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news & views
© 1999 Nature America Inc. • http://genetics.nature.com
different (adjacent) genes, in contrast with
Neurospora crassa, where a single gene
encodes a product with both activites6.
Last year Peer Bork and colleagues published a report7 showing that interacting
proteins could be identified simply on the
basis of gene order, at least in Bacteria and
Archaea. They searched genomes for evidence of conserved gene order as a clue to
interaction, identifying 301 proteins in
the process, of which more than threequarters were found to have been
reported to interact by direct experiment.
They suggested that the temporal coordination of synthesis is advantageous
for protein-protein interactions, and critical to interdependent co-translational
folding8. Ultimately, the new findings of
Marcotte et al. are quite similar to those
of Bork and colleagues, although in the
latter case, the genes are adjacent instead
of fused. Clearly the thermodynamic
advantage of the tether cannot be the
only issue here. Perhaps the explanation
has to do with differences in bacterial and
eukaryotic assembly processes9, although
some ‘Rosetta Stones’ are provided by
bacterial genomes.
One is left wondering why some genes
sit next to each other in some bacteria but
not in others. Even the gyrase A and B
genes, while widely separated in E. coli,
are adjacent in many other bacteria.
Clearly, genomes are in constant foment,
and gene order is continually reshuffled11.
The evolutionary advantages of recombination are widely accepted. So there may
be two opposing forces at play here: one
that shuffles the genome, and another,
that preserves gene associations for functional reasons.
This is not to say that these new
genomic approaches aren’t both useful
and exciting. It seems certain that they
will generate valuable data. The fact that
all the data presented by Marcotte et al.
are freely available on the internet will
probably set off a rush of activity (their
web site at www.doe-mbi.ucla.edu has
already hosted more than 1,500 visits).
Where to look first? I think that most of
the significant connections involving
known genes will turn out to be of the
sort where the functional relationships
were already apparent from ordinary
homology searches—with occasional
help from gene-order considerations. The
most useful ‘connections’ will be those
where the function of only one of the separated components (equivalent to half of
the fused ‘pair’) is known; in these cases a
genuine new finding—a clue to the function of a previously ‘anonymous’ gene—is
possible (Fig. 2). Even if no functions
have been ascribed to either component,
the very existence of the fused gene product implies some kind of connection
between two separated genes in the
organism under scrutiny, which should
get at least some biologists grooving. 1.
2.
3.
Doolittle, R.F. Nature 392, 339–342 (1998).
Marcotte, E.M. et al. Science 285, 751–753 (1999).
Corpet, F., Gouzy, J. & Kahn, D. Nucleic Acids Res.
26, 323–326 (1998).
4. Pellegrini, M., Marcotte, E.M., Thompson, M.J.,
Eisenberg, D. & Yeates, T.O. Proc. Natl Acad. Sci.
USA 96, 4285–4288 (1999).
5. Gaertner, F.H., Ericson, M.C. & DeMoss, J.A. J. Biol.
Chem. 245, 595–600 (1970).
6. DeMoss, J. Biochem. Biophys. Res. Comm. 18,
850–857 (1965).
7. Dandekar, T., Snel, B., Huynen, M. & Bork, P. Trends
Biochem. Sci. 23, 324–328 (1998).
8. Thanaraj, T.A. & Argos, P. Protein. Sci. 5, 1594–1612
(1996).
9. Netzer, W.J. & Hartl, F.U. Nature 388, 343–349
(1997).
10. Lawrence, J.G. & Roth, J.R. Genetics 143, 1843–1860
(1996).
11. Siefert, J.L. et al. J. Mol. Evol. 45, 467–472 (1997).
Baby, don’t stop!
Alexander S. Mankin1 & Susan W. Liebman2
1Center for Pharmaceutical Biotechnology and 2Department of Biological Sciences, University of Illinois at Chicago,
900 South Ashland Avenue, Chicago, Illinois 60607, USA. e-mail: [email protected] and [email protected]
A large number of human genetic diseases
result from mutations that cause the premature termination of the synthesis of the
protein encoded by the mutant gene1. A
study by Elisabeth Barton-Davis and colleagues2, exploring antibiotic effect on a
mouse model of Duchenne muscular dystrophy (DMD) and published in a recent
issue of the Journal of Clinical Investigation, suggests that gentamicin may provide a readily accessible treatment for
disease caused by mutant stop codons.
