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J Biol Chem, Vol. 273, Issue 45, 29764-29769, November 6, 1998
From The Scripps Research Institute,
La Jolla, California 92037
The information that targets a protein to the
nucleus often consists of a short cluster of basic amino acids called a
nuclear localization sequence (NLS). Since a wide range of sequences
rich in basic amino acid residues function as NLSs, we postulated that an NLS-like sequence composed exclusively of D-amino
acids might have biological activity. We synthesized peptides
corresponding to the c-Myc NLS composed of either all L or
D-amino acids, both in the forward and reverse order. We
tested these peptides for nuclear import activity in a
digitonin-permeabilized cell assay. All four peptide-bovine serum
albumin conjugates localized to the nucleus with similar efficiency,
and each conjugate competed for import with an SV40 large T
antigen-derived NLS conjugate. Cross-linking experiments with free NLS
peptides in HeLa cytosol indicated that each peptide bound to a protein
that migrated at the molecular weight of importin A great deal of research is currently focused on the development
of small synthetic peptides that mimic the biological activity of
natural proteins. Small peptides offer a variety of advantages as
therapeutic agents. Their small size facilitates absorption compared
with large proteins (1), and they are more easily synthesized and,
thus, less expensive to develop as therapeutic agents. Examples of
successful small peptide drugs include anti-microbial agents (2),
competitive inhibitors of angiotensin converting enzyme (3) and of the
EPO receptor (4), and immunogenic agents (5, 6). With the number of
published three-dimensional protein structures increasing, rational
drug design based on molecular recognition may find its most direct
application in the development of peptide mimetics.
One of the chief obstacles to the use of therapeutic peptide mimetics
is their susceptibility to proteolysis (7). To address this problem,
some research has been directed toward the development of
D-amino acid peptide mimetics, which are resistant to
proteolytic degradation (8). This resistance stems from the highly
site-specific recognition required for protease binding (9). For
example, a synthetic human immunodeficiency virus protease composed
exclusively of D-amino acids can cleave
D-peptide substrates but not L-peptide substrates (10). Similarly, the L-amino acid version of the protease cleaves L-peptides but not D-peptides.
Unfortunately, the promise of this approach is compromised by the fact
that relatively few D-amino acid peptides possess
biological activity. Ironically, it is likely that the vice of
D-peptides arises from their virtue; although proteases
cannot recognize these peptides, neither can intended target proteins.
A promising solution to the D-peptide conundrum was
proposed by Chorev et al. (8), who suggested that
D-reverse peptides might have greater biological activity than D-peptides by more closely mimicking the side chain to
main chain orientation of L-peptides (for example, see Fig.
1B under "Results"). However, the efficacy of particular
peptides synthesized as D or D-reverses is
difficult to predict and must be determined empirically.
The nuclear protein import pathway offers an appealing model system for
analyzing the relative activities of chiral peptides. Nuclear proteins
are imported into the nucleus through aqueous channels that span the
nuclear envelope called nuclear pore complexes. Although ions and
molecules less than ~20-40 Da can diffuse passively through the
nuclear pore complexes, larger proteins are transported by saturable
pathways that are energy- and signal-dependent. The signals
that specify nuclear protein import
(NLSs)1 are commonly short
stretches of amino acids rich in basic amino acid residues, although
other classes of NLSs have been described recently (11, 12). The
initial step in the import of proteins containing basic amino acid-type
NLSs occurs in the cytosol, where the NLS-containing proteins are bound
to a receptor (variously called the NLS receptor, importin Several properties of basic amino acid-type NLSs make them particularly
suitable for chiral analysis. The minimal length required for activity
is conveniently small (6 to 10 amino acids), and they retain their
nuclear targeting function when synthesized as discrete peptides and
chemically coupled to carrier proteins such as BSA. The target
receptors are apparently forgiving, for other than a preponderance of
basic amino acids, neither the precise number nor sequence of amino
acids defines a consensus required for function. For example, in the
case of the prototypical basic amino acid-type NLS found in the SV40
large T antigen, only the second lysine in a continuous stretch of five
basic amino acids is absolutely required for function (15). The NLS
receptors for basic amino acid-type NLSs comprise a group of several
proteins containing a central core of eight degenerate armadillo-type
repeats of 42 amino acids flanked by N- and C-terminal nonrepeat
regions (16). Although their precise role is unknown, the NLS binding site appears to occur within these multiple armadillo repeats.
