Impaired Chaperone Activity of Human Heat Shock Protein Hsp27 Site-Specifically Modified with Argpyrimidine

Article abstract of DOI:10.1002/anie.201605366

Non-enzymatic posttranslational modifications (nPTMs) affect at least ～30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site-specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site-selective protein modification.

Full text of DOI:10.1002/anie.201605366

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International Edition: DOI: 10.1002/anie.201605366
German Edition: DOI: 10.1002/ange.201605366
Posttranslational Modifications
Impaired Chaperone Activity of Human Heat Shock Protein Hsp27
Site-Specifically Modified with Argpyrimidine
Maria Matveenko, Elena Cichero, Paola Fossa, and Christian F. W. Becker*
Abstract: Non-enzymatic posttranslational modifications
(nPTMs) affect at least ~ 30% of human proteins, but our
understanding of their impact on protein structure and function
is limited. Studies of nPTMs are difficult because many
modifications are not included in common chemical libraries
or protein expression systems and should be introduced site-
specifically. Herein, we probed the effect of the nPTM
argpyrimidine on the structure and function of human protein
Hsp27, which acquires argpyrimidine at residue 188 in vivo.
We developed a synthetic approach to an argpyrimidine
building block, which we then incorporated at position 188
of Hsp27 through protein semisynthesis. This modification did
not affect the protein secondary structure, but perturbed the
oligomeric assembly and impaired chaperone activity. Our
work demonstrates that protein function can be altered by
a single nPTM and opens up a new area of investigation only
accessible by methods that allow site-selective protein modifi-
cation.
catalysis, and reaction time, and thus the resulting distribution
pattern of nPTMs under native conditions is not random[7] but
determined by protein structure, microenvironment, and half-
life. On the other hand, the majority of biochemical studies of
nPTM-derivatized proteins have been conducted by incubat-
ing proteins with high concentrations of electrophiles, often
precluding selectivity observed in vivo and favoring crosslink
formation. Major differences may exist in the nature and
location of nPTMs formed in this way compared to those
found in vivo.[8] In addition, there are no effective muta-
genesis-based strategies for introducing or mimicking nPTMs,
and thus very little is known about the structural and
functional consequences of selective, biologically-relevant
nPTM formation. Therefore, new methods are required that
can introduce nPTMs in a site-directed manner and provide
homogenous modified proteins to pinpoint the biological
effect of particular nPTMs. Promising steps toward this goal
have recently been made by establishing chemical access to
several nPTMs in the context of small peptides.[9]
P
rotein structure and function are often modulated by
Here, we focused on the effect of the formation of
argpyrimidine (Apy), a nPTM that has been detected, to
varying extents, in healthy and cataractous lenses,[10] in
amyloid fibers from patients with familial amyloidotic poly-
neuropathy,[11] and in tissues from diabetic and cancer
patients.[12] Apy is a fluorescent adduct formed between
arginine and methylglyoxal (MG, Scheme 1),[13] a highly
reactive electrophile generated as an unavoidable by-product
of glycolysis and responsible for the modification of at least
~ 30% of human proteins.[14]
enzyme-controlled chemical reactions with small molecules
that result in covalent posttranslational modifications
(PTMs).[1] In addition, protein modification can proceed in
an enzyme-independent fashion through the binding of
electrophilic metabolites to nucleophilic groups in protein
side chains.[2] The ensuing non-enzymatic posttranslational
modifications (nPTMs) comprise a large and expanding group
of chemical moieties that include the so-called AGEs
(advanced glycation end products)[3] and ALEs (advanced
lipoxidation end products),[4] caused by metabolites originat-
ing from carbohydrate or lipid sources.[5] These modifications
have become prominently linked to aging, diabetes, uremia,
vascular disease, inflammatory disorders, neurodegenerative
diseases, cancer, and cataract.[3,4,5b,6] They have been recog-
nized not only as markers of pathology, but also as promoting
disease initiation and progression.[5b] nPTM formation is
subject to the general determinants of nucleophilic additions,
such as reactant concentration, steric accessibility, acid-base
Scheme 1. Modification of arginine with methylglyoxal (MG) can
produce argpyrimidine (Apy), a non-enzymatic posttranslational modi-
fication (nPTM).