The stop (nonsense) codons UAA, UAG
and UGA signal the termination of protein synthesis. While the anticodons of
aminoacyl transfer RNAs (tRNAs) recognize sense codons, leading to the incorporation of a specific amino acid, there are
normally no tRNAs with anticodons that
precisely match any of the three nonsense
codons. Rather, these codons are recognized by proteins that promote the release
of the completed polypeptide chain
8
(Fig. 1a). When a nonsense codon in a
structural gene results from mutation, the
protein product is incomplete (or truncated; Fig. 1b) and the messenger RNA
(mRNA) often rapidly degraded3.
Start making sense
The synthesis of complete protein from
such a mutant gene can be partially
restored by unlinked mutations that
affect the translational apparatus. For
example, tRNAs that have been mutated
so that their anticodon can recognize a
stop codon will bind to the stop codon in
competition with release factors, thereby
preventing premature chain termination
some fraction of the time. Alternatively,
aminoglycoside antibiotics allow normal
tRNAs to recognize ‘incorrect’ codons,
including stop codons4 (Fig. 1c). Aminoglycosides interact with the highly conserved decoding centre of the ribosomal
RNA (rRNA). This centre normally fac-
ilitates accurate codon-anticodon pairing, but when bound with a drug, RNA
conformation is altered and accuracy
reduced. Depending upon the dose, these
drugs may inhibit protein synthesis.
An ideal treatment of genetic disease
would be to replace or supplement the
mutant gene with a wild-type copy. An
alternative approach to treating human
genetic diseases caused by premature stop
codons would be to engineer mutant
human tRNA genes capable of decoding
stop codons5. A less ideal, but accessible
treatment is the use of aminoglycosides6,
as suggested in 1985 by Burke and Mogg.
Using cultured mammalian cells, they
showed that the aminoglycoside antibiotics paromomycin and G-418 could
partially restore the synthesis of a full-size
protein from a mutant gene with a premature UAG mutation. Later, G-418 and gentamicin (another aminoglycoside) were
shown to restore the expression of the cysnature genetics • volume 23 • september 1999
© 1999 Nature America Inc. • http://genetics.nature.com
wild type
a
start codon
AUG
mRNA
CAA
b
stop codon
UAG
AUG
mutant
premature
stop codon
UAA
c
UAG
news & views
mutant in the presence
of gentamycin
AUG
UAA
UAG
protein
product
dystrophin
nascent
peptide
aminoacyl
tRNA
glutamine
RF
tRNA
© 1999 Nature America Inc. • http://genetics.nature.com
ribosome
RF
CAA
UAA
UAA
dystrophin
mRNA
gentamicin
Trumping termination. a, Normal dystrophin mRNA encodes the complete dystrophin protein. The ‘correct’ tRNA binds to the dystrophin CAA sense codon.
b, A mutant mRNA with a premature stop codon and the truncated polypeptide it encodes. Release factor (RF) proteins bind to the premature UAA stop codon
within the mutant dystrophin mRNA. c, Gentamicin occasionally allows the incorporation of an amino acid at the internal stop codon of mutant dystrophin
mRNA. The truncated protein is still made, but in some cases a full-length protein results from read-through and has an amino acid substitution at the site corresponding to the premature stop codon.
tic fibrosis transmembrane conductance
regulator (CFTR) protein in a cell line carrying a nonsense mutation in CFTR
(refs 7,8). As gentamicin is currently used
to treat bacterial infections in cystic fibrosis (CF) patients and about 10% of all CF
patients carry a nonsense codon in CFTR,
it should be possible to determine the clinical efficacy of gentamicin in suppressing
CFTR nonsense mutations.
