Masayori Inouye Professor UMDNJ Dept. Biochemistry 675 Hoes Lane Piscataway. NJ 08854 (732) 235-4540 FAX - 4783 inouye@umdnj.edu

Molecular biology of cellular adaptation to stresses

Cold-shock Adaptation - When E. coli cells are exposed to low temperatures. a number of cytoplasmic proteins (cold-shock proteins) are specifically induced. Among them. CspA. CspB and CsrA have been extensively studied in our laboratory. Our data indicate that CsrA is a new ribosomal protein which functions as an ATP-dependent RNA helicase to unwind stable secondary structure in mRNAs formed at low temperatures. CspA is an RNA chaperone which prevents the formation of secondary structures in mRNA. and CspB is a DNA chaperone which plays an important role in transcription as well as in DNA replication.

Osmoregulation and Transmembrane Signal Transduction - Adaptive responses of E. coli to medium osmolarity provides an ideal system in which to study signal transduction across the membrane as well as the transcriptional regulation of two porin genes (ompF and ompC) by a transcriptional factor (OmpR). Its function is modulated by a transmembrane signal transducer (EnvZ) which functions not only as kinase but also as phosphatase for OmpR.

Molecular Biology of Aging - One of the major causes of aging is known to be oxidative damage due to various oxidants produced during respiration. We have constructed transgenic mice overexpressing human glutathione peroxidases and superoxide dismutase to study oxidative stress on aging and the effect of antioxidant enzymes on radical-generation chemicals and carcinogens.

Development of Myxococcus xanthus - M. xanthus is a Gram-negative bacterium which can undergo cellular differentiation upon nutrient starvation. M. xanthus provides an excellent system in which to study the morphogenesis and molecular mechanism of control of gene expression during development. Attempts are being made to characterize several development-specific proteins. including a number of eukaryotic-like protein serine/threonine kinases.

GTP Metabolism - GTP plays key roles in various cellular functions. We are investigating a number of GTP-associated proteins in E. coli such as Era (an essential GTP-binding protein of unknown function). EF-Tu. and nucleoside diphosphate kinase.

Protein Folding - We are attempting to decipher the precise process of protein folding by molecular genetic as well as biochemical approaches using pro-subtilisin as a model system. In this system the propeptide of 77 amino acid residues functions as an intramolecular chaperone which is essential for the folding of active subtilisin.

Bacterial Retroelements - About 10% of the natural population of E. coli contain a highly diverse retroelement called 'retron'. which is responsible for the production of a peculiar RNA-DNA complex (msDNA). We are attempting to determine the biological function of the retron. the biosynthetic mechanism of msDNA. and to search for its origin.

Selected Publications

Mao L, Tang Y, Vaiphei ST, Shimazu T, Kim SG, Mani R, Fakhoury E, White E, Montelione GT, Inouye M. (2009) Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system. J Struct Funct Genomics. Oct 24. [Epub ahead of print] PMID: 19856129

Mao L, Vaiphei ST, Shimazu T, Schneider WM, Tang Y, Mani R, Roth MJ, Montelione GT, Inouye M. (2009) The E. coli single protein production system for production and structural analysis of membrane proteins. J Struct Funct Genomics. Oct 15. [Epub ahead of print]
PMID: 19830594

Yamaguchi Y, Park JH, Inouye M. (2009) MqsR, a crucial regulator for quorum sensing and biofilm formation, is a GCU-specific mRNA interferase in Escherichia coli. J Biol Chem. 284(42):28746-53.

Schneider WM, Inouye M, Montelione GT, Roth MJ. (2009) Independently inducible system of gene expression for condensed single protein production (cSPP) suitable for high efficiency isotope enrichment. J Struct Funct Genomics. 10(3):219-25.

Baik S, Inoue K, Ouyang M, Inouye M. (2009) Significant bias against the ACA triplet in the tmRNA sequence of Escherichia coli K-12. J Bacteriol. 191(19):6157-66.

