4. Devolución creativa
4.2 Análisis del cumplimiento del Objetivo General
5.1.1
Ancient and recent adaptive evolution in the antiviral TRIM22
gene: identification of a single nucleotide polymorphism that
impacts TRIM22 function
The evolution of TRIM22 in mammals was examined using 29 evolutionarily diverse mammalian TRIM22 sequences. These sequences were aligned using COBALT and a phylogenetic tree was generated using EvolView software. Site-specific evolutionary analysis of TRIM22 with the Selecton program identified multiple amino acid sites in TRIM22 that have evolved under strong positive selection. The majority of these sites were located in the PRY/SPRY (B30.2) domain; however, there was also evidence for positive selection in other TRIM22 domains. Many positively selected sites clustered around putative functional motifs in TRIM22, such as the zinc-finger motif in the BB2 domain and the NLS in the SP2 domain. Moreover, a number of sites corresponded in location and spacing to sites that have evolved under positive selection in the closely- related TRIM5α protein. For example, in both TRIM22 and TRIM5α, many positively selected sites are located in one of four variable regions (v1-v4) in the B30.2 domain. In addition to sites undergoing positive selection among mammals, nsSNPs in human TRIM22 were investigated. A total of 64 nsSNPs and 2 indels were obtained from the NCBI dbSNP database for the human TRIM22 gene, including 56 missense mutation-
inducing nsSNPs and 8 frameshift mutation-inducing nsSNPs. To identify nsSNPs in TRIM22 that may be functionally relevant, an in silico prediction program (SIFT) was
used to analyze the 56 missense mutation-inducing nsSNPs. SIFT predicted that 23 of these nsSNPs were deleterious to TRIM22 function and 33 were tolerated. Notably, 2 potentially deleterious nsSNPs were located at sites that evolved under strong positive
selection in mammals. One of these nsSNPs, rs1063303:G>C, was selected for further analysis because of its high prevalence in the human population.
Large frequency differences were observed for nsSNP rs1063303:G>C among distinct ethnic populations, including AFR, AMR, ASN, and EUR 1000 Genomes cohorts. Of interest, an excess of intermediate frequency rs1063303 alleles was identified in AFR, AMR, and EUR populations, indicating a decrease in population size and/or balancing selection. To assess potential functional consequences of nsSNP rs1063303:G>C, its RNA and protein expression, sub-cellular localization pattern, and anti-HIV-1 activity were determined and compared to the wild type TRIM22 protein. Surprisingly, nsSNP rs1063303:G>C significantly increased both RNA and protein expression of TRIM22, but disrupted its anti-HIV-1 activity. nsSNP rs1063303:G>C also obstructed TRIM22 sub-cellular localization (localized diffusely in both the cytoplasm and nucleus, not in punctate NBs). Taken together, these results describe multiple sites that have evolved under positive selection in TRIM22 and identify a highly prevalent functional nsSNP (rs1063303:G>C) with a complex evolutionary history.
5.1.2
In silico analysis of functional single nucleotide
polymorphisms in the human TRIM22 gene
To identify additional nsSNPs that may alter TRIM22 function, and to examine amino acid sites in TRIM22 that may be subject to post-translational modification (PTM), an extensive in silico analysis was performed on the protein coding region of the TRIM22
gene. All missense mutation-inducing nsSNPs (56) in TRIM22 were analyzed using 6 different nsSNP prediction algorithms, including Polyphen-2, PhD-SNP, SIFT, nsSNP Analyzer, PMUT, and SNPs&GO. Since these algorithms use different parameters to evaluate and rank nsSNPs, multiple algorithms were used to increase the accuracy and power of prediction. A total of 14 nsSNPs were predicted to be deleterious to TRIM22 function by ≥ 4 nsSNP prediction algorithms. These 14 nsSNPs (L68R, H73R, E135K, I234K, S244L, G346S, K364N, P403T, L432W, R442C, F456I, T460I, C494F) were classified as high-risk deleterious and selected for further in silico analysis.
Highly conserved amino acids tend to be required for important protein functions. As such, nsSNPs that are located at highly conserved sites are often deleterious to protein function 1. ConSurf analysis revealed that 13 of the 14 sites occupied by the high-risk nsSNPs were highly conserved (conservation score of 7-9). In addition, by combining evolutionary conservation data and solvent accessibility predictions, the ConSurf web server predicted that T460 was a key structural residue and that L68, K364, and P403 were key functional residues. Structural analysis of the 9 high-risk deleterious nsSNPs located in the B30.2 domain, including K364N, P403T, and T460I, showed that all of these nsSNPs markedly altered the putative 3D structure of TRIM22’s B30.2 domain, particularly the surface-exposed v2 and v3 regions. These same regions are critical for HIV-1 restriction in the closely-related TRIM5α protein 2,3.
A number of putative PTM sites were also identified in the TRIM22 protein, including multiple ubiquitylation (3), sumoylation (1), and phosphorylation (21) sites that were predicted to undergo PTM by two or more in silico programs. Moreover, 7 SIMs were
identified in TRIM22, 2 of which are also present in TRIM5α (ILGV and VIGL) and were previously shown to be required for its antiviral activity 4,5. These 2 SIMs, plus 3 additional SIMs (5/7), are highly conserved among TRIM22 orthologues. Of interest, several PTM sites coincide with the location of nsSNPs, including 2 high-risk nsSNPs (S244L and T460I, which are both predicted to undergo phosphorylation). This study comprises the first systematic in silico analysis of functional sites in the TRIM22 gene
and will be a valuable resource for future studies.
5.1.3
The TRIM22 nsSNP rs1063303:G>C is not evolving under
balancing selection in the Inuit and is associated with low
serum TG and high serum HDL levels in the Canadian Inuit
To determine the frequency of TRIM22 nsSNP rs1063303:G>C in the Inuit and examine
the selective forces acting on this site, we genotyped TRIM22 rs1063303:G>C in two
different Inuit populations and one non-Inuit population (Canadian Inuit, Greenlandic Inuit, and Canadian population of European Caucasian descent). Interestingly, we found that the TRIM22 rs1063303:C allele is inordinately prevalent in both Inuit populations
and that unlike in the AFR, AMR, and EUR cohorts from the 1000 Genomes project, the Canadian and Greenlandic Inuit populations do not contain an excess of intermediate frequency TRIM22 rs1063303:G>C alleles. The latter indicates that in these two Inuit
populations TRIM22 nsSNP rs1063303:G>C is not evolving under balancing selection.
We also found an unexpected, but interesting association between the TRIM22 nsSNP
rs1063303:G>C and serum lipoprotein levels. Specifically, the TRIM22 rs1063303:C
allele was associated with significantly lower serum TG levels and significantly higher serum HDL levels in the Canadian Inuit population. The effect on TRIM22 on TG and HDL levels is unprecedented in the TRIM family and may represent an exiting new avenue of research for TRIM22 and other TRIM family members.