Going live
The work of Barton-Davis et al.2 represents an important step towards the use of
aminoglycosides in the treatment of
genetic diseases; it brings drug-mediated
bypass of premature stop codons from cell
culture to the organismal level. DMD is
caused by inactivation of the human dystrophin gene—which is effected by a premature stop codon in 5–15% of DMD
patients. The mdx mouse, an animal
model of DMD, carries a mutant UAA
stop codon in place of a glutamine CAA
codon in its dystrophin gene. BartonDavis and colleagues found that injecting
mdx mice with high doses of gentamicin
for two weeks restored the amount of dystrophin in muscles to up to 20% of the
normal level. Furthermore, post-mortem
examination of these mice following the
treatment period revealed a decrease in
some symptoms of the disease.
nature genetics • volume 23 • september 1999
Most aminoglycosides are highly active
against bacterial ribosomes, but do not
affect the cytoplasmic ribosomes in
human cells. The sensitivity of eukaryotic
ribosomes to some aminoglycosides, such
as gentamicin, G-418, paromomycin,
hygromycin and a few others, has been
viewed as an unwanted side effect associated with these antibacterial drugs. But
it is precisely that ‘side’ effect that opens
the possibility of using these drugs for the
treatment of human genetic diseases. So
far, the clinical application of aminoglycosides has been limited to their use as
antibacterials; no effort has been made to
optimize their ability to cause translation
errors in eukaryotic cells. This leaves hope
that their capacity to ‘bypass’ stop-codons
could be improved by screening combinatorial chemical libraries or rational drug
design—especially as the structures of
several aminoglycosides complexed with
their target sites have been solved9.
At the moment, aminoglycosides are
the only drugs known to affect translational fidelity in eukaryotes. However,
alterations in several different rRNA
regions (both in small and large ribosomal subunits), ribosomal proteins and
translation factors can affect accuracy of
translation. So, in addition to the decoding centre, other ribosomal sites and
translation-elongation and termination
factors could be viewed as potential targets for drugs that would allow the cell to
synthesize a full-length protein from a
gene with a mutant stop codon.
On the downside, such approaches are
expected to be effective only against a small
fraction of deleterious mutations. The
effects of some genetic alterations, such as
deletions, will not be ameliorated by drugs
affecting translational accuracy. It is also
not clear if any missense or frameshift
mutants could be suppressed, but this
remains a possibility. Furthermore, not all
nonsense mutations are likely to be countered by this approach. This is because
competition between release factors and
aminoacyl tRNA depends on the composition of sequence flanking the stop codon10.
In model studies with eukaryotic cells,
bypass of stop codons only within specific
‘contexts’ could be stimulated by aminoglycoside antibiotics11,12. Other factors
may further reduce the spectrum of genetic
diseases amenable to aminoglycoside treatment. The activity of the protein containing an altered amino acid in place of the
premature stop, the amount of the protein
required to carry out its cellular function
and its stability will clearly impose additional limitations. Finally, the increased
frequency of translation errors will result
in the production of aberrant proteins,
which, together with the other known side
9
news & views
© 1999 Nature America Inc. • http://genetics.nature.com
effects of gentamicin (and yet unknown
consequences of the long-term use of the
drug), will make it tricky to find effective
treatment regimens. Nevertheless, as there
is no effective treatment for most genetic
disorders, this readily accessible approach
is exciting.
More than ten years elapsed before the
idea of treating genetic diseases with
aminoglycosides6 was tested in an animal
model2. Hopefully, it will take less time to
extrapolate this approach to the point
where it can actually help patients.
1.
2.
3.
4.
5.
Atkinson, J. & Martin, R. Nucleic Acids Res. 22,
1327–1334 (1994).
Barton-Davis, E.R., Cordier, L., Shoturma, D.I.,
Leland, S.E. & Sweeney, H.L. J. Clin. Invest. 104,
375–381 (1999).
Culbertson, M.R. Trends Genet. 15, 74–80 (1999).
Davies, J., Gilbert, W. & Gorini, L. Proc. Natl Acad.
Sci. USA 51, 883–890 (1964).
Temple, G.F., Dozy, A.M., Roy, K.L. & Kan, Y.W.
Nature 296, 537–540 (1982).
Burke, J.F. & Mogg, A.E. Nucleic Acids Res. 13,
6265–6272 (1985).
7. Bedwell, D.M. et al. Nature Med. 3, 1280–1284
(1997).
8. Howard, M., Frizzell, R.A. & Bedwell, D.M. Nature
Med. 2, 467–469 (1996).
9. Fourmy, D., Recht, M.I., Blanchard, S.C. & Puglisi,
J.D. Science 274, 1367–1371 (1996).
10. Mottagui-Tabar, S., Tuite, M.F. & Isaksson, L.A. Eur. J.
Biochem. 257, 249–254 (1998).
