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Paul I. Freimuth

Research Interests

Our current studies focus on toxic gain-of-function by proteins that misfold during recombinant expression in microbial cells. Errors in folding of endogenous proteins occur frequently, and cells have several independent quality control pathways to monitor protein folding status and mitigate the potentially toxic effects associated with accumulation of misfolded protein species. Failure of these QC pathways to restore protein folding homeostasis can result in cell death and disease. A number of factors in addition to amino acid sequence can influence folding, including temperature fluctuations, global and local rates of translation elongation, and availability of molecular chaperones and other enzymes for post-translational modification. These factors may be changed radically when polypeptides are expressed in heterologous host cells using recombinant expression technology, which likely contributes to the frequent production of misfolded protein species in such experiments. Misfolded polypeptides produced by these methods often accumulate as insoluble aggregates that have little or no apparent deleterious effects on cell physiology. However, we have encountered several instances where misfolded proteins gain toxic functions that strongly inhibit cell growth. In earlier work, for example, we found that the head domain of the serotype 2 adenovirus fiber protein is conditionally toxic when expressed in thi- strains of E. coli, and that the essential vitamin thiamine diphosphate is tightly sequestered in the protein subunit interface. By contrast, fiber head domains derived from other adenoviruses closely related to serotype 2 do not bind thiamine diphosphate and are not toxic for thi- strains. Results of this study provide the important insight that the toxic activity of misfolded proteins or misassembled protein oligomers can result from highly specific mechanisms. The toxicity of amyloid fibrils, in contrast, has been proposed to result from nonspecific sequestration of numerous essential cell proteins.

The conclusion that the toxic activity of misfolded proteins can result from highly specific mechanisms is also supported by our current investigation of the toxic activity of the Arabidopsis Gld protein, which misfolds during expression in E. coli. Like the fiber head domain, the toxic activity of Gld is conditional, in this case depending on which RNA polymerase is used for expression of the plasmid-borne Gld gene in E. coli. When gld is transcribed by the E. coli RNA polymerase, cell growth is arrested immediatedly following induction of expression, and cell viability plummets. Minute but detectable quantities of Gld protein are produced in growth-arrested cells. Importantly, the Gld protein is found in the soluble fraction of cell lysates. When gld is transcribed by the T7 phage RNA polymerase, by contrast, cell growth proceeds following induction, and Gld protein accumulates to high concentration in cells as insoluble aggregates. These results are in striking parallel to recent studies of amyloidogenic proteins, which show that toxicity is associated primarily with soluble, pre-aggregated oligomers of misfolded protein, whereas the insoluble amyloid fibrils themselves were found to be relatively nontoxic. Additional studies indicate that an internal region 26-residues in length is both necessary and sufficient for the toxic activity of the 325-residue Gld polypeptide. The corresponding synthetic peptide inhibits transcription in vitro by purified E. coli RNA polymerase. Results of our current study thus support the conclusion that the toxic activity of misfolded Gld also results from a highly specific mechanism, in this case inhibition of transcription by E. coli RNA polymerase. Our results also provide the insight that the aggregation-prone Gld polypeptide can only exert its toxic effect over short distances, for example only when the host RNA polymerase is physically coupled to ribosomes translating the Gld polypeptide. In future studies we will determine the molecular mechanism of the toxic Gld peptide interaction with RNA polymerase.

