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.
- 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).
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).
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).
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).
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).
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).
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).
Howitt J., Anderson C.W. and FreimuthP.
Adenovirus interaction with its cellular receptor CAR.
Curr. Top. Microbiol. Immunol., 272:331-364 (2003).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
A human cell line selected for resistance to adenovirus infection has reduced
levels of the virus receptor.
J. Virol., 70:4081-4085 (1996).
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).
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).
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).
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).