Department of Biochemistry and Molecular Biology
RBHS, Robert Wood Johnson Medical School
Cancer Institute of New Jersey
Ph.D., 1963, Osaka University
Telephone: (848) 445-9813
Fax: (732) 235-4559
Cell growth and cell death regulation in bacteria; functional and structural characterization of apoptotic proteins in E. coli and pathogenic bacteria; mRNA interferase; single protein production systems; cold-shock adaptation; propeptide-mediated protein folding
Cell growth and cell death regulation by apoptotic toxin-antitoxin (TA) systems: Almost all bacteria including human pathogens contain suicide or toxin genes, which are induced under stress conditions leading to cell growth arrest and eventual cell death in a way similar to apoptosis or programmed cell death in higher eukaryotic systems. Bacterial physiology is tightly regulated by the networks of these toxins and their cognate antitoxins under stress conditions. Some toxins are equally toxic in eukaryotic cells causing programmed cell death and therefore they can be used as a tool for gene therapy. The study of these toxins may also provide important insights into development of novel antibiotics against human pathogens. Since Escherichia coli contains more than 30 TA systems, it serves as ideal paradigm for the study of not only bacterial toxins targeting DNA, RNA, protein and cell wall synthesis, but also previously unknown aspects of bacterial physiology governed by the toxin networks. Our goal is to decipher structures and functions of the E. coli toxins and study their cellular network. In addition, we are also studying the TA systems in Staphylococcus aureus by identifying their cellular targets and their possible roles in the pathogenecity of this bacterium in human tissues.
mRNA interferases: mRNA interferases are sequence-specific endoribonucleases encoded by the TA systems in bacteria. MazF from E. coli is the first mRNA interferase discovered in our laboratory and functions as an ACA-specific mRNA interferase. Its induction in E. coli cells results in cell growth arrest and eventual cell death. We also observed that its induction in mammalian cells effectively causes Bak (a pro-apoptotic protein)-dependent programmed cell death. We explore the application of bacterial mRNA interferases for mammalian cell growth regulation to develop an effective method for treatment of cancer cells. We also use E. coli MazF for regulation of HIV-1 infection. In addition to E. coli MazF, we have identified a large number of mRNA interferases having different RNA cleavage specificity including one cleaving a specific seven base sequence. On the basis of these findings we also attempt to engineer mRNA interfereases with different RNA cleavage specificity.
Single protein production in living cells: MazF, an ACA-specific mRNA interferase expression, results in nearly complete degradation of cellular mRNAs, leading to severe reduction of protein synthesis in conjunction with growth arrest. However, most intriguingly, MazF-induced cells are still fully capable of producing a protein at a high level if the mRNA for that protein is engineered to be devoid of all ACA sequences without altering its amino acid sequence. Therefore, we are able to convert E. coli cells into a bioreactor producing a single protein of interest. This system termed “single-protein production” (SPP) system is a unprecedented novel technology for structural biology, as using this technology one does not have to purify a protein of interest for NMR structural study. Thus, this system is especially powerful for structural study of membrane proteins. We are exploiting the SPP system for NMR protein structural study in a most cost effective manner (less than 0.5% of the conventional methods) and to study protein dynamics in living cells (in-cell NMR).
Cold-shock response and adaptation: When an organism senses downshift in temperature, it responds by eliciting cold-shock response for its survival and growth. We use E. coli as a model system to study cold shock response. When the cells encounter cold shock, there is a lag period of growth termed acclimation phase, in which cellular synthesis of most of the proteins is inhibited as opposed to that of a select group of proteins, termed cold-shock proteins. These proteins help the cells counteract various detrimental cellular changes triggered by the temperature downshift. This is followed by resumption of the cell growth with restoration of synthesis of normal cellular proteins and decrease in the rate of synthesis of cold-shock proteins. E. coli contains a family of nine CspA homologues, CspA being the major cold shock protein. We found that CspA plays an important role as an RNA chaperone to unfold mRNAs which otherwise cannot be translated at low temperature. Our group is currently focusing on low temperature RNA metabolism involving cold shock proteins such as PNPase, CsdA, and RNase R, as these RNases and helicases play essential roles in maintenance of RNA function at low temperature.
Propeptide-mediated protein folding: The propeptide is an N-terminal extension of a protein and essential for the folding of the protein. Thus, the propeptides are termed as ‘intramolecular chaperones’. It is known that the propeptide-mediated protein folding plays a crucial role in many important proteins in humans and mutations in the propeptide can lead to serious human diseases; for example Val66 to Met mutation in the propeptide of BDNF (brain derived neurotrophic factor) causes misfolding of mature BDNF resulting in a serious defect in human memory. Research on the essential role of the propeptide in protein folding, originally discovered in our laboratory in 1987, is thus very important not only for our understanding the basic principle of protein folding, but also for our understanding of the human diseases caused by propeptide mutations. Using subtilisin as a model system, we study the role of the 77-residue propetide in subtilisin folding by genetic, biochemical and biophysical (including NMR) methods. We are particularly interested in the mechanisms of misfolding caused by mutations in the propeptide termed as ‘protein-memory mutations’.
Park JH, Kwon M, Yamaguchi Y, Firestein BL, Park JY, Yun J, Yang JO, Inouye M. (2017) Preferential use of minor codons in the translation initiation region of human genes. Hum Genet 136:67-74
Inouye M, Ishida Y, Inouye K. (2017) Designing of a single gene encoding four functional proteins. J Theor Biol 419:266-8
Ishida Y, Inouye K, Inouye M. (2017) The role of the loop 1 region in MazFbs mRNA interferase from Bacillus subtilis in recognition of the 3' end of the RNA substrate. Biochem Biophys Res Commun 483:403-8
Inouye M. (2017) The first demonstration of the existence of reverse transcriptases in bacteria. Gene 597:76-7
Inouye M. (2017) The first discovery of RNA interference by RNA restriction enzymes to inhibit protein synthesis. Gene 597:78-9
Monneau YR, Ishida Y, Rossi P, Saio T, Tzeng SR, Inouye M, Kalodimos CG. (2016) Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications. J Biomol NMR 65:99-108
Miyanoiri Y, Ishida Y, Takeda M, Terauchi T, Inouye M, Kainosho M. (2016) Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain. J Biomol NMR 65:109-19
Masuda H, Awano N, Inouye M. (2016) ydfD encodes a novel lytic protein in Escherichia coli. FEMS Microbiol Lett 363
Ishida Y, Inouye M. (2016) Suppression of the toxicity of Bac7 (1-35), a bovine peptide antibiotic, and its production in E. coli. AMB Express 6:19
Yamaguchi Y, Inouye M. (2015) An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli. Nucleic Acids Res 43:10387-96
Yamaguchi Y, Tokunaga N, Inouye M, Phadtare S. (2014) Characterization of LdrA (long direct repeat A) protein of Escherichia coli. J Mol Microbiol Biotechnol 24:91-7
Shimazu T, Mirochnitchenko O, Phadtare S, Inouye M. (2014) Regression of solid tumors by induction of MazF, a bacterial mRNA endoribonuclease. J Mol Microbiol Biotechnol 24:228-33