[*] Dr. M. Matveenko, Prof. Dr. C. F. W. Becker
University of Vienna, Faculty of Chemistry
Institute of Biological Chemistry
Wꢀhringer Str. 38, 1090 Vienna (Austria)
E-mail: christian.becker@univie.ac.at
Apy has been co-immunoprecipitated with human small
heat shock protein Hsp27 (HspB1) in a number of diseased
human samples including diabetic heart,[15] brunescent cata-
ractous lenses,[16] hyperglycemic endothelial cells,[17] and
several cancer cells and tissues.[12b,18] Hsp27 is an ATP-
independent chaperone that is upregulated in response to
a variety of stress conditions.[19] In cancer cells, expression of
Dr. E. Cichero, Prof. Dr. P. Fossa
University of Genoa, Department of Pharmacy
Viale Benedetto XV n.3, 16132 Genoa (Italy)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under
http://dx.doi.org/10.1002/anie.201605366.
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Hsp27 is abnormally high, and the protein may participate in
oncogenesis and resistance to chemotherapy.[20] The 205-
amino acid protein has 16 arginines (Figure 1a), but while all
of these are in principle susceptible to Apy formation, only
mutation of Arg188 (to Gly) eliminated the recognition of the
protein by an anti-argpyrimidine antibody and the take-up of
14C-labeled MG.[18d]
(Cys173), which can be reverted, after the ligation, to the
native Ala173 by chemical desulfurization.[25] While the
desulfurization step is not selective and thus also reduces
the only native cysteine 137 to alanine, this Cys137Ala
conversion is functionally neutral in the context of our
characterization.[26]
For our approach to Apy-modified Hsp27, we required an
efficient, multigram-scale synthetic route that affords Apy as
a building block for standard Fmoc-based SPPS. By contrast,
the syntheses of Apy published so far are limited to providing
protecting-group-free compounds on analytical scale.[9b,13,27]
Our synthesis commenced with the construction of the
aminopyrimidine ring of Apy by subjecting readily available
guanidine sulfate to an excess of the known 1,3-diketone 2[28]
in methanesulfonic acid (Scheme 2). TBS-protection of the
Figure 1. a) Amino acid sequence of Hsp27, indicating the position
that has been observed to undergo the Apy modification in human
cancer cells (R188, magenta); the only cysteine residue (C137) is
shown in italic; the ligation site (see below) is underlined. b) Overview
of the strategy for the semisynthesis of Hsp27 bearing the Apy188
modification.
Scheme 2. Chemical synthesis of multi-gram quantities of Apy as
a building block for Fmoc-based SPPS and access to the unprotected
amino acid, Apyfree
.
Despite the biological significance of Apy formation at
position 188 of Hsp27, no functional consequences of this
modification at the molecular level have been reported to
date. The required homogeneously Apy-modified protein is
not attainable by incubation with MG, as it gives rise to
mixtures of differentially modified and cross-linked proteins.
Likewise, there are no amino acids that can mimic the unique
electronic and structural features of Apy for use in muta-
genesis. To address this limitation, we devised a robust
semisynthetic strategy to introduce Apy site-specifically that
requires the synthesis of a suitably protected Apy building
block, its incorporation into a peptide, and subsequent
ensuing 2-aminopyrimidinol 3, followed by the activation of
the amine function as the corresponding 2,2,4,6,7-pentam-
ethyldihydrobenzofuran-5-sulfonyl
(Pbf)
sulfonamide,
allowed the resulting intermediate 5 to engage in a smooth
Mitsunobu reaction[29] with the glutamate derivative 6.[30]
Gentle hydrogenolysis of the resulting benzyl ester 7 then
furnished the desired Apy building block 8. Our synthesis
allowed us to prepare over 5 g of this valuable material in five
steps with an overall yield of 37%. We then went on to obtain
pure samples of the free amino acid Apy (Apyfree, 9) for
analytical purposes through global deprotection of 8 (see
Figure S1 and the Supporting Information). The spectral and
physical data derived from this amino acid were in excellent
agreement with the literature values.[9b,13,27] In particular, the
UV/Vis spectrum of this material displays the expected
absorption maxima at 230 and 320 nm (Figure 2a), consistent
with the values reported by Nagaraj et al.[13] Likewise, the
characteristic Apy excitation and emission maxima at 320 and
385 nm, respectively,[13] are observed in the fluorescence
spectrum of our synthetic Apyfree (Figure S2).