The fact that the receptors for basic amino acid-type NLSs can
functionally interact with a broad range of different sequences encouraged us to believe that chiral variants of this signal might be
active in nuclear import. In this study, we have examined the effect of
chirality on NLS function using an in vitro nuclear import
assay with synthetic peptide conjugates. We have found that the basic
amino acid-type NLS of the c-Myc protein is functional in both
L- and D-forward and reverse configurations.
Although the L configurations are proteolyzed in a
cytosolic extract of cells, the D configuration is
protease-resistant. This work presents results useful for functional
studies of NLS-mediated transport and therapeutic applications and
provides insight for understanding the nature of NLS ligand-receptor
binding interactions.
Preparation of Transport Substrate (FITC-BSA-NLS)--
The
peptide-BSA conjugates used as transport substrates were prepared as
described in Adam et al. (17). The activated BSA-FITC was
mixed with 1 mg of either the L-forward,
L-reverse, D-forward, or D-reverse
NLS peptide and incubated overnight at 4°C. Peptide concentrations
were determined by spectrophotometry absorbance at 205 and 214 and
normalized. The recombinant import factors used to reconstitute import
were 200 nM importin Nuclear Protein Import Assay--
Nuclear protein import in
digitonin-permeabilized HeLa cells was carried out as described by Adam
et al. (17) and Paschal and Gerace (18). HeLa cytosol was
prepared as described by Adam et al. (17). The peptide-BSA
conjugates used as transport substrates were prepared as described in
Adam et al. (17). The activated BSA-FITC was mixed with 1 mg
of either the L-forward, L-reverse, D-forward, or D-reverse NLS peptide and
incubated overnight at 4°C. Peptide concentrations were determined by
spectrophotometry absorbance at 205 and 214 and added to BSA-FITC at
normalized levels.
Peptide Synthesis--
Synthesis of peptides was carried out
manually by established solid-phase synthesis protocol (19) on either
(4-(hydroxymethyl) phenyl)-methyl or
t-butoxycarbonyl-S-aminoacyl (20) resins. Coupling was monitored by quantitative ninhydrin assay (21). Upon
completion the final peptides were deprotected, cleaved, and purified
by standard procedures (21).
SDS Gel Electrophoresis and Peptide Cross-linking--
Chemical
cross-linking of NLS peptides to importin was carried out as described
by Adam et al. (14), resolved by SDS-polyacrylamide gel
electrophoresis, and monitored by autoradiography on Kodac XAR film.
Peptide Stability in Cytosol--
1 mg of L-forward,
D-forward, L-reverse, and D-reverse
c-Myc NLS peptide was dissolved in 100 µl of cytosol transport buffer (20 mM HEPES, pH 7.4, 100 mM potassium acetate,
2 mM magnesium acetate. 50 µl of this solution was mixed
with 20 µl of purified HeLa cytosol, and the mixture was incubated at
37°C. Degradation of the NLS was monitored by analytical reversed
phase HPLC with a gradient of 0-67% solution B in solution A over 30 min (solution A = 100% water, 0.1% trifluoroacetic acid;
solution B = 90% acetonitrile, 10% water, 0.09% trifluoroacetic
acid). Isolated peaks were analyzed by electrospray ionization mass
spectrometry to determine their identity.
A schematic diagram depicting the chiral relationships between
L- and D-forward and reverse peptides is shown
in Fig. 1A. This figure
illustrates the close structural similarity between the
L-forward and D-reverse peptides. To
investigate whether D-reverse peptides are biologically
active as NLSs, we initially compared the nuclear import activity of a
D-reverse peptide based on the SV40 T antigen NLS with its
natural (L) counterpart (sequence shown in Fig.
