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Biology subjects

Hitomi, K.

Publications and source records attributed to Hitomi, K..

3 recordsLinked to original sources

Diversity for commonality in the evolutionary changes of the reduced genome to regain the growth fitness

As the genome encodes the information crucial for cell growth, a sizeable genomic deficiency often causes a significant decrease in growth fitness. Whether and how the decreased growth fitness caused by genome reduction could be compensated by evolution was investigated here. Experimental evolution with an Escherichia coli strain carrying a reduced genome was conducted in multiple lineages for approximately 1,000 generations. The growth rate, which was largely declined due to genome reduction, was considerably recovered, associated with the improved carrying capacity. Genome mutations accumulated during evolution were significantly varied across the evolutionary lineages and were randomly localized on the reduced genome. Transcriptome reorganization showed a common evolutionary direction and conserved the chromosomal periodicity, regardless of highly diversified gene categories, regulons, and pathways enriched in the differentially expressed genes. Genome mutations and transcriptome reorganization caused by evolution, which were found to be dissimilar to those caused by genome reduction, must have followed divergent mechanisms in individual evolutionary lineages. Gene network reconstruction successfully identified three gene modules functionally differentiated, which were responsible for the evolutionary changes of the reduced genome in growth fitness, genome mutation, and gene expression, respectively. The diversity in evolutionary approaches improved the growth fitness associated with the homeostatic transcriptome architecture as if the evolutionary compensation for genome reduction was like all roads leading to Rome.

evolutionary biology↗

Substrate profiling of human Transglutaminase 1 using cDNA display and next-generation sequencing

Human transglutaminase 1 regulates skin development and is linked with several disease conditions with yet fully unknown mechanisms. To uncover all of its roles in health and disease, the understanding of protein substrates and their reactivity with transglutaminase 1 is necessary. To gain insight into the substrate profile of human transglutaminase 1, this study uses an in vitro selection system based on the cDNA display technology to screen two displayed peptide libraries differing in the number and position of mutated sites relative to the reactive glutamine, in terms of their reactivity to transglutaminase 1. Analysis of the selected DNA pools of by next-generation sequencing and in-house bioinformatics methods revealed a detailed transglutaminase 1 substrate profile indicating preferred and non-preferred amino acid sequences. We have identified a peptide with sequence AEQHKLPSKWPF showing high reactivity to human transglutaminase 1 and low reactivity to transglutaminase 2 and transglutaminase 3. The position weight matrix consisting of per residue amino acid enrichment factors of all selected peptides was used to search human proteins by our in-house search algorithm and identify highly scoring sequence motifs in their primary structure. The search identified six already known transglutaminase 1 substrate proteins as highly scored hits as well as a list of candidate substrates that are under investigation.

bioengineering↗

cDNA display coupled with next-generation sequencing for rapid activity-based screening: Comprehensive analysis of transglutaminase substrate preference

cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, where a stable complex is formed suitable for a wide range of selection conditions. A great advantage of cDNA display is the ability to handle enormous library size (1012) in a microtube scale, in a matter of days. To harness its benefits, we aimed at developing a platform which combines the advantages of cDNA display with high-throughput and accuracy of next-generation sequencing (NGS) for the selection of preferred substrate peptides of transglutaminase 2 (TG2), a protein cross-linking enzyme. After the optimization of the platform by the repeated screening of binary model libraries consisting of the substrate and non-substrate peptides at different ratios, screening and selection of combinatorial peptide library randomized at positions -1, +1, +2, and +3 from the glutamine residue was carried out. Enriched cDNA complexes were analyzed by NGS and bioinformatics, revealing the comprehensive amino acid preference of the TG2 at targeted positions of the peptide backbone. This is the first report on the cDNA display/NGS screening system to yield comprehensive data on TG substrate preference. Although some issues remain to be solved, this platform can be applied to the selection of other TGs and easily adjusted for the selection of other peptide substrates and even larger biomolecules.

bioengineering↗