ligation to
a recombinantly produced Hsp27 segment
(expressed protein ligation, EPL[21]; Figure 1b). EPL is
a powerful combination of recombinant protein expression
and solid phase peptide synthesis (SPPS), which, along with
chemical protein synthesis,[22] has enabled deciphering the
impact of enzymatic PTMs on a large variety of proteins.[23]
Herein, the selectively modified C-terminal peptide could be
appended to the longer segment produced recombinantly as
a protein a-thioester covering amino acids 1–172. In the
absence of a strategically useful cysteine residue for the
pivotal native chemical ligation (NCL),[24] peptide Hsp27
(174–205) was extended with an N-terminal cysteine
With the Apy building block in hand, we proceeded to
synthesize the 33-mer peptide that comprises the C-terminus
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Figure 2. Semisynthesis of Hsp27Apy. a) Fmoc-based SPPS afforded Apy-modified peptide that was characterized by analytical HPLC, ESI-MS, and
UV/Vis spectroscopy. b) Thiolysis of the expressed fusion protein Hsp27 (1–172)-Mxe GyrA intein-His6 afforded Hsp27 (1–172) a-thioester.
c) NCL of the two segments followed by radical desulfurization provided Hsp27Apy that was characterized by analytical HPLC and ESI-MS: i) 6m
Gdn·HCl, 0.2m NaPi, 250 mm MESNA, 50 mm TCEP, pH 7.8; ii) 6m Gdn·HCl, 0.2m NaPi, 200 mm TCEP, 16 mm V-50, pH 6.7.
of Hsp27 including Apy188 and an N-terminal cysteine
residue (Apy-modified peptide; Figure 2a). As anticipated,
automated Fmoc-SPPS using the building block 8 proceeded
readily to afford sufficient quantities (~ 20 mg) of the required
peptide in a purity of > 95% and in 6% yield (based on the
crude material) after HPLC. Notably, global deprotection and
cleavage of the peptide from the resin went smoothly and did
not require additional steps or unconventional reagents. The
corresponding peptide without the Apy modification (non-
modified peptide) was prepared as well in a similar purity and
yield (> 95% and 10%, respectively; Figure S4). Fluores-
cence properties of Apy were retained after its incorporation
into the peptide with an absorption and emission maxima at
320 and 385 nm, respectively (Figure S5), along with the
expected absorptions in the UV region around 230 and
320 nm (Figure 2a).
In parallel experiments, protein a-thioester encompassing
the bulk of Hsp27 was generated (Figure 2b). Hsp27 (1–172)
segment was expressed in E. coli as an N-terminal fusion with
Mxe GyrA intein and a polyhistidine purification tag, and this
protein was then harvested within inclusion bodies. Solubi-
lized proteins were subjected to nickel affinity chromatog-
raphy to obtain the required fusion protein accompanied by
traces of prematurely excised intein-His6-containing segments
(Figure 2b, SDS-PAGE analysis, lane 1). Thiolysis of this
material using an excess of sodium 2-mercaptoethanesulfo-
nate (MESNA; Figure 2b, SDS-PAGE analysis, lane 2),
followed by HPLC purification, afforded sufficient amounts
of the protein a-thioester (3.3 mgliterÀ1 of E. coli culture) in
high purity. The only inhomogeneity here was due to the
inconsequential partial removal of the N-terminal methionine
(DMW 131 Da), as often observed for proteins overexpressed
in bacteria[31] (Figure S7).
With the ligation partners in hand, we focused on the
assembly of full-length Hsp27 by the key NCL reaction
(Figure 2c). Submitting Hsp27 (1–172) a-thioester to a 2-fold
excess of the Apy-modified or non-modified synthetic peptide
under denaturing and reducing conditions (6m Gdn·HCl,
50 mm tris(2-carboxyethyl)phosphine (TCEP)) in the pres-
ence of MESNA as a mediator afforded the desired ligation
products after 48 h in high conversion (Figures 2c, S8). The
subsequent in situ radical desulfurization,[32] mediated by
additional TCEP as the thiyl radical acceptor, MESNA as the
hydride source and the water-soluble radical initiator V-
50,[25b,33] proceeded smoothly in 16 h (Figure S9) to afford,
after HPLC purification including separation of traces of
hydrolyzed a-thioester, the required semisynthetic Hsp27
variants in milligram amounts. These two proteins featured
the native Ala173 at the ligation site as well as the Cys137Ala
conversion, and differed only in residue 188 being Apy
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(Hsp27Apy) or Arg (Hsp27NM). The compounds were obtained
in high purity (Figures 2c, S10, S11) and with isolated yields of
~ 45% over two steps. Successful folding by dilution with
appropriate buffer was indicated by circular dichroism (CD)
(Figure 3a and see below). The fluorescent properties of Apy
allowed us to directly observe its incorporation by the
characteristic absorption and emission maxima at 320 nm
and 380 nm, respectively (Figure 3b).