Selected Publications

  • Graziano V., McGrath W.J., Suomalainen M., Greber U.F., Freimuth P., Blainey P.C., Luo G., Xie X.S., and Mangel W.F. Regulation of a viral proteinase by a peptide and DNA in one-dimensional space. I. Binding to DNA and to hexon of the precursor to protein VI, pVI, of human adenovirus. J. Biol. Chem., 288(3):2059-2067 (2013).
  • Tawde M.D. and Freimuth P. Toxic misfolding of Arabidopsis cellulases in the secretory pathway of Pichia pastoris. Protein Expression and Purification, 85(2):211-217 (2012).
  • Freimuth P., Philipson L. and Carson S.D.
    The coxsackievirus and adenovirus receptor.
    Current Topics in Microbiology and Immunology: Group B Coxsackieviruses, S. Tracy, M. S. Oberste, and K. M. Drescher, Editors, Vol. 323, Chapter 4, pp. 67-87, Springer-Verlag Berlin Heidelberg (2008).
  • Maye M.M., Freimuth P., and Gang O.
    Adenovirus knob trimers as tailorable scaffolds for nanoscale assembly.
    Small, 4(11):1941-1944 (November, 2008).
  • Freimuth P.
    Protein overexpression in mammalian cell lines.
    Genetic Engineering: Principles and Methods, J. K. Setlow, Editor, Vol. 28, pp. 95-104, Springer Science + Business Media, LLC, New York, NY (2007).  PubMed
  • Schulz R., Zhang Y.-B., Liu C.-J. and Freimuth P.
    Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli.
    Protein Science, 16(12):2684-2693 (2007).  PubMed
  • Zhang Y.-B., Kanungo M., Ho A.J., Freimuth P., van der Lelie D., Chen M., Khamis S.M., Datta S.S., Charlie Johnson A.T., Misewich J.A. and Wong S.S.
    Functionalized carbon nanotubes for detecting viral proteins.
    Nano Letters, 7(10):3086-3091 (2007).  PubMed
  • Awasthi V.D., Meinken G., Springer K., Srivastava S.C. and Freimuth P.
    Biodistribution of radioiodinated adenovirus fiber protein knob domain after intravenous injection in mice.
    J. Virol., 78(12):6431-6438 (2004).   PubMed   Full Text
  • Howitt J., Bewley M., Graziano V., Flanagan J. and P. Freimuth.
    Structural basis for variation in adenovirus affinity for the cellular coxsackievirus and adenovirus receptor.
    J. Biol. Chem., 278(28):26208-15 (2003).   PubMed   Full Text
    PDB files 1P69 1P6A   Jmol viewer
  • Zhang Y.-B., Howitt J., McCorkle S., Lawrence P., Springer K. and Freimuth P.
    Protein aggregation during overexpression limited by peptide extensions with large net negative charge.
    Protein Expr Purif., 36(2):207-216 (2004).   PubMed
  • Howitt J., Anderson C.W. and FreimuthP.
    Adenovirus interaction with its cellular receptor CAR.
    Curr. Top. Microbiol. Immunol., 272:331-364 (2003).   PubMed
  • Walters R.W., Freimuth P., Moninger T.O., Ganske I., Zabner J. and Welsh M.J.
    Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape.
    Cell, 110:789-799 (2002).   PubMed
  • Anderson C.W., Dunn J.J., Freimuth P.I., Galloway A.M. and Allalunis-Turner M.J.
    Frameshift mutation in PRKDC, the gene for DNA-PKcs, in the DNA repair-defective, human, glioma-derived cell line M059J.
    Radiat. Res., 156:2-9 (2001).   PubMed
  • He Y., Chipman P.R., Howitt J., Bator C.M., Whitt M.A., Baker T.S., Kuhn R.J., Anderson C.W., Freimuth P. and Rossmann M.G.
    Interaction of coxsackievirus B3 with the full-length coxsackievirus-adenovirus receptor.
    Nat. Struct. Biol., 8(10):874-878 (2001).   PubMed
    PDB file 1JEW   Jmol viewer
  • Thanos P.K., Volkow N.D., Freimuth P., Umegaki H., Ikari H., Roth G., Ingram D.K. and Hitzemann R.
    Overexpression of dopamine D2 receptors reduces alcohol self-administration.
    J. Neurochem., 78:1094-1103 (2001).   PubMed
  • Bewley M.C., Springer K., Zhang Y.-B., Freimuth P. and Flanagan J.M.
    Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR.
    Science, 286:1579-1583 (1999).   PubMed   Full Text
    PDB files 1KAC 1NOB   Jmol viewer
  • Freimuth P., Springer K., Berard C., Hainfeld J., Bewley M. and Flanagan J.
    Coxsackievirus and adenovirus receptor amino-terminal immunoglobulin V related domain binds adenovirus type 2 and fiber knob from adenovirus type 12.
    J. Virol., 73:1392-1398 (1999).   PubMed   Full Text
  • Hainfeld J.F., Liu W., Halsey C.M.R., Freimuth P. and Powell R.D.
    Ni-NTA-gold clusters target His-tagged proteins.
    J. Struct. Biol., 127:185-198 (1999).   PubMed
  • Schaack J., Ho W.Y., Tolman S., Ullyat E., Guo X., Frank N., Freimuth P.I., Roovers D.J. and Sussenbach J.S.
    Construction and preliminary characterization of a library of "lethal" preterminal protein mutant adenoviruses.
    J. Virol., 73(11):9599-9603 (1999).   PubMed   Full Text
  • Mayr G.A. and Freimuth P.
    A single locus on human chromosome 21 directs the expression of a receptor for adenovirus type 2 in mouse A9 cells.
    J. Virol., 71:412-418 (1997).   PubMed   Full Text
  • Zabner J., Freimuth P., Puga A., Fabrega A. and Welsh M.J.
    Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection.
    J. Clin. Invest., 100:1144-1149 (1997).   PubMed   Full Text
  • Freimuth P.
    A human cell line selected for resistance to adenovirus infection has reduced levels of the virus receptor.
    J. Virol., 70:4081-4085 (1996).   PubMed   Full Text
  • Agger R. and Freimuth P.
    Purification and cDNA sequence of a murine protein homologous to the human p62 tyrosine phosphoprotein that associates with the Ras GTPase-acivating protein p120 GAP.
    Gene, 158:307-308 (1995).   PubMed
  • Bai M., Campisi L., and Freimuth P.
    Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2.
    J. Virol., 68:5925-5932 (1994).   PubMed   Full Text
  • Bai M., Harfe B. and Freimuth P.
    Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells.
    J. Virol., 67:5198-5205 (1993).   PubMed   Full Text
  • Freimuth P. and Anderson C.W.,
    Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.
    Virology, 193:348-355 (1993).   PubMed