1B). An L-reverse peptide based on the SV40 T
antigen NLS has previously been shown to be largely inactive. For this analysis, fluorescent BSA-peptide conjugates were prepared and analyzed
in a permeabilized cell assay for nuclear protein import. Although the
wild type SV40 NLS peptide conjugate had the expected high level of
nuclear import activity, the D-reverse peptide conjugate was essentially inactive (data not shown). We reasoned that the lack of
activity in the D-reverse peptide conjugate could be due to
the presence of a proline in the SV40 T antigen NLS. This is because
proline, unlike other amino acids, forms a covalent bond with the main
chain and is not topologically equivalent with respect to the N
terminus in the forward and reverse directions. Most documented basic
amino acid-type NLSs have a proline that closely flanks the core of
basic residues (22), presumably to contribute to some contextual
structure in the folded proteins. However, the c-Myc NLS without its
proline is known to have activity comparable with the SV40 signal (23,
24). Based upon these data, we decided to explore the chiral properties
of the c-Myc NLS.
We synthesized the c-Myc NLS sequence in all four homochiral peptide
orientations: all L-amino acids, all D-amino
acids, all L-amino acids synthesized in reverse, and all
D-amino acids synthesized in reverse (Fig. 1A).
These four peptides were coupled to FITC-conjugated BSA at an average
ratio of five peptides/BSA molecule (as estimated by a size shift on
SDS-polyacrylamide gel electrophoresis), and the conjugates (at 100 mM) were tested for their activity in the permeabilized
cell import assay. To our surprise, all four c-Myc peptide-BSA
conjugates were transported into the nucleus at similar levels as seen
by fluorescence microscopy (Fig.
2A). The transport of these
peptide conjugates occurred at a level comparable with a BSA conjugate
containing the SV40 T antigen NLS coupled to BSA at a similar level
(Fig. 2A). Quantitative analysis of the nuclear import of
the c-Myc conjugates by flow cytometry (18) confirmed that all four
conjugates were virtually identical in their transport activity and
equivalent in activity to the SV40 T antigen conjugate (Fig.
2B). Control (not shown) experiments demonstrated that the nuclei of the permeabilized cells were almost all intact, as shown by
their ability to exclude trypan blue (which is too large to diffuse
into intact nuclei), and thus, the conjugates are unable to enter the
nucleus by a nonphysiological diffusion route. Furthermore, nuclear
accumulation of all four c-Myc conjugates was blocked by well
characterized inhibitors of NLS-mediated nuclear import, including
incubation at 0 °C (L- and D-reverse
conjugates shown in Fig. 2B; others not shown), depletion of
ATP with hexokinase/glucose, and addition of GTP
All Four Homochiral Enantiomers of a Nuclear Localization
Sequence Derived from c-Myc Serve as Functional Import Signals*
,, and
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
. Recombinant
importin , importin 
, Ran, and NTF2 alone were sufficient to
support the import of both L-form and D-form
conjugates in permeabilized cells. This indicates that both
D- and L-form NLS peptides use the same import machinery. Although the free D-forms of the NLS were
proteolytically resistant in cytosol, the L-forms were
rapidly degraded. To our knowledge, this is the first example of an
intracellular pathway in which the receptor is insensitive to the
chirality of the ligand.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, and
karyopherin (13). The substrate-receptor complex then associates
with the cytoplasmic face of the nuclear pore complexes, and with the
participation of other cytosolic factors, is transported through a
gated channel in the nuclear pore complexes to the nuclear interior.
The in vivo events of NLS-mediated nuclear import can be
duplicated in an in vitro system using
digitonin-permeabilized cells supplemented with cytosolic extracts and
ATP (14). Transport in this in vitro assay is blocked by the
same inhibitors that block in vivo import, is rapid, and is
easily quantified.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, 200 nM importin ,
200 nM NTF2, and 1 µM Ran with 1% BSA in
transport buffer.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (20K):
[in a new window]
Fig. 1.
