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CONCLUSIONS


To sum up, we want to emphasize that the aim of this project was to identify the selenoproteome and selenoprotein machinery of a recently sequenced specie: Craseonycteris thonglongyai. Forty-one proteins from Homo sapiens were used to align with the Craseonycteris thonglongyai’s genome in order determine if these proteins were found in the tested specie. The human proteome was chosen because of its phylogenetic proximity with Craseonycteris thonglongyai as they are both placental mammalians. Moreover, it has the most accurately sequenced genome among mammals, together with Mus Musculus.

In order to decide whether the studied proteins were found in Craseonycteris thonglongyai, the deduced proteins had to meet with some requirements. First of all, the predicted protein had to be well aligned with the reference protein, meaning that the coverage had to be 60% or greater. Then, to consider that the predicted protein was a selenoprotein, one of the exons of the gene had to present a UGA codon that corresponded to a selenocysteine. Moreover, a SECIS element should be predicted in the 3’ UTR. And finally, the SECIS element should be located in the same chain where we found the Sec.

We focus on the discussion of the conservation and evolution of the proteins that contain a Sec amino acid in Craseonycteris thonglongyai, as well as the predicted cysteine containing homologs. Finally, we also describe the predicted proteome related to the selenoprotein machinery and the proteins that have not been predicted or that have no enough evidence to be determined as selenoproteins.

We show below a classification of the obtained results:



As it can be seen, 22 selenoproteins are preserved in Craseonycteris thonglongyai. 7 proteins have been described as part of the selenoproteins machinery. And 8 proteins have been predicted as cysteine containing homologs. Finally, the results obtained from TR1, SelR3, MsrA and DI3 were not conclusive to determine whether these proteins are conserved in Craseonycteris thonglongyai genome.

About the UGA-to-SECIS distances in the predicted proteins, although their relationship appears to be complex (Turanov A et al, 2013), the observed results (an average distance of 1192 nucleotides) are coherent with the determined distances in previous studies (Mariotti M et al, 2008), where the average distance for all mammalian selenoproteins was determined to be 872 nucleotides.

Thioredoxin reductases (TRs)

In mammals, three TR isozymes are found that have the Sec residue in its penultimate position of the C-terminus (Labunskyy V et al, 2014). In our organism, TR2 and TR3 have been identified and described as selenoproteins. However, in TR1 no Sec residue was found. Since it has been reported that these proteins are highly important and conserved in mammalian organisms (Labunskyy V et al, 2014), and in our predicted TR1 protein the C-terminus was lacking, we hypothesized that this part of the gene could be in another scaffold that we could not detect. This makes sense because we have found several proteins that were found truncated in different scaffolds due to the short length of the scaffolds in Craseonycteris thonglongyai. Therefore, we believe that a more exhaustive investigation should be performed in order to decide whether this protein is find or not in our organism.

SelWTH (SelW, SelT, SelH and SelV)

We conclude that SelW1, SelT, SelH and SelV are conserved selenoproteins in Craseonycteris thonglongyai. Regarding to SelW2, we understand from our results that the selenocysteine has been lost in both of the species and it is SelW1 that carries the essential functions of this member of the SelWTHV family. This is congruent with the findings made by Mariotti M et al (2008) in which they argue that SelW2 was lost in the division between bony fishes and mammals.

Sep15 and SelM

From our results we have found that Sep15 and SelM are conserved selenoproteins in both human and Craseonycteris thonglongyai.

SelS and SelK

SelS and SelK are conserved selenoproteins in both human and Craseonycteris thonglongyai.

SelU

This family of proteins has undergone a selenocysteine to cysteine transformation in superior mammal species. This occurrence has been found in our organism since any selenocysteine has been found in any of the three members of the family. Moreover, we have seen that in Craseonycteris thonglongyai, SelU1 still preserves a SECIS element suggesting that this protein was a selenoprotein in the past.