Zhang Y, Yamaguchi Y, Inouye M. (2009) Characterization of YafO, an Escherichia coli toxin.
J Biol Chem. 284(38):25522-31.

Yoshizumi S, Zhang Y, Yamaguchi Y, Chen L, Kreiswirth BN, Inouye M. (2009) Staphylococcus aureus YoeB homologues inhibit translation initiation. J Bacteriol. 191(18):5868-72.

Phadtare S, Zhu L, Uemori T, Mukai H, Kato I, Inouye M. (2009) Applications of nucleic acid chaperone activity of CspA and its homologues. J Mol Microbiol Biotechnol. 17(3):110-7.

Kobayashi H, Yoshida T, Inouye M. (2009) Significant enhanced expression and solubility of human proteins in Escherichia coli by fusion with protein S from Myxococcus xanthus. Appl Environ Microbiol. 75(16):5356-62.

Yoshizumi A, Yu Z, Silva T, Thiagarajan G, Ramshaw JA, Inouye M, Brodsky B. (2009)
Self-association of streptococcus pyogenes collagen-like constructs into higher order structures. Protein Sci. 18(6):1241-51.

Li GY, Zhang Y, Inouye M, Ikura M. (2009) Inhibitory mechanism of Escherichia coli RelE-RelB toxin-antitoxin module involves a helix displacement near an mRNA interferase active site. J Biol Chem. 284(21):14628-36.

Zhu L, Inoue K, Yoshizumi S, Kobayashi H, Zhang Y, Ouyang M, Kato F, Sugai M, Inouye M. (2009) Staphylococcus aureus MazF specifically cleaves a pentad sequence, UACAU, which is unusually abundant in the mRNA for pathogenic adhesive factor SraP. J Bacteriol. 191(10):3248-55.

Yamaguchi Y, Inouye M. (2009) mRNA interferases, sequence-specific endoribonucleases from the toxin-antitoxin systems. Prog Mol Biol Transl Sci. 85:467-500.

Prysak MH, Mozdzierz CJ, Cook AM, Zhu L, Zhang Y, Inouye M, Woychik NA. (2009) Bacterial toxin YafQ is an endoribonuclease that associates with the ribosome and blocks translation elongation through sequence-specific and frame-dependent mRNA cleavage. Mol Microbiol. 71(5):1071-87.

Zhang Y, Inouye M. (2009) The inhibitory mechanism of protein synthesis by YoeB, an Escherichia coli toxin. J Biol Chem. 284(11):6627-38.

Chen YJ, Wu KP, Kim S, Falzon L, Inouye M, Baum J. (2008) Backbone NMR assignments of DFP-inhibited mature subtilisin E. Biomol NMR Assign. 2(2):131-3.

Chen YJ, Inouye M. (2008) The intramolecular chaperone-mediated protein folding. Curr Opin Struct Biol. 18(6):765-70.

Aramini JM, Rossi P, Huang YJ, Zhao L, Jiang M, Maglaqui M, Xiao R, Locke J, Nair R, Rost B, Acton TB, Inouye M, Montelione GT. (2008) Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad. Biochemistry. 47(37):9715-7.

Awano N, Inouye M, Phadtare S. (2008) RNase activity of polynucleotide phosphorylase is critical at low temperature in Escherichia coli and is complemented by RNase II. J Bacteriol. 190(17):5924-33.

Li GY, Zhang Y, Inouye M, Ikura M. (2008) Structural mechanism of transcriptional autorepression of the Escherichia coli RelB/RelE antitoxin/toxin module. J Mol Biol. 380(1):107-19.

Zhu L, Phadtare S, Nariya H, Ouyang M, Husson RN, Inouye M. (2008) The mRNA interferases, MazF-mt3 and MazF-mt7 from Mycobacterium tuberculosis target unique pentad sequences in single-stranded RNA. Mol Microbiol. 69(3):559-69.

Liu M, Zhang Y, Inouye M, Woychik NA. (2008) Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit. Proc Natl Acad Sci U S A. 105(15):5885-90.