Schematic representation of the four possible side
chain to main chain configurations (A). Each side chain of
the purely hypothetical peptide is numbered to denote a different
residue, so that one can compare the orientation with respect to the N
terminus. The peptides in this study are comprised of all
L-amino acids (L-forward) or all
D-amino acids (D-forward) or all
D-amino acids synthesized in reverse
(D-r, reverse) or all L-amino acids
synthesized in reverse order (L-r,
reverse). Note that the D and L
peptides are mirror images. More interestingly, note the similarity
between the L peptide and the D-reverse
peptide. B, the primary structure of each NLS peptide and
that of SV40 L-forward.
S to the medium
(data not shown). Together, these results indicate that the nuclear
import of the c-Myc conjugates occurs by a physiological pathway
(13).
View larger version (35K):
[in a new window]
Fig. 2.
All four chiral variants of the c-Myc peptide
serve as functional nuclear localization sequences. Accumulation
of nuclear fluorescence in the permeabilized cell protein import assay
in the presence of cytosol (A) or with recombinant factors
alone (B). C, quantitative comparison of absolute
import levels as measured by fluorescence-activated cell sorter
analysis. 4° controls with either SV40 NLS conjugate or c-Myc NLS
conjugate represent the background fluorescence.
Dr and Lr, D
and L reverse.
We performed competition experiments to investigate whether these
substrates enter the nucleus by the same pathway as the SV40 T antigen
NLS. For this experiment, import assays with fluorescently labeled BSA
coupled with the SV40 T antigen NLS were carried out in the presence of
increasing concentrations of unlabeled c-Myc peptide-BSA conjugates. We
chose to use BSA-NLS conjugates rather than free peptides because they
are more efficient competitors (25), perhaps due to an increased
avidity for receptor or because tethering of the NLS peptide at one end
constrains its conformations to those more appropriate for receptor
recognition. Although uncoupled BSA alone had little effect on import,
each of the four chiral c-Myc-BSA conjugates competed with the import
of the SV40 T antigen NLS conjugate, as seen both by fluorescence
staining (Fig. 3A) and by flow
cytometry (Fig. 3B). The level of import competition with
various unlabeled c-Myc peptide conjugates was similar to the
competition obtained with an SV40 T antigen NLS peptide conjugate. For
example, in a separate experiment from that shown in Fig. 3, the SV40 T
antigen and the c-Myc L peptide conjugates, both, gave
approximately 50% competition when present at 20 mM
concentration. We reproducibly observed that the L-reverse
version of the c-Myc NLS was a somewhat less effective competitor of T
antigen NLS-mediated nuclear import than the other three chiral
versions (Fig. 3B). Note that the weaker activity of the
L-reverse signal was not evident by fluorescence staining
(Fig. 3A), which is included only to show a gross
correspondence to the more sensitive fluorescence-activated cell sorter
assay. To directly examine whether the transport competition by the
c-Myc NLS peptide conjugates was due to binding to the NLS receptor, we
carried out peptide cross-linking and competition experiments to
measure the NLS receptor interaction of these peptides. Free
unconjugated 125I-labeled SV40 T antigen NLS peptide was
incubated with a cytosolic fraction enriched in the NLS
receptor2 in the presence of
a 200-fold excess of each of the four unlabeled c-Myc NLS peptides
(Fig. 4), followed by cross-linking of
the radiolabeled peptide to bound protein with
2,2-dimethyl-2-silapentanesulfonic acid and analysis of the products by
SDS-polyacrylamide gel electrophoresis. As shown previously (14), we
found that the SV40 T antigen NLS peptide was specifically cross-linked
to the ~55-kDa NLS receptor in this assay (Fig. 4, lane 1)
and was strongly competed by the homologous unlabeled NLS peptide (data
not shown). Each of the free c-Myc peptides also competed with
radiolabeled SV40 T antigen NLS for cross-linking to the NLS receptor
(Fig. 4, lanes 2-3 and 5-6), although the
L-reverse c-Myc peptide was somewhat less effective than
the other three chiral versions (Fig. 4). To verify that the c-Myc NLS
peptide conjugates utilizing the known basic-type NLS receptor pathway,
all four conjugates were incubated with permeabilized cells
supplemented solely with the recombinant import factors importin ,
importin , NTF2, and Ran. These factors alone were sufficient to
reconstitute the import of the c-Myc NLS peptide conjugates in
vitro (Fig. 2B).