SelR

This family of proteins consist of three members: SelR1, SelR2 and SelR3. Regarding SelR1, we found it to have a conserved structure. On the other hand, for SelR2 and SelR3, we could not find any selenocysteine residue in SelR2, and the one found in SelR3 was discarded. However, the structures of both proteins were conserved.

Therefore, for SelR2, we concluded that it was conserved as a cysteine containing homolog in Craseonycteris thonglongyai. However, for SelR3, more studies should be performed since a selenocysteine residue was found only when one of the two scaffolds considered was analyzed.

Our findings seem to be coherent since there is not much literature involving the function of this protein family, but it seems that they might play a role in methionine sulfoxide reduction like MsrA. Moreover, they seem to be present in all organisms except parasites and hyperthermophiles (Kryukov G et al, 2002).

Glutathione peroxidases (GPx)

It is known that mammals have eight GPx homologs and only five of them are selenoproteins (GPx 1-4 and GPx6). (Mariotti M et al, 2008). Moreover, phylogeny studies have defined three evolutionary groups (Mariotti M et al, 2008; Margis R et al, 2008; Toppo S et al, 2008): GPx1 and GPx2, originated by a duplication before the divergence of birds and mammals, but after the origin of amphibians and fishes; GPx3, GPx5 and GPx6, found only in mammals and being GPx5 and GPx6 closer than GPx3; GPx4, GPx7 and GPx8. In the study of Matiotti M et al separate this group in two different groups.

In our study, we have predicted all the selenoproteins from the GPx family that are described to conserve the Sec amino acid in mammals (GPx1, GPx2, GPx3 and GPx6). Moreover, as Matiotti M et al observed, all of them had great nucleotide sequence identity. At the same time, the proteins that have converted the Sec to Cys (GPx5, GPx7 and GPx8) have also been predicted as no Sec-containing in Craseonycteris thonglongyai.

Finally, we have also generated a phylogenetic tree using our predicted proteins (figure shown below) and we have seen that the evolutionary relationship between them is similar to the one found in the study of Mariotti M et al (2008). We clearly see the three groups described before: GPx1 and GPx2 are closely related, then we see GPx3 with GPx6 and GPx5, being the two last ones more related between each other than with GPx3 and finally GPx4 with GPx7 and GPx8, again being the to last ones evolutionary closer to each other than with GPx4.



Iodothyronine deiodinases (DI)

The iodothyronine deiodinases family consists of three paralogous proteins in mammals (Labunskyy V et al, 2014). In our study, all of them were deduced in Craseonycteris thonglongyai. DI1 and DI2 have been well identified in the studied organism. However, in DI3 we did not find the SECIS element. We hypothesized that it could be due to the length of the scaffold where the gene was deduced. We saw that the last exon was predicted almost at the end of the scaffold. Therefore, we thought that maybe the SECIS element could not be predicted because it was found in another scaffold. This would be coherent with the bibliography related to selenoproteins since this family is known to be well conserved in mammals (Mariotti M et al, 2008) and we found that DI3 deduced in Craseonycteris thonglongyai genome conserved the selenocysteine in the same place as humans.

SELENOPROTEIN MACHINERY

Regarding to the selenoprotein machinery, we conclude that all of the proteins described in mammals are found in Craseonycteris thonglongyai. Generally, these proteins do not have Sec or SECIS elements. However, we see in SPS family, which has been defined as selenium donor proteins for selenocysteine biosynthesis, that, as it was expected, SPS2 contains Sec and a SECIS element. Therefore it is well conservated in Craseonycteris thonglongyai. However, as in Homo sapiens, SPS1 does not have Sec or SECIS element. These results are are congruent with the fact that SPS1 does not have an essential role in the metabolism of selenoproteins, as well as with the findings of Labunskyy V et al, 2014: “further analysis revealed that SPS2 could generate selenophosphate in vitro, whereas SPS1 could not [...] SPS2 is required for de novo synthesis of selenophosphate, while SPS1 may have a possible role in Sec recycling through a Se salvage system”.