Evolution of Specificity in Protein-Protein Interactions

Hub proteins are proteins that maintain promiscuous molecular recognition. Because they are reported to play essential roles in cellular control, there has been a special interest in the study of their structural and functional properties, yet the mechanisms by which they evolve to maintain functional interactions are poorly understood. By combining biophysical simulations of coarse-grained proteins and analysis of proteins-complex crystallographic structures, we seek to elucidate those mechanisms. We focus on two types of hub proteins: Multi hubs, which interact with their partners through different interfaces, and Singlish hubs, which do so through a single interface. We show that loss of structural stability is required for the evolution of protein-protein-interaction (PPI) networks, and it is more profound in Singlish hub systems. In addition, different ratios of hydrophobic to electrostatic interfacial amino acids are shown to support distinct network topologies (i.e., Singlish and Multi systems), and therefore underlie a fundamental design principle of PPI in a crowded environment. We argue that the physical nature of hydrophobic and electrostatic interactions, in particular, their favoring of either same-type interactions (hydrophobic-hydrophobic), or opposite-type interactions (negatively-positively charged) plays a key role in maintaining the network topology while allowing the protein amino acid sequence to evolve.     

Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production

Sucrose is one of the most promising carbon sources for industrial fermentation. To achieve sucrose catabolism, the sucrose utilization operons have been introduced into microorganisms that are not able to utilize sucrose. However, the rates of growth and sucrose uptake of these engineered strains were relatively low to be successfully employed for industrial applications. Here, we report a practical example of developing sucrose-utilizing microorganisms using Escherichia coli K-12 as a model system. The sucrose utilizing ability was acquired by introducing only β-fructofuranosidase from three different sucrose-utilizing organisms (Mannheimia succiniciproducens, E. coli W, and Bacillus subtilis). Among them, the M. succiniciproducens β-fructofuranosidase was found to be the most effective for sucrose utilization. Analyses of the underlying mechanism revealed that sucrose was hydrolyzed into glucose and fructose in the extracellular space and both liberated hexoses could be transported by their respective uptake systems in E. coli K-12. To prove that this system can also be applied for the production of useful metabolites, the M. succiniciproducens β-fructofuranosidase was introduced into the engineered l-threonine production strain of E. coli K-12. This recombinant strain was able to produce 51.1 g/L l-threonine by fed-batch culture, resulting in an overall yield of 0.284 g l-threonine per g sucrose. This simple approach to make E. coli K-12 to acquire sucrose-utilizing ability and its successful biotechnological application can be employed to develop sustainable bioprocesses using renewable biomass.     

Overexpression of Newcastle disease virus (NDV) V protein enhances NDV production kinetics in chicken embryo fibroblasts

Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established. The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strains.     

Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40°C and 7.5–8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0–7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.     

MT2 Melatonin Receptor Immunoreactivity in Neurons is Very High in the Aged Hippocampal Formation in Gerbils

Melatonin exerts many physiological functions via its G protein-coupled receptors. In the present study, we investigated age-related changes in MT2 melatonin receptor immunoreactivity and its levels in the gerbil hippocampus during normal aging. In the postnatal month 1 (PM 1) group, MT2 immunoreaction was well observed in neurons in all subregions of the gerbil hippocampus. In the PM 3 and 6 groups, MT2 immunoreactivity in neurons was decreased compared to that in the PM 1 group. Thereafter, MT2 immunoreactivity in neurons was increased. In the PM 18 and 24 groups, MT2 immunoreactivity in neurons was strong in all subregions of the gerbil hippocampus. In addition, the number of MT2 immunoreactive cells was lowest at PM 3 and highest at PM 24. From western blot analysis, age-dependent change pattern in MT2 level in the gerbil hippocampus was similar to the immunohistochemical result. These results indicate that MT2 immunoreactivity and levels are altered in the gerbil hippocampus during normal aging; lowest at young adult stage and highest at aged stage.     

Suppression of Glutamate-Induced Excitotoxicity by 2-Cyclopropylimino-3-methyl-1,3-thiazoline Hydrochloride in Rat Glial Cultures

We have screened new drugs with a view to developing effective drugs against glutamate-induced excitotoxicity. In the present work, we show effects of a new drug, 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride against glutamate-induced excitotoxicity in primary rat glial cultures. Pretreatment of glial cells with 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride for 2 h significantly protected glial cells against glutamate-induced excitotoxicity in a time- and dose-dependent manner with an optimum concentration of 100 μM. The drug significantly reduced production of proinflammatory cytokines, tumor necrosis factor-α, and interlukin-1β in glutamate-induced excitotoxicity. The drug also prevented glutamate-induced intracellular Ca²⁺ influx and reduced the subsequent overproduction of nitric oxide and reactive oxygen species. Furthermore, the drug preserved the mitochondrial potential and inhibited the overproduction of cytochrome c. In addition, the drug effectively attenuated the protein level changes of β-catenin and glycogen synthase kinase-3β. These results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride effectively protected primary cultures of rat glial cells against glutamate-induced excitotoxicity.