Nephrol Dial Transplant (2004) 19: 1029-1032
Nephrol Dial Transplant Vol. 19 No. 5 © ERA-EDTA 2004; all rights reserved
Editorial Comment
Rescue of protein mutants: why?
Peter Gross1 and
Torsten Schöneberg2
1Nephrologie, Universitätsklinikum C.G. Carus, Dresden and 2Abteilung für Molekulare Biochemie, Medical Faculty, University of Leipzig, Germany
Correspondence and offprint requests to: Peter Gross, MD, Nephrologie, M.K. III, Universitätsklinikum C.G. Carus, Fetscherstrasse 74, D-01307 Dresden, Germany. Email: peter.gross{at}mailbox.tu-dresden.de
Keywords: chemical chaperones; congenital diseases; nephrogenic diabetes insipidus; protein synthesis; rescued mutations
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Introduction
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Inherited renal disease can change the patient's life in many
ways and, as a rule, medicine has very little to offer. Until
recently, that is; however: ‘
tempora mutantur’ (‘
times are changing’).
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Mutations: do they really matter?
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A 23-year-old male outpatient of ours has congenital nephrogenic
diabetes insipidus (nDI). For such a young man, the ‘personal
city map’ normally would be marked by shops for certain
types of sportswear, ‘power-food’ stations, information-technology
outlets and discos. Not so in nDI. Our patient's city map is
highlighted by lots of toilets. Areas of reduced density (>25
min between two ‘pit stops’) are off-limits. Needless
to say, our prescription of thiazides barely added a minute
to his critical interval. Can you imagine the ordeal of going
to the movies or of attending school under such circumstances?
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Why are they making all these new noises?
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A recent landmark article by Morello
et al. [
1] from Montreal
says that there is hope for improvement on the horizon. In the
article they report successful experiments to ‘rescue’
mutated human collecting duct V
2 vasopressin receptor (V
2R)
protein [the cause of most congenital nDI (X chromosome-linked
nDI)] and turn it functional. In this issue of
Nephrology Dialysis Transplantation, de Jong
et al. [
2] take interest in Gitelman's
syndrome and report another fascinating experiment in kind.
So, how do they pull the rabbit out of the hat?
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At which site do mutations matter?
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Secretory and membrane proteins are synthesized in the cell
from messenger RNA (mRNA), by a process called translation (
Figure 1).
It involves close co-operation between the mRNA template
and ‘catalytic bodies’ named ribosomes. The latter
are located at the membrane of the endoplasmic reticulum (ER;
Figure 1). In this way, the elongating chain of amino acids
forming the nascent protein is forwarded into the lumen of the
ER, much like a nascent noodle drops from the noodle machine
into the boiling pot. In the next step it is paramount that
the nascent protein is protected from spontaneous aggregation
with other similar protein molecules and that it assumes its
correct length and configuration rapidly. Remember the noodle:
can you imagine what would happen if the noodles in the boiling
pot clumped together or, worse, if instead of becoming spaghetti
they turned into cannelloni? If you can, you may stop reading
here, because apparently you understand what all this is about.
View larger version (19K):
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Fig. 1. Fate of most mutated proteins in the ER. In the upper part of the diagram, an mRNA chain is seen being translated into nascent protein by a ribosome in the membrane of the ER. In the case of a mutated protein (right side of the diagram), misfolding ensues together with prolonged binding of the mutant to a selection of some, but not all, physiological chaperones. This is eventually followed by degradation in the cytoplasm, even though the functional site of the protein is usually intact throughout. (Redrawn from Science 2000; 287: 816.)
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Protection from aggregation, trimming of oligosaccharide side-chains
and folding into the proper conformation are all achieved within
the ER by a series of enzymes and ‘molecular chaperones’
working on the nascent protein in a multi-step process (
Figure 1).
It resembles the fabrication of an industrial product along
an assembly line. The names of some of these chaperones are
grp78, grp170, grp94, ERp72, grp58, calreticulin, calnexin,
FKBp-13, etc. (simply to give you some terms and cheer you up).
The final result will be the mature protein. Only this variety
will be recognized by the ER as complete and correct and only
under these conditions is it transported out of the ER and to
its site of destination (
Figure 1). Therefore, it is easy to
see how mutations may confuse the machinery and stop production.
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What is quality control?