Hwang J, Inouye M. (2008) RelA functionally suppresses the growth defect caused by a mutation in the G domain of the essential Der protein. J Bacteriol. 190(9):3236-43.

Nariya H, Inouye M. (2008) MazF, an mRNA interferase, mediates programmed cell death during multicellular Myxococcus development. Cell. 132(1):55-66.

Inoue K, Basu S, Inouye M. (2007) Dissection of 16S rRNA methyltransferase (KsgA) function in Escherichia coli. J Bacteriol. 189(23):8510-8.

Mohs A, Silva T, Yoshida T, Amin R, Lukomski S, Inouye M, Brodsky B. (2007) Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline. J Biol Chem. 282(41):29757-65.

Suzuki M, Mao L, Inouye M. (2007) Single protein production (SPP) system in Escherichia coli. Nat Protoc. 2(7):1802-10.

Kishii R, Falzon L, Yoshida T, Kobayashi H, Inouye M. (2007) Structural and functional studies of the HAMP domain of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli. J Biol Chem. 282(36):26401-8.

Yoshida T, Phadtare S, Inouye M. (2007) Functional and structural characterization of EnvZ, an osmosensing histidine kinase of E. coli. Methods Enzymol. 423:184-202.

Yoshida T, Phadtare S, Inouye M. (2007) The design and development of Tar-EnvZ chimeric receptors. Methods Enzymol. 423:166-83.

Awano N, Xu C, Ke H, Inoue K, Inouye M, Phadtare S. (2007) Complementation analysis of the cold-sensitive phenotype of the Escherichia coli csdA deletion strain. J Bacteriol. 189(16):5808-15.

Shimazu T, Degenhardt K, Nur-E-Kamal A, Zhang J, Yoshida T, Zhang Y, Mathew R, White E, Inouye M. (2007) NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition. Genes Dev. 21(8):929-41.

Falzon L, Patel S, Chen YJ, Inouye M. (2007) Autotomic behavior of the propeptide in propeptide-mediated folding of prosubtilisin E. J Mol Biol. 366(2):494-503.

Suzuki M, Roy R, Zheng H, Woychik N, Inouye M. (2006) Bacterial bioreactors for high yield production of recombinant protein. J Biol Chem. 281(49):37559-65.

Inouye M. (2006) The discovery of mRNA interferases: implication in bacterial physiology and application to biotechnology. J Cell Physiol. 209(3):670-6. Review.

Inouye M. (2006) Signaling by transmembrane proteins shifts gears. Cell. 126(5):829-31.

Hwang J, Inouye M. (2006) The tandem GTPase, Der, is essential for the biogenesis of 50S ribosomal subunits in Escherichia coli. Mol Microbiol. 61(6):1660-72.

Falzon L, Suzuki M, Inouye M. (2006) Finding one of a kind: advances in single-protein production. Curr Opin Biotechnol. 17(4):347-52.

Inoue K, Chen J, Tan Q, Inouye M. (2006) Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli. J Mol Microbiol Biotechnol. 11(1-2):41-52.

Yoshida T. Qin L. Egger LA. Inouye M. (2006) Transcription regulation of ompF and ompC by a single transcription factor. OmpR. J Biol Chem. 281(25):17114-23.

Zhang Z. Emanetoglu NW. Saraf G. Chen Y. Wu P. Zhong J. Lu Y. Chen J. Mirochnitchenko O. Inouye M. (2006) DNA immobilization and SAW response in ZnO nanotips grown on LiNbO3 substrates. IEEE Trans Ultrason Ferroelectr Freq Control. 53(4):786-92.

Mineno J. Okamoto S. Ando T. Sato M. Chono H. Izu H. Takayama M. Asada K. Mirochnitchenko O. Inouye M. Kato I. (2006) The expression profile of microRNAs in mouse embryos. Nucleic Acids Res. Mar 31;34(6):1765-71.

Li GY. Zhang Y. Chan MC. Mal TK. Hoeflich KP. Inouye M. Ikura M. (2006) Characterization of dual substrate binding sites in the homodimeric structure of Escherichia coli mRNA interferase MazF. J Mol Biol. 357(1):139-50.