Next, we examined the degree of oligomerization of the
semisynthetic proteins using size exclusion chromatography
(SEC). Hsp27WT can exist as a multimer of 12 to 40 subunits,
and the quaternary structure of the protein greatly influences
its activity.[19] The elution profile of semisynthetic Hsp27NM is
dominated by a sharp peak at the void volume of the column
(~ 8 mL, Figure 3c), indicating the formation of very large
oligomers with an apparent molecular weight of more than
1,000 kDa. As expected, the Hsp27D analogue behaved
identically (Figure S15b). On the other hand, Hsp27Apy
eluted predominantly as oligomers of ~ 600 kDa (peak at
~ 9 min, Figure 3c) corresponding to a complex of ~ 26
monomers, accompanied by a decreased peak at 8 mL at
the same protein concentration. In contrast to the spectrum of
the non-modified counterpart, the monomer peak is clearly
visible at ~ 16 mL elution volume of Hsp27Apy. These results
indicate that the Apy modification destabilizes the formation
of very large oligomers or aggregates of the protein, and
induces the dissociation to monomers.
Higher order oligomers formed by interaction of mono-
meric or dimeric subunits, as well as binding to client proteins
(see below), have been previously attributed to the C-
terminal domain of Hsp27.[35] Here, homology modeling
revealed a change in the surface charges within this region
upon Apy188 modification (see Table S2, Figure S16, and the
Supporting Information), providing a possible mechanism for
the observed changes.
Figure 3. Structural and functional characterization of Hsp27Apy. a) Far-
UV CD spectra of folded semisynthetic proteins, Hsp27Apy and
Hsp27NM. b) Fluorescence spectrum derived from the above proteins.
To study the potential effect of the Apy modification on
the function of Hsp27, we analyzed the in vitro activity of the
folded semisynthetic variants as molecular chaperones using
the standard non-native client protein citrate synthase
(CS).[36] We examined the ability of the proteins to suppress
the aggregation of CS that was denatured at 458C by
monitoring UV absorbance at 400 nm as a measure of
amorphous precipitation of this client protein. In the presence
of 0.2 molar equivalents of Hsp27NM, the expected suppres-
sion of aggregation was observed (Figure 3d), identical to the
activity of the analogue Hsp27D (Figure S17b), indicating that
the semisynthetic proteins were functional. On the other
hand, Hsp27Apy was less effective in preventing CS aggrega-
tion, observed by both the faster onset and higher level of
precipitation of the client protein (Figure 3d). At maximum
aggregation (~ 21 min), there was a 22% decrease in chaper-
one activity for Hsp27Apy relative to the control protein. Our
results suggest that a single Apy modification that occurs in
the C-terminal domain of Hsp27 had a significant detrimental
effect on the chaperone function of the protein. Our site-
specifically modified Hsp27 behaved very differently to the
MG-modified mixtures reported by Nagaraj et al. that
showed a strong enhancement of chaperone activity, which
the authors themselves found counterintuitive.[16,37]
c) Size exclusion chromatogram obtained for Hsp27Apy and Hsp27NM
d) Functional assay of Hsp27Apy and Hsp27NM using citrate synthase
(CS) as a client protein.
.