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Most, though not all, mutations result in a nascent protein
that is not amenable to its designated folding and trimming
machinery, as described before. The result will be a misfolded
polypeptide-chain that will remain bound to one or more of the
chaperone molecules (
Figure 1), whereas under normal circumstances
the protein dissociates rapidly from the chaperone site after
folding. Such proteins will not be transported out of the ER.
The impasse leads to clogging of the ER and an absence of mature
protein from the cell. In other ‘milder’ cases of
mutation, the polypeptide may still be processable but not exactly
in the correct way. Then, the resultant protein usually shows
lack of fidelity with the cell's master plan for it. Consequently,
it will not be recognized as fit for transport out of the ER
either. All such molecules will end up being degraded. Basically,
this process is called ‘quality control by the ER’.
An important point to note is that retention by the ER does not depend much on the location of the mutation along the polypeptide chain; the only important aspect is that a mutation is present at all. In other words, although it is quite common that a mutated protein's receptor binding site, its catalytic site or its channel-pore forming site are all normal and intact, the mutated protein is retained in the ER simply because of a mutation elsewhere, e.g. in its tail or in its structural backbone.
This realization has led researchers to assume that such proteins would often function just fine if only one could get the ER to send them onto their route of transport and to their site of destination. This assumption is increasingly demonstrated to be valid. Some examples of disturbances where they are now trying to do such tricks are cystic fibrosis, von Willebrand's disease, alpha-1-antitrypsin deficiency, congenital nDI, Gitelman's syndrome and a number of others. So, what precisely are the tricks?
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How to outsmart quality control
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It had been known since 1966 that certain chemical agents (e.g.
glycerol) could be used to stabilize the native structure of
proteins and the activity of enzymes [
3]. The observations were
explained thermodynamically by a protein's propensity to minimize
its surface contact with glycerol in a 10% glycerol/water mixture.
This would result in a stabilization of the globular structure
of the protein [
4]. In 1996, Sato
et al. [
5] were the first
to show proof of the principle
in vivo using an addition of
10% glycerol to the cell culture medium. They demonstrated an
enhanced transport of
F508 polypeptide [a mutation of the cystic
fibrosis transmembrane conductance regulator (CFTR), causing
cystic fibrosis] out of the ER and into the cell membrane where
the
F508 polypeptide was functionally active [
5]. The importance
of that study is related to the high incidence of cystic fibrosis
in the paediatric population and the fact that most cystic fibrosis
is caused by a single mutation of the CFTR, i.e.
F508. In other
disorders, such as Gitelman's syndrome, many different mutations
have been detected in various locations all over the protein.
In 1997, Brown et al. [6] took this kind of experimentation one step further. They were able to demonstrate that in addition to glycerol, other agents, such as trimethylamine N-oxide (TMAO) and deuterated water, were also able to correct folding defects in cell culture of mutants of tumour suppressor gene p53 product, of viral oncogene protein pp60 or of the ubiquitin activating enzyme E1. In their experiments they brought about a ‘wild-type’ cellular phenotype of these proteins. Bebök et al. [7] in 1998 tested if this approach would also work in human cells. They cultured nasal polyp epithelial cells from a cystic fibrosis patient homozygous for the F508 mutation. They treated the cells with yet another agent, dimethyl sulfoxide (DMSO) 2%, for 4 days. They were then able to show an increase of F508 CFTR in the plasma membrane, which was also functionally active. Verkman and co-workers [8] took interest in renal disturbances and studied several mutants of aquaporin-2 (AQP2) obtained from patients with an autosomal form of nDI. Using CHO cells in culture they showed that most of their mutations had resulted in misfolded proteins that were retained in the ER. Retention was followed by rapid degradation, although it was possible to demonstrate that the misfolded proteins were fully functional as water channels. In additional experiments [9], the authors used the ‘chemical chaperones’ glycerol, TMAO or DMSO in a variety of cells (Xenopus oocytes, MDCK cells and CHO cells) to demonstrate successful redistribution of mutant AQP2 from the ER to cell membrane fractions under these conditions. Again, measurements of water permeability indicated that functional correction had been achieved.
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When will the new strategies help real patients?