11. Singh, A., Ursic, D. & Davies, J. Nature 277, 146–148
(1979).
12. Palmer, E., Wilhelm, J.M. & Sherman, F. Nature 277,
148–150 (1979).
6.
© 1999 Nature America Inc. • http://genetics.nature.com
gadzooks!
Marcy E. MacDonald
Molecular Neurogenetics Unit, Massachusetts General Hospital, 13th Street, Charlestown, Massachusetts 02129, USA.
e-mail: [email protected]
The ubiquitin-proteasome pathway of
protein degradation is essential for
eukaryotes and involves cellular machinery as elaborate as that required by protein
synthesis. Critical molecular steps, however, are emerging from the discovery of
mutations that cause problems ranging
from cancer to hypertension to fat facets1.
On page 47 of this issue, Kazumasa Saigoh
and colleagues2 broaden the spectrum by
discovering that a mutation in the ubi-
Ub
quitin carboxy-terminal hydrolase gene
(Uchl1) causes gracile axonal dystrophy
(gad) in mice.
The gad mouse is a simple genetic
model with features reminiscent of inherited neurodegenerative disease in human3.
It develops subtle sensory ataxia, progressing to motor ataxia. Later, axon terminals
of the dorsal root ganglia degenerate, followed by neurons that comprise the
gracile nucleus of the medulla oblongata
Ub Ub
1
poly-ubiquitin
Ub
ubiquitin
monomer
2
E1
3
E2
The ubiquitin-proteasome system.
A stepwise model of the ubiquitin-proteasome pathway1 depicting (step 1)
release of ubiquitin from pro-ubiquitin
during biosynthesis (steps 2–5) conjugation of ubiquitin to (a) substrate(s) and
(steps 6,7) degradation of the polyubiquitinated-substrate by the 26S proteasome
complex
(19S,
20S,
19S).
Conjugation involves activation of ubiquitin by ubiquitin-activating enzyme E1
(step 2), transfer of activated-ubiquitin
to a specific E2 ubiquitin-conjugating
enzyme (UBC; step 3), transfer of activated ubiquitin from E2 to a substratespecific E3 ubiquitin-ligase (step 4) and
formation of a substrate-E3 complex and
biosynthesis of the polyubiquitin chain
(step 5). Degradation includes binding of
the polyubiquitinated-substrate to the
19S complex (step 6), proteolysis to short
peptides by the 20S complex, and release
of recycled ubiquitin (step 7) from ‘end’
proteolytic products.
10
4
E3
E1
Ub
E2
Ub
E3
Ub
s
Ub
5
Ub
Ub
Ub
s
Ub
Ub
E3
Ub
s
19S
6
20S
7
Ub
19S
Ub
peptides
Ub
recycled
ubiquitin
region4. The brain of the gad mutant contains ‘spheroid bodies’ and dots of ubiquitin-conjugated protein in ‘dying’ terminals
and axons5, reminiscent of the often beautiful, varied dots and swirls of abnormal
ubiquitin-conjugated protein found in the
post-mortem brains of people with inherited neurodegenerative diseases. The dots
seem to hold a clue to the pathogenic
process1,6–8, but the discovery of unrelated
genes which, when mutated, cause more
than a dozen of these disorders has failed,
in each instance, to directly link disease
mechanism to defects in the cellular
machinery that degrades proteins.
The identity of the gad gene defect,
therefore, comes as a welcome surprise.
Saigoh et al. homed in on the gad locus on
chromosome 5 by following co-inheritance
of the gad phenotype with polymorphic
DNA markers in a classic genetic linkagebased cloning strategy. Their reward is an
in-frame deletion in Uchl1 that results in a
truncated form of ubiquitin hydrolase, a
thiol protease that participates in ubiquitin
biosythesis and in protein degradation.
This discovery raises new issues. As
usual, the molecular consequence of the
mutation as mode of action needs to be
delineated. A simple loss of UCH-L1 activity is the most parsimonious explanation.
But, as the authors point out, mRNA is synthesized from the mutant Uchl1 allele at
normal levels, which makes it hard to reject
a mechanism involving the predicted truncated UCH-L1 product. The larger issue,
which remains a key puzzle for cloned
genes whose mutation causes most neurodegenerative disease, is to discover why
the gad genetic defect affects neurons
nature genetics • volume 23 • september 1999