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To investigate the extent to which the D-forward and D-reverse peptides were proteolytically resistant in cytosol as compared with the L-peptides, we incubated each chiral variant of the c-Myc NLS as a free peptide in cytosol in the absence of protease inhibitors for varying times and analyzed the mixture by high performance liquid chromatography fractionation (Fig. 5). The peak corresponding to the L-peptide was degraded to a considerable extent by 15 min and was completely degraded at 4 h. By contrast, intact D-peptide was present at undiminished levels up to the longest time point tested (20 h). The high performance liquid chromatography fractions shown were taken at 1 min and 4 h for both the L-forward and D-reverse c-Myc. The later eluting L-forward peak is due to peptide dimer formation by a disulfide bond. This confirms that free L-forward peptides are quite susceptible to proteolytic degradation in cell cytosol. It also demonstrates that the D-peptide analogues of the c-Myc NLS would be useful as proteolytically resistant tools for therapeutic applications and for studies involving analysis of NLS function.
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DISCUSSION |
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Discussion
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Our data indicate that all four chiral variants of the c-Myc NLS
are equally active as nuclear import directing signals when analyzed as
BSA-peptide conjugates in a permeabilized cell nuclear import assay.
Furthermore, all four peptide-BSA conjugates compete for the import
pathway used by the SV40 T antigen NLS in vitro as shown by
substrate competition experiments, and all four variants specifically
bind to the same NLS receptor as demonstrated by cross-linking
competition. The observation that recombinant import factors alone are
sufficient to direct the import of all four chiral conjugates in
permeabilized cells supports the notion that these chiral NLS peptides
interact with the same import machinery utilized by the SV40 NLS. The
fact the importin supported import of these conjugates, combined
with our cross-linking data, strongly suggest that importin is the
receptor responsible for the observed import of the four chiral
conjugates. Since the D-reverse and D-forward
forms of the c-Myc NLS are functionally active (like the
L-forward peptide) but are proteolytically resistant
(unlike the L-peptide), these D-form NLSs will
provide effective tools for analyzing the pathway of nuclear import
in vivo and in vitro, where they could serve as
stable competitive inhibitors. This is especially significant given the
marked sensitivity to degradation we observed for the
L-forward peptides. The D-form NLSs also could prove useful for gene delivery applications (26).
Although the L-reverse peptide conjugate of the c-Myc NLS was as active as the other three chiral forms in nuclear import, this peptide was somewhat less active than the other three chiral peptides in competing with the SV40 T antigen NLS peptide conjugate for import and for cross-linking to the NLS receptor. It must be emphasized that each BSA molecule (on average) contained five NLS peptides, which were coupled to a heterogeneous range of sites that could influence the affinity of the immobilized NLS peptide for the NLS receptor (25). The rate of import could be determined by the highest affinity NLS site on a particular BSA molecule, whereas competition for import and peptide cross-linking would reflect an average affinity over the whole NLS population. It must be acknowledged that the diminished activity of the free L-reverse peptide during cross-linking could also be attributed to a lower resistance to proteolytic degradation.
Although there is no strict consensus sequence for basic-type NLS signals, there appear to be certain requirements that govern their activity. Previous work together with our own suggests that in addition to the need for basic amino acids, subtle structural requirements for activity must also be satisfied. In the case of the SV40 NLS, the presence of a Lys at position 128 is absolutely required for activity (15). An extensive study probing the functional requirements of this signal using conservative substitutions at position 128 revealed that a single D-Lys placed within the context of an otherwise L-NLS significantly reduced activity (27). In light of our own observation that an NLS peptide made entirely of D-amino acids is active, the disruptive effect of the above substitution suggests that the relative position of the charged side chains is important for activity and suggests that certain amino acid side chains must share the same relative orientation with respect to the main chain as their neighbors. Evidently, the activity of a NLS peptide depends less on absolute chirality than on the need for chiral uniformity.
