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It is important to note that the administration of THA significantly increased the area immunostained by the anti-pVEGFR2 antibody in medial septal cholinergic neurons. The same treatment slightly increased the expression levels of VEGFR2 in cholinergic neurons; however, this change was not confirmed by the qPCR analysis targeting the gene coding VEGFR2. Since p-VEGFR2 is an auto-phosphorylated form of VEGFR2 that is produced via the activation of VEGFR2 by VEGF (Takahashi et al., 2001), the present results suggest that the THA treatment increased the expression levels of VEGF in cholinergic neurons via the endogenous ACh stimulation of nicotinic AChR, thereby facilitating VEGF-VEGFR2 transmission on medial septal cholinergic neurons via autocrine and/or paracrine mechanisms. Lin et al. indicated that VEGF is expressed in the cytoplasm, whereas VEGFR2 is distributed in the membrane and cytoplasm, and also that pVEGFR2 partly localized to the cytoplasm and nucleus in gastric cancer cells after VEGFR2 was activated by VEGF secreted via an autocrine mechanism (Lin et al., 2017).
In these studies, we focused on VEGF expression by THA-induced endogenous ACh elevation, because we previously revealed that THA administration increased VEGF expression in several animal models (Zhao et al., 2011, 2012). However, in order to elucidate the mechanism of the VEGF expression via Ach receptor stimulation, it is also very important to analyze the effects of direct stimulation of Ach receptor by CCh in vivo. The studies of CCh on VEGF expression in medial septal cholinergic neurons are now underway.
Conflicts of interest
Acknowledgments This work was in part supported by Grant-in-aid for the 2012 and 2013 Cooperative Research Project I from Institute of Natural Medicine, University of Toyama (to J.-I. O. and K. M.).
Introduction A major goal of biochemistry is to understand the amino Ozagrel HCl origins of protein function. This stems from the seminal observation that a protein's three-dimensional structure, and biological function, is ultimately determined by its amino acid sequence . The functional importance of a particular amino acid residue is usually inferred from its proximity to an active site in a three-dimensional protein structure, or from its degree of conservation in an amino acid sequence alignment. While these familiar approaches have successfully identified functionally relevant amino acids in a wide array of proteins, they have their limitations. Not all structure-function relationships are obvious from structure gazing, and lack of conservation of an amino acid in a sequence alignment does not necessarily mean that the amino acid is unimportant for protein function. Furthermore, many protein functions are not imparted by a single amino acid residue, and instead emerge from an entangled network of residues. To pinpoint these residues, a number of bioinformatic approaches have been developed that exploit the growing fund of protein sequences . Experimentally validating a putative network often requires mutation of a large number of residues, in multiple combinations, which can be experimentally impractical. The problem is best illustrated in the context of amino acid sequence space, which encompasses all possible combinations of polypeptide sequences containing the twenty naturally occurring amino acids. Even for a putative network with just five residues, testing all possible combinations of amino acids in these five positions means that, 205, or 3.2 million combinations would have to be examined. Clearly such a strategy is not feasible. What is needed is a way of identifying combinations of amino acids that may be biologically relevant, by mapping meaningful sequences within a protein's amino acid sequence space. Evolution has been exploring amino acid sequence space for billions of years, and the innovations in amino acid sequence that led to modern protein functions are embedded within each protein's evolutionary history . If this evolutionary history could be rewound, and then replayed, it would be possible to unravel the series of mutations that accrued throughout a protein's evolution, and unveil the amino acid origins of the protein's modern-day functions . In the 1960s, Zuckerkandl and Pauling recognized that the amino acid sequences of modern proteins could be used to document evolution, and reconstruct phylogenetic relationships . These “molecular phylogenies”, combined with models of how amino acid sequences evolve, now make it possible to reconstruct the sequences of ancestral proteins . This ability, combined with the declining costs of DNA synthesis, make experiments tracing the evolution of protein structure and function possible. The result is not only an emerging understanding of protein evolution, but also insight into the amino acid origins of protein functions . Here we outline how such a strategy can be used to unveil enigmatic structure-function relationships in the muscle-type nicotinic acetylcholine receptor (AChR), an excellent model for understanding both protein evolution and biophysics.