Alternative Splice Variants in TIM Barrel Proteins from Human Genome Correlate with the Structural and Evolutionary Modularity of this Versatile Protein Fold

<div><p>After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα)₈ barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα)₂, (βα)₄, (βα)₅, (βα)₆, and (βα)₇ subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to diversify the proteins encoded in the human genome.</p></div

Biochemical characterization of a chimeric variant.

<p>The overall standard errors of enzyme kinetic parameters are less than 20%.</p>a<p>The apparent thermal melting temperature (°C).</p>b<p>Data obtained from Ochoa-Leyva et al., 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070582#pone.0070582-OchoaLeyva1" target="_blank">[25]</a>.</p

Pipeline of bioinformatics analysis to identify the (βα)₈ barrel proteins with splice variants in the human genome.

<p>Summary of our bioinformatics data flow to extract the 135 experimentally confirmed (βα)₈ barrel splice variants in the human genome.</p

(βα)₈ subdomains and barrel modifications reported by protein engineering experiments.

<p>NA: Not assigned because the corresponding structure forms the overall barrel domain.</p

Functional annotations of human splice variants.

a<p>Reported by <i>in vitro</i> translation.</p><p>ND: Not determined.</p

Sequence analysis under selective pressure at variable positions in chimera enzymes.

<p>Amino acids are represented by the one code letter. The histograms represent the relative frequencies of the selected versus unselected libraries. The red colored histograms (*) represent those amino acids at the variable positions that had a frequency either significantly higher (2 standard deviations) or lower than expected by chance. The sequence analysis for the α-helix 3 and β-strand 7 from MetR swapped into the TrpF scaffold are shown in A and B, respectively. The variable positions are represented by the NNS codon. The three-dimensional structure of the <i>E. coli</i> enzyme (PDB: 1PII) was used to identify the variable positions. The amino acid numbering of TrpF is according to gene reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070582#pone.0070582-OchoaLeyva1" target="_blank">[25]</a>.</p

Quantitative multiplexed proteomics of <i>Taenia solium</i> cysts obtained from the skeletal muscle and central nervous system of pigs

<div><p>In human and porcine cysticercosis caused by the tapeworm <i>Taenia solium</i>, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst’s proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, <i>i</i>.<i>e</i>., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the ‘optimal’ tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.</p></div

Peptide mixtures as potential diagnostic agents for <i>Taenia solium</i> cysticercosis.

<p>Several peptide mixtures were used: A) The best four individual peptides (using 500 ng or 1 μg to coat each microtiter plate well). The rest of the peptides combinations were used at 500 ng. B) Mixture of all 14 peptides synthesized in this study, C) Mixture of the peptides derived of skeletal muscle (SM) abundant proteins, D) Mixture of one peptide of a central nervous system (CNS) abundant protein and one of a constitutive protein and E) Mixture of peptides from SM and CNS cysts. The normalized optical density was calculated by dividing each individual O.D. by the cut-off value (mean value of non cysticercotic pigs plus two standard deviations). P-values are shown at the top of each figure. F) Heat map showing the individual response to antigenic peptide mixtures. The normalized optical density was transformed using Log₂. White represents values near to the cut-off point, red represents values over the cut-off point (positive samples) and blue represents values below the cut-off point (negative samples).</p

Protein abundance of several well-studied antigens in <i>T</i>. <i>solium</i> cysts antigens.

<p>A) Relative abundance of cyst’s antigens obtained from the skeletal muscle (SM) and central nervous system (CNS); B) Relative abundance of tetraspanin proteins quantified in our proteomic analysis for SM and CNS cysts; C) Alignment of SM- and CNS-abundant tetraspanins. The extracellular loops are bold in red and blue, selected peptides are marked by a square. Listed at the bottom are the selected peptide sequences and length. D) Recognition of the synthetic peptides by non cysticercotic and cysticercotic pig sera. The normalized optical density (O.D.) is the result of dividing each individual O.D. by the cut-off value (mean value of non cysticercotic pigs plus two standard deviations). P-values are shown at the top of each figure.</p

<i>Taenia solium</i> peptide recognition by cysticercotic and non cysticercotic pig sera through ELISA.

<p>A) Peptides from central nervous system, B) skeletal muscle abundant proteins, C) constitutive proteins and D) cysts protein extracts. Microtiter plates were coated with the peptides or extracts and incubated with sera from non cysticercotic (n = 15) and cysticercotic (n = 15) pigs, all bred in rural endemic areas. The normalized optical density (O.D.) is the result of dividing each individual O.D. by the cut-off value (mean value of non cysticercotic pigs plus two standard deviations). P-values are shown at the top of each figure.</p