Extracción del contraste de Michelson

The results from the protein modeling analyses were examined in the biological context to confirm the biological reliability of these in silico results. The amino acid change from threonine to proline, caused by the variant at 7718bp within COX II, is likely to be biologically reliable in terms of its prediction on protein dysfunction (Figure 3.9). Threonine is a hydrophilic molecule due to possession of a hydroxyl group, and structurally can reside on either the interior or surface sides of a protein. Any substitution involving an amino acid change into proline, will impact on protein structure. Since the side chain in proline connects twice to the protein backbone, introduction of this imino acid induces rigidity to the local protein region. As a result, proline preferably resides on the protein surface.

While variants present in loop regions are not likely to impact significantly on the overall protein conformation, flexible loop regions remain important for protein-ligand and protein-protein interactions essential for mediating biochemical signal transduction230. For the substitution involving proline to leucine (P9L) at 14772bp within CYT B (Figure 3.10), it is possible that further flexibility of the local region will be induced, as proline is lost in this case. In addition, the L150W protein mutation reported in COX I, assigned a RI value of 2, is likely to be biologically reliable as the variant residing in the loop region is unlikely to initiate disease (Figure 3.12).

The RI value of 8, given to the second COX I mutation H291D, appears to be acceptable as the location of the variant appears buried within the interior of COX I (Figure 3.12). Furthermore, the H291D mutation is biochemically

unfavourable. Although histidine and aspartate are both polar molecules, the latter is also negatively charged. While histidine can reside within the interior or the surface of a protein, aspartate displays preference to reside on the protein surface. If however, aspartate is present on the interior side, it often participates in the formation of salt-bridges with other positively charged residues to create hydrogen bonds that assist in maintaining protein stability231.

Histidine at position 291 is also a copper B metal binding site (Uniprot P00395). Histidine is commonly found in enzyme reactive centers, as the imidazole ring present in histidine allows it to accept or donate protons near to physiological pH, making these amino acids ideal for enzyme catalysis reactions232. Substitution of histidine is likely to interfere with the iron present in heme a3, which functions to form a heterobimetallic site with the CuB centre233, since histidine 291 represents one of three histidine residues participating in the stabilization of the CuB centre234. Altogether, these observations suggest that loss of the conserved histidine at position 291 can disrupt functionality of the catalytic cycle. This will interfere with the transfer of electrons from the hydrophobic carrier ferrocytochrome C to the heme a3/CuB centre.

The amino acid substitution V142G in COX II represents another mutation that is likely to biologically induce disease (Figure 3.13). Valine is limited in the conformations it can form, since there is the presence of two non-hydrogen side chains. While valine is unreactive, its hydrophobic property allows it to participate in interactions with other hydrophobic molecules, for example, lipids231_{. Glycine on the other hand, carries only hydrogen on its side chain. Its}

small size means that presence of glycine will confer flexibility of the protein structure where it resides. The loss of rigidity in protein structure from a substitution involving valine to glycine, could interfere with stability of the beta barrel structure of COX II, a consequence which was reflected in the in silico

predictions made in Table 3.13 (P = 0.002).

With reference to the P145S mutation also in the COX II protein (Figure 3.13), the substitution event may be unfavorable due to serine primarily being found in functional centers, as its hydroxyl group is capable of hydrogen bonding with a range of polar molecules231_{. Both V142G and P145S were found to be present in} both the CD45+ and CD45– cells. The combination of both is likely to severely reduce stability of the beta barrel structure. The final COX II mutation, P223T, was predicted to induce a neutral effect (Table 3.13), most likely due to its location being relatively distant from the main catalytic core center (Figure 3.13).

It is probable that the two mutations residing adjacent to each other on the helical segment of CYT B (Figure 3.11) exert a greater effect on protein function than the presence of a single mutation. The biochemical effect of a transition from proline to leucine has been discussed previously. On the other hand, a change from threonine to serine at position 174 on the protein sequence is considered a favourable substitution. Both residues are polar in nature, with the chemical difference between them being a hydrogen group in serine, whereby a methyl group is found at the same location in threonine. However, together these mutations may disrupt overall structure to CYT B. Given that variants 15264

and 15267bp appear common across the myeloma tumors screened, their joint presence is likely detrimental to protein stability.

Not surprisingly, all non-synonymous ATP6 mutations (F78C, P134H, I138N and S99P) were predicted to impair protein function. Due to its essential role in the production of ATP, loss of ATP6 function is likely to disrupt respiratory capacity of a cell, thereby promoting aerobic glycolysis. According to the Warburg theory, under these conditions, proliferation of the tumor cells will increase, therefore enhancing malignancy of the tumor.

A study performed in the Chlamydomonas reinhardtii model have presented results to support the theory that suppression of ND4L polypeptide production impairs assembly of the entire Complex I protein structure235. Moreover, ND4 participates in ubiquinone-binding236 as well as in the translocation of protons across the mitochondrial inner membrane237_{. Therefore, incomplete translation of} both these gene regions, caused by the introduction of pre-mature stop codons in the mtDNA sequence, are likely to impose adverse implications.