Our study also suggests, in agreement with other work, that topology,
rather than simply positive charge, plays a role in recognition of NLS
signals. The widely reported inactivity of the reverse SV40 NLS and our
observed inactivity of the D-reverse SV40 NLS suggests that
the presence of a structurally incongruous proline overrides simple
charge interactions. Supporting this notion is the observation that
even the position of the proline in an all L-forward NLS
influences activity (28). Furthermore, we observed that a
D-reverse SV40 NLS peptide with an alanine in place of the
proline is an active NLS
signal.3 Brady and Dodson
(29) suggest that prolines, in particular, may present a major obstacle
to the widespread application of D-reverse technology.
Unlike all the other natural amino acids, the proline side chain forms
a covalent bond with the main chain. The resulting structure is not
topologically equivalent in the forward and reverse directions.
Anchoring the side chain to the main chain restricts the 
angle to
60°, which favors the formation of turns, and for this reason,
proline commonly occupies the i + 1 position in turns. The presence
of a proline may be particularly significant to the activity of NLS
signals, since almost all identified basic-type NLS signals possess a
flanking proline. Indeed, Chou Fasman predictions of 17 known nuclear
and nucleolar sequences reveal a propensity to form turns (30).
Crystallographic evidence that NLS sequences form turns via prolines
can be seen in the 3-dimensional crystal structure of the human
immunodeficiency virus matrix protein, where a proline plays a critical
role in the formation of a loop structure that projects the NLS
sequence above the plane of a surrounding strand (31). Although
free NLS peptides probably lack secondary structure, the restricted conformation of proline may impart some structural character to these
peptides that influences receptor binding.
A rather general question arises from our study. How could the NLS
receptor-mediated pathway recognize D-form peptides when they do not arise commonly in the intracellular environment? We suggest
that the observed chiral promiscuity is a consequence of the fact that
hundreds of different nuclear proteins must be accommodated by the NLS
receptor-mediated import pathway. The NLS is usually located in an
internal portion of the primary structure and must function without
impinging upon the folding and functional requirements of the specific
protein in which it resides. The compromise between the need for an
easily recognized signal, and the need for that signal to be an
integral part of structurally unrelated nuclear proteins necessitates
the lack of consensus seen in NLS sequences. But this signal diversity
presents a problem for receptor recognition. Since it would be too
energetically expensive to produce a receptor that is tailor-made for
each nuclear protein, we suggest that the signals are accommodated by a
small group of fairly permissive receptors. Our study shows that this permissiveness extends to include signals whose chirality is entirely opposite to that of naturally occurring NLSs. By limiting signal recognition to a combination of charge and side chain topology, a few
types of receptor could accommodate the plethora of structurally diverse proteins that must cross the nuclear envelope. Other
intracellular signaling pathways that must process diverse proteins
also exploit the use of signals with very loose consensus sequences,
such as mitochondrial import (32), endoplasmic reticulum translocation (33), and lysozomal import pathways (34). Our study suggests that
D-forward and D-reverse peptides may also be
functional in these pathways, although these alternate chiral versions
have yet to be tested. The structure of the NLS receptor
co-crystallized with these chiral peptides should help to reconcile the
apparent paradox of a structurally discerning, yet chirally promiscuous system.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stephen Kent for critical discussions and the technical support of his lab, in which the peptides were synthesized, Dr. Frauke Melchior, Dr. Bryce Paschal, and Dr. Tom Muir for their intellectual input, and Dr. Susie Lyman for her critical reading of the manuscript.
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FOOTNOTES |
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* Supported by National Institutes of Health Grant GM41955 (to L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Equal contributors.
§ To whom correspondence should be addressed: Depts. of Cell and Molecular Biology, 10550 N. Torrey Pines Rd./IMM 10, La Jolla, CA 92037. Tel.: 619 784 8514; Fax: 619 784 9132; E-mail: lgerace{at}scripps.edu.
The abbreviations used are:
NLS, nuclear
localization sequence; BSA, bovine serum albumin; FITC, fluorescein
isothiocyanate; GTPS, guanosine
5'-3-O-(thio)triphosphate.
2 S. Adam, personal communication.
3 A. C. S. Saphire, S. J. Bark, and L. Gerace, unpublished data.
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