To investigate the effect of Apy188 on the structural
properties and stability of Hsp27, we analyzed the folded
semisynthetic proteins by CD spectroscopy. The far-UV CD
spectra of Hsp27Apy and the non-modified variant are
characterized by a broad negative maximum between 207
and 220 nm (Figure 3a), and the position and the amplitude of
this maximum are consistent with those of the recombinant
wild-type Hsp27 (hereafter, Hsp27WT; Figure S12b), indicat-
ing the correct folding of the semisynthetic proteins at the
secondary structure level. The spectrum of Hsp27NM was
indistinguishable from that of its analogue that had been
obtained by subjecting Hsp27WT to the same procedure of
desulfurization, purification, and refolding as for the semi-
synthetic proteins (Hsp27D; Figures S12b, S13). Notably,
Figure 3a shows that the control Hsp27NM exhibits a practi-
cally identical CD spectrum to that of Hsp27Apy, dominated by
the b-sheet structural element as for Hsp27WT (Table S1).
Variable temperature CD analysis (Figure S14) indicated
similar transition temperatures for both variants (59 and 608C
for Hsp27Apy and Hsp27NM, respectively) that are only slightly
lower than the previously measured values of 62–648C for
recombinant Hsp27.[34] Together, these results suggest that the
Arg188Apy modification did not cause significant changes in
the secondary structure and the overall stability of Hsp27.
In summary, we were able to obtain molecular level
details of the impact of the formation of the nPTM Apy on
human Hsp27. Combining organic chemistry and protein
semisynthesis, we produced Hsp27 bearing the pathologically
relevant Apy188 modification that showed impaired chaper-
one activity. To our knowledge, this work represents the first
instance of the generation of a site-specifically nPTM-
modified, functional protein, and may serve as a paradigm
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[12] a) T. Oya, N. Hattori, Y. Mizuno, S. Miyata, S. Maeda, T. Osawa,
for obtaining molecular insights into the influence of nPTMs
on proteins.
K. Uchida, J. Biol. Chem. 1999, 274, 18492 – 18502; b) J. W. J.
van Heijst, H. W. M. Niessen, K. Hoekman, C. G. Schalkwijk,
Ann. N. Y. Acad. Sci. 2005, 1043, 725 – 733.
[13] I. N. Shipanova, M. A. Glomb, R. H. Nagaraj, Arch. Biochem.
Biophys. 1997, 344, 29 – 36.
Acknowledgements
[14] P. J. Thornalley, S. Battah, N. Ahmed, N. Karachalias, S. Agalou,
R. Babaei-Jadidi, A. Dawney, Biochem. J. 2003, 375, 581 – 592.
[15] T. Gawlowski, B. Stratmann, I. Stork, B. Engelbrecht, A.
Brodehl, K. Niehaus, R. Korfer, D. Tschoepe, H. Milting,
Horm. Metab. Res. 2009, 41, 594 – 599.
[16] T. Oya-Ito, B. F. Liu, R. H. Nagaraj, J. Cell. Biochem. 2006, 99,
279 – 291.
[17] C. G. Schalkwijk, J. van Bezu, R. C. van der Schors, K. Uchida,
C. D. A. Stehouwer, V. W. M. van Hinsbergh, FEBS Lett. 2006,
580, 1565 – 1570.
[18] a) J. W. J. van Heijst, H. W. M. Niessen, R. J. Musters, V. W. M.
van Hinsbergh, K. Hoekman, C. G. Schalkwijk, Cancer Lett.
2006, 241, 309 – 319; b) W. B. Bair, C. M. Cabello, K. Uchida,
A. S. Bause, G. T. Wondrak, Melanoma Res. 2010, 20, 85 – 96;
c) T. Oya-Ito, Y. Naito, T. Takagi, O. Handa, H. Matsui, M.
Yamada, K. Shima, T. Yoshikawa, Biochim. Biophys. Acta Mol.
Basis Dis. 2011, 1812, 769 – 781; d) H. Sakamoto, T. Mashima, K.
Yamamoto, T. Tsuruo, J. Biol. Chem. 2002, 277, 45770 – 45775.
[19] M. Haslbeck, T. Franzmann, D. Weinfurtner, J. Buchner, Nat.
Struct. Mol. Biol. 2005, 12, 842 – 846.
We thank M. Felkl, A. Kçrner, and G. Niederacher for
technical assistance; Dr. A. Kravchuk for expert advice and
assistance; Dr. M. Haslbeck (Technische Universitꢀt Mꢁn-
chen) for the Hsp27 expression plasmid; A. Roller and Dr. V.