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In 2000, Morello and co-workers [
1], also studying X chromosome-linked
nDI, for the first time used (more or less) ‘natural ligands’
(i.e. something that can presumably be applied in patients)
as chemical chaperones to rescue misfolded V
2R protein to the
cell surface of model cells (COS-1 cells, HEK-293 cells), where
they were functionally active. The apparent breakthrough in
their study was the successful use of selective, non-peptidic,
orally available (i.e. cell-membrane permeant) V
2R antagonists
[
1], which are now becoming generally available for the first
time [
10]. In contrast, additional work with cell-membrane impermeant
V
2R antagonist failed to mimic the previous effects. The authors
hypothesized that binding of vasopressin antagonist to partially
folded mutants of the V
2R in the ER stabilized or corrected
their configuration enough to permit release from the ER and
transport to the plasma membrane. A subsequent report [
11] has
shown this new medically relevant strategy to be generally applicable,
e.g. to mutations in other G-protein-coupled receptors as well
as mutations in the V
2R. Such an example is presented by the
paper by de Jong
et al. [
2] reporting work in Gitelman's syndrome
in this issue of
NDT.
Has this already had effects in patients? No published results have appeared and we have to wait for pilot observations in patients with congenital nDI utilizing oral V2R antagonists. Conceivably, the use of V2R antagonists, although apparently increasing V2R in the basolateral cell membrane of collecting duct principal cells of congenital nDI patients, still cannot be overly productive in decreasing the 24 h urinary volume, such as in our patient, because of the antagonistic action on the receptor much of the time. However, clearly the race is on. It is possible that agents like orally available V2R agonists currently in development will turn out to be the next step and the solution to congenital nDI.
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Conclusion
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Inherited mutations of kidney proteins usually cause major and
even disabling diseases, such as autosomal dominant polycystic
kidney disease or Bartter's syndrome. In many cases the defect
caused by the mutation is eventually attributable to misfolding
of a nascent protein inside the ER. Very often the mutated protein's
functional sites are still intact. Misfolded proteins are generally
retained by the ER and degraded. Basic science research has
recently uncovered ways to correct the misfolding by using so-called
chemical chaperones. In this way, recovery of physiological
function is often achievable. This progress is presently reaching
the doorstep of clinical nephrology.
Conflict of interest statement. None declared.
[See related article by de Jong et al. (this issue, pp. 1069–1076)]
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References
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- Morello JP, Salahpour A, Laperrière A et al. Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J Clin Invest 2000; 105: 887–895[ISI][Medline]
- de Jong JC, Willems PHGM, Goossens M. et al. Effects of chemical chaperones on partially retared NaCl cotransporter mutants associated with Gitelman's syndrone in a maise cortical collecting duct cell line. Nephol Dial Transplant 2004; 19: 1069–1076[Abstract/Free Full Text]
- Jarabak J, Seeds E, Talaly P. Reversible cold inactivation of a 17ß-hydroxysteroid dehydrogenase of human placenta: protective effect of glycerol. Biochemistry 1966; 5: 1269–1279[CrossRef]
- Gekko K, Timasheff SN. Mechanism of protein stabilization by glycerol: preferential hydration in glycerol–water mixtures. Biochemistry 1981; 20: 4667–4676[CrossRef][Medline]
- Sato S, Ward CL, Krouse ME et al. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem 1996; 271: 635–638[Abstract/Free Full Text]
- Brown CR, Hong-Brown LQ, Welch WJ. Correcting temperature-sensitive protein folding defects. J Clin Invest 1997; 99: 1432–1444[ISI][Medline]
- Bebök Z, Venglarik CJ, Pánczél Z et al. Activation of F508 CFTR in an epithelial monolayer. J Am Physiol 1998; 275: C599–C607
- Tamarappoo BK, Yang B, Verkman AS. Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus. J Biol Chem 1999; 274: 34 825–34 831
- Tamarappoo BK, Verkman AS. Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones. J Clin Invest 1998; 101: 2257–2267[ISI][Medline]
- Gerbes AL, Gülberg V, Ginès P et al. Therapy of hyponatremia in cirrhosis with a vasopressin receptor antagonist: a randomized double-blind multicenter trial. Gastroenterology 2003; 124: 933–939[CrossRef][ISI][Medline]
- Petäjä-Repo UE, Hogue M, Bhalla S et al. Ligands act as pharmacological chaperones and increase the efficiency of
opioid receptor maturation. EMBO J 2002; 21: 1628–1637[CrossRef][ISI][Medline]
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Introduction
Mutations: do they really...