Foronjy RF. Mirochnitchenko O. Propokenko O. Lemaitre V. Jia Y. Inouye M. Okada Y. D'Armiento JM. (2006) Superoxide dismutase expression attenuates cigarette smoke- or elastase-generated emphysema in mice. Am J Respir Crit Care Med. 173(6):623-31.

Hong M. Xu W. Yoshida T. Tanaka K. Wolff DJ. Zhou F. Inouye M. You G. (2005) Human organic anion transporter hOAT1 forms homooligomers. J Biol Chem. 280(37):32285-90.

Jiang W. Prokopenko O. Wong L. Inouye M. Mirochnitchenko O. (2005) IRIP. a new ischemia/reperfusion-inducible protein that participates in the regulation of transporter activity. Mol Cell Biol. 25(15):6496-508.

Zhang Y. Zhu L. Zhang J. Inouye M. (2005) Characterization of ChpBK. an mRNA interferase from Escherichia coli. J Biol Chem. 280(28):26080-8.

Sone M. Falzon L. Inouye M. (2005) The role of tryptophan residues in the autoprocessing of prosubtilisin E. Biochim Biophys Acta. 1749(1):15-22.

Suzuki M. Zhang J. Liu M. Woychik NA. Inouye M. (2005) Single protein production in living cells facilitated by an mRNA interferase. Mol Cell. 18(2):253-61.

Acton TB. Gunsalus KC. Xiao R. Ma LC. Aramini J. Baran MC. Chiang YW. Climent T. Cooper B. Denissova NG. Douglas SM. Everett JK. Ho CK. Macapagal D. Rajan PK. Shastry R. Shih LY. Swapna GV. Wilson M. Wu M. Gerstein M. Inouye M. Hunt JF. Montelione GT. (2005) Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium. Methods Enzymol. 394:210-43.

Zhang Y. Zhang J. Hara H. Kato I. Inouye M. (2005) Insights into the mRNA cleavage mechanism by MazF. an mRNA interferase. J Biol Chem. 280(5):3143-50.

Phadtare S. Inouye M. wild-type and cold-sensitive. quadruple-csp-deletion strains of Escherichia coli. J Bacteriol. (2004) Genome-wide transcriptional analysis of the cold shock response in 186(20):7007-14.

Inouye M. Ke H. Yashio A. Yamanaka K. Nariya H. Shimamoto T. Inouye S. (2004) Complex formation between a putative 66-residue thumb domain of bacterial reverse transcriptase RT-Ec86 and the primer recognition RNA. J Biol Chem. 279(49):50735-42.

Khorchid A. Inouye M. Ikura M. (2005) Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization.
Biochem J. 385(Pt 1):255-64.

Zhu Y. Inouye M. (2004) The HAMP linker in histidine kinase dimeric receptors is critical for symmetric transmembrane signal transduction. J Biol Chem. 279(46):48152-8.

Inouye M. Phadtare S. (2004) Cold shock response and adaptation at near-freezing temperature in microorganisms. Sci STKE. 2004(237):pe26.

Qing G. Ma LC. Khorchid A. Swapna GV. Mal TK. Takayama MM. Xia B. Phadtare S. Ke H. Acton T. Montelione GT. Ikura M. Inouye M. (2004) Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol. 22(7):877-82.

Qing G. Xia B. Inouye M. (2003) Enhancement of translation initiation by A/T-rich sequences downstream of the initiation codon in Escherichia coli. J Mol Microbiol Biotechnol. 6(3-4):133-44.

Zhang J. Zhang Y. Zhu L. Suzuki M. Inouye M. (2004) Interference of mRNA function by sequence-specific endoribonuclease PemK. J Biol Chem. 279(20):20678-84.

Phadtare S. Inouye M. Severinov K. (2004) The mechanism of nucleic acid melting by a CspA family protein. J Mol Biol. 337(1):147-55.