Arion for X-ray crystallographic analysis; and Dr. E. Mymri-
kov (Technische Universitꢀt Mꢁnchen) for helpful discus-
sions. This work was supported by the Austrian Academy of
Sciences. M.M. is a recipient of an APART fellowship of the
Austrian Academy of Sciences.
Keywords: argpyrimidine · biological activity ·
chaperone proteins · protein modifications ·
protein semisynthesis
[20] C. G. Concannon, A. M. Gorman, A. Samali, Apoptosis 2003, 8,
61 – 70.
[21] T. W. Muir, D. Sondhi, P. A. Cole, Proc. Natl. Acad. Sci. USA
1998, 95, 6705 – 6710.
[1] C. T. Walsh, S. Garneau-Tsodikova, G. J. Gatto, Angew. Chem.
Int. Ed. 2005, 44, 7342 – 7372; Angew. Chem. 2005, 117, 7508 –
7539.
[22] S. Bondalapati, M. Jbara, A. Brik, Nat. Chem. 2016, 8, 407 – 418.
[23] a) T. W. Muir, Annu. Rev. Biochem. 2003, 72, 249 – 289; b) T.
Durek, C. F. W. Becker, Biomol. Eng. 2005, 22, 153 – 172;
c) T. W. Muir, Biopolymers 2008, 90, 743 – 750; d) D. P. Gamblin,
S. I. van Kasteren, J. M. Chalker, B. G. Davis, FEBS J. 2008, 275,
1949 – 1959; e) P. Siman, A. Brik, Org. Biomol. Chem. 2012, 10,
5684 – 5697; f) M. Holt, T. Muir, Annu. Rev. Biochem. 2015, 84,
265 – 290.
[2] R. E. Moellering, B. F. Cravatt, Science 2013, 341, 549 – 553.
[3] a) R. Singh, A. Barden, T. Mori, L. Beilin, Diabetologia 2001, 44,
129 – 146; b) N. Nass, B. Bartling, A. Navarrete Santos, R. J.
Scheubel, J. Borgermann, R. E. Silber, A. Simm, Z. Gerontol.
Geriatr. 2007, 40, 349 – 356.
[4] A. Negre-Salvayre, C. Coatrieux, C. Ingueneau, R. Salvayre, Br.
J. Pharmacol. 2008, 153, 6 – 20.
[5] a) T. Miyata, K. Kurokawa, C. V. De Strihou, J. Am. Soc.
Nephrol. 2000, 11, 1744 – 1752; b) S. Jaisson, P. Gillery, Clin.
Chem. 2010, 56, 1401 – 1412; c) G. Vistoli, D. De Maddis, A.
Cipak, N. Zarkovic, M. Carini, G. Aldini, Free Radical Res. 2013,
47, 3 – 27.
[6] a) S. Y. Goh, M. E. Cooper, J. Clin. Endocrinol. Metab. 2008, 93,
1143 – 1152; b) N. Rabbani, P. J. Thornalley, Biochem. Biophys.
Res. Commun. 2015, 458, 221 – 226; c) J. Li, D. Liu, L. Sun, Y. Lu,
Z. Zhang, J. Neurol. Sci. 2012, 317, 1 – 5; d) Z. Hegab, S.
Gibbons, L. Neyses, M. A. Mamas, World J. Cardiol. 2012, 4, 90 –
102.
[24] P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science
1994, 266, 776 – 779.
[25] a) L. Z. Yan, P. E. Dawson, J. Am. Chem. Soc. 2001, 123, 526 –
533; b) Q. Wan, S. J. Danishefsky, Angew. Chem. Int. Ed. 2007,
46, 9248 – 9252; Angew. Chem. 2007, 119, 9408 – 9412.
[26] a) N. Pasupuleti, M. Gangadhariah, S. Padmanabha, P. San-
thoshkumar, R. H. Nagaraj, J. Cell. Biochem. 2010, 110, 408 –
419; b) A. Zavialov, R. Benndorf, M. Ehrnsperger, V. Zav’yalov,
I. Dudich, J. Buchner, M. Gaestel, Int. J. Biol. Macromol. 1998,
22, 163 – 173.
[27] a) N. Sreejayan, X. Yang, K. Palanichamy, K. Dolence, J. Ren,
Eur. J. Pharmacol. 2008, 593, 30 – 35; b) B.-L. Renard, B.
Boucherle, B. Maurin, M.-C. Molina, C. Norez, F. Becq, J.-L.
Dꢂcout, Eur. J. Med. Chem. 2011, 46, 1935 – 1941.
[28] L. Valgimigli, G. Brigati, G. F. Pedulli, G. A. DiLabio, M.
Mastragostino, C. Arbizzani, D. A. Pratt, Chem. Eur. J. 2003, 9,
4997 – 5010.
[29] K. C. K. Swamy, N. N. B. Kumar, E. Balaraman, K. V. P. P.
Kumar, Chem. Rev. 2009, 109, 2551 – 2651.
[30] J. Broddefalk, K. E. Bergquist, J. Kihlberg, Tetrahedron 1998, 54,
12047 – 12070.
[7] N. Ahmed, D. Dobler, M. Dean, P. J. Thornalley, J. Biol. Chem.
2005, 280, 5724 – 5732.
[8] R. A. Gomes, L. M. A. Oliveira, M. Silva, C. Ascenso, A.
Quintas, G. Costa, A. V. Coelho, M. S. Silva, A. E. N. Ferreira,
A. P. Freire, C. Cordeiro, Biochem. J. 2008, 416, 317 – 326.
[9] a) P. Gruber, T. Hofmann, J. Pept. Res. 2005, 66, 111 – 124; b) M.
Hellwig, S. Geissler, R. Matthes, A. Peto, C. Silow, M. Brandsch,
T. Henle, ChemBioChem 2011, 12, 1270 – 1279; c) T. N. Wang, R.
Kartika, D. A. Spiegel, J. Am. Chem. Soc. 2012, 134, 8958 – 8967;
d) T. M. Woods, M. Kamalov, P. W. R. Harris, G. J. S. Cooper, M.
Brimble, Org. Lett. 2012, 14, 5740 – 5743.
[31] P. H. Hirel, M. J. Schmitter, P. Dessen, G. Fayat, S. Blanquet,
Proc. Natl. Acad. Sci. USA 1989, 86, 8247 – 8251.
[32] P. Siman, O. Blatt, T. Moyal, T. Danieli, M. Lebendiker, H. A.
Lashuel, A. Friedler, A. Brik, ChemBioChem 2011, 12, 1097 –
1104.
[10] a) N. Ahmed, P. J. Thornalley, J. Dawczynski, S. Franke, J.
Strobel, G. Stein, G. M. Haik, Invest. Ophthalmol. Vis. Sci. 2003,
44, 5287 – 5292; b) P. S. Padayatti, A. S. Ng, K. Uchida, M. A.
Glomb, R. H. Nagaraj, Invest. Ophthalmol. Vis. Sci. 2001, 42,
1299 – 1304.
[11] R. Gomes, M. S. Silva, A. Quintas, C. Cordeiro, A. Freire, P.
Pereira, A. Martins, E. Monteiro, E. Barroso, A. P. Freire,
Biochem. J. 2005, 385, 339 – 345.
[33] V-50, unlike the more commonly applied counterpart VA-044, is
readily commercially available in Europe through Sigma–
Aldrich.
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[34] T. Rogalla, M. Ehrnsperger, X. Preville, A. Kotlyarov, G. Lutsch,
C. Ducasse, C. Paul, M. Wieske, A. P. Arrigo, J. Buchner, M.
Gaestel, J. Biol. Chem. 1999, 274, 18947 – 18956.
[37] R. H. Nagaraj, T. Oya-Ito, P. S. Padayatti, R. Kumar, S. Mehta,
K. West, B. Levison, J. Sun, J. W. Crabb, A. K. Padival,
Biochemistry 2003, 42, 10746 – 10755.
[35] M. V. Sudnitsyna, E. V. Mymrikov, A. S. Seit-Nebi, N. B. Gusev,
Curr. Protein Pept. Sci. 2012, 13, 76 – 85.
[36] F. Narberhaus, M. Haslbeck in Protein Folding Handbook (Eds.:
J. Buchner, T. Kiefhaber), Wiley-VCH, Weinheim, 2005,
pp. 830 – 857.
Received: June 1, 2016
Revised: June 28, 2016
Published online: && &&, &&&&
6
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Communications
Posttranslational Modifications
Underinvestigated protein modifications:
The non-enzymatic posