Family history LVEDD EF (%) Coronary ECG Outcome (gender) of DCM (mm) angiography ............................................................................................................................................................................... Patient 1 (male) 175G . A (Ala59Thr) Positive 64 18 Normal Left bundle branch block Died at the age of 68 Patient 2 (male) 175G . A (Ala59Thr) Negative 66 25 Normal AV block 1st degree Died at the age of 65 Patient 3 (male) 1124G . A (Gly375Arg) Negative 68 27 Normal Right bundle branch block Died at the age of 83 DCM, dilated cardiomyopathy; EF, ejection fraction; LVEDD, left ventricular end-diastolic diameter; ECG, electrocardiogram. answered, whether the mutations act in a dominant-negative fashion, thus impairing the function of the heterozygous wt allele. The protein structure of TXNRD2 and the FAD-binding domain To gain better insight into a possible adverse role of the mutations for TXNRD2 function, we followed two complementary approaches: structural modelling of TXNRD2 and integrated analysis of the evolutionary conservation of FAD-binding domains in thioredoxin reductases and GSH reductases (GR), which are evolutionary highly related enzymes. Although the crystal structure of murine Txnrd2 as well as rat and human Txnrd1 have been resolved, 24 – 26 the molecular nature of the FAD-binding domain has remained poorly defined in thioredoxin reductases. The FADbinding domain is essential for enzyme function since the reducing equivalents from NADPH are first transferred to FAD, from where they are passed on to the N-terminal redox-reactive centre within the same molecule and eventually to the Sec-containing C-terminal catalytic site of the second monomer. 27 Close inspection of the structure revealed that the human TXNRD2-binding pocket for FAD is formed by the N-terminal parts of four helices (h1: 49– 61, h2: 92–106, h4: 228–240, and h6: 368–382), the N-terminally adjacent amino acids, and eight non-contiguous, non-helical short stretches of amino acids. The mutations A59T and G375R are located in helices 1 and 6, respectively, that both contribute to the formation of the FAD-binding pocket (Figure 2A). As FAD binding is common to thioredoxin reductases and GR, we reasoned that amino acids conserved among both types of enzymes would participate in FAD binding. To address this, we made two sequence alignments, one comparing human thioredoxin reductase 2 with human GR (Figure 2B), and a second that included 36 thioredoxin reductases (including 13 thioredoxin reductase 2 genes) and 10 GR genes (see Supplementary material online, Figure S1 and Table S1). Amino acids shared among all or almost all enzymes across a large number of species were defined and marked in the alignment of TXNRD2 and GR. The overlay of the structural and evolutionary analysis clearly demonstrated that the regions participating in FAD binding are evolutionary highly conserved. We then compared the FAD-binding domain, which had been previously determined for GR, 28 with the structural information obtained from the analysis of the modelled TXNRD2 structure. Notably, the FAD-binding domain of GR as defined by Schulz et al. 28 is virtually identical to that of TXNRD2 as defined by inspection of the TXNRD2 structure (Figure 2B). The four helices as well as the non-contiguous, non-helical stretches of amino acids are highly conserved in evolution. In GR, a fifth helix participates in the formation of the FAD-binding pocket. This helix is somewhat disturbed in TXNRD2 (amino acids 208–212), but the corresponding conserved residues contribute to the binding pocket in a similar manner. Implications of the mutations for TXNRD2 protein structure D. Sibbing et al. G375R is located in the middle of helix 6. Not only is arginine much larger than glycine, it is also a polar amino acid. Our model indicates that the charged side chain points into the core of the enzyme (Figure 2A). This makes it likely that the bulky charged side chain disrupts the hydrophobic interaction with the neighbouring helix 1 and thus destroys the FAD-binding pocket. A59T (Figure 2A) is located at the end of helix 1 and is thus not directly involved in FAD binding. Our sequence alignments (see Supplementary material online, Figure S1) including 46 enzymes revealed only alanine and valine at this position. Two explanations may be given that are not mutually exclusive. First, an unpolar amino acid at this position may be required in this region. In our model, the side chain of threonine causes clashes with A55, located on helix1 or with V65 in the adjacent b-sheet (Figure 2A). Proper backfolding of this b-sheet towards FAD is required for hydrogen bonding of D69 to the ribose of FAD (as shown for E50 in GR 28 ) and for bringing helix 2 into the proper position relative to the other helices forming the FAD-binding pocket. Furthermore, the polar threonine may disturb the movement of the second redox centre, which is located on the flexible C-terminal part of the second subunit. In addition, we took a close look at the position of the five nonsynonymous variants that were identified in patients as well as in controls. Four of them (A66S, R286S, S299R, and G384S) are located more in peripheral regions of TXNRD2 facing the solvent and are neither involved in FAD/NADPH binding nor enzymatic function (Figure 2A). The fifth non-synonymous variant I370T is located at the beginning of helix 6, but it points away from FAD and is thus not involved in FAD binding. It points into a pocket formed by residues P365, L367, M390, Y392, and V395 of one subunit and V494’ of the other. All amino acids of this pocket are strictly conserved in mouse Txnrd2, which harbours threonine at this position. This shows that threonine is tolerated. Moreover, amino acid sequence alignment of orthologous TXNRD2 sequences revealed that 8 of Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
TXNRD2 and dilated cardiomyopathy 1127 Figure 2 The identified mutations localize to highly conserved amino acid residues in the FAD-binding domain of TXNRD2. (A) Overview of one TXNRD2 molecule (grey). The surfaces of FAD and NADP are shown in yellow and blue, respectively. Amino acid variants [red (observed in patients); blue and green (observed in patients and controls, not tolerated or tolerated according to SIFT analysis, respectively)] and relevant amino acids (grey) are shown as a ball and stick model. N- and C-termini are marked by N (grey) and C (orange). Molecular graphics images were produced using the UCSF Chimera package. (B) Alignment between human thioredoxin reductase 2 and human glutathione reductase. The alignment was performed using NCBI Blast. The numbering of amino acids is taken from the protein structures of mouse Txnrd2 (1ZDL) and human glutathione reductase (3DJG). Amino acids shown in bold are conserved to more than 80% in 46 FAD- and NADPH-binding oxidoreductases (36 thioredoxin reductases and 10 glutathione reductases) across a large number of species (see also Supplementary material online, Figure S1). Helices in TXNRD2 are underlined in green and helices in glutathione reductases in yellow. Amino acids in close contact with FAD as revealed by analysis of the modelled hTXNRD2 structure are shown in magenta. Corresponding amino acids forming the FAD-binding domain of human glutathione reductase 28 are underlined in cyan. The binding pocket for FAD is formed by four conserved helices, amino acids Nterminally adjacent to these helices, and eight non-helical, non-contiguous stretches of highly conserved amino acids. In glutathione reductases, a fifth helix participates in the formation of the FAD-binding pocket. This helix is somewhat disturbed in TXNRD2 (amino acids 208–212), but the corresponding conserved residues contribute to the binding pocket in a similar manner. The mutations G375R and A59T are shown as red letters and the non-synonymous variants observed in patients as well as in controls in blue letters. Note that mutations G375 and A59T are located in helices contributing to the formation of the FAD-binding pocket. Of the non-synonymous variants, only I370T is located in a helix contributing to FAD binding, but threonine at this position is structurally tolerated and represents the evolutionary more ancient allele (see also Supplementary material online, Figure S1, Figure S2, and Table S1). 11 species harbour threonine at this position, providing evidence that threonine is the evolutionary older variant. Taken together, G375 and A59 are highly conserved across a wide range of species, whereas the five other non-synonymous variants are conserved to a much lower degree in evolution and can be predicted not to interfere with FAD binding (for a summary, see Supplementary material online, Table S2). Both mutations abolish the function of Txnrd2 We reasoned that if the mutations were functionally silent and did not impact on Txnrd2 function, they would be able to rescue the phenotype of Txnrd2 2/2 cells in a manner similar to the wt Txnrd2 gene. To address this question experimentally, we cloned mouse wt Txnrd2 and the two mutants Txnrd2-A59T and Txnrd2-G375R into a bicistronic lentiviral vector and expressed them stably in primary Txnrd2 2/2 MEFs. Immunoblotting of cellular lysates with a Txnrd2-specific antibody showed that all three variants were expressed in knockout MEFs, albeit, to varying extent that was highly reproducible (Figure 3A). Double immunocytochemical staining of these cells with a FLAG-specific antibody for the reconstituted wt Txnrd2 and a peroxiredoxin III (Prx III)-specific antibody for mitochondria 29 revealed the expected mitochondrial localization of wt Txnrd2 in Txnrd2 2/2 MEFs (Figure 3B). Likewise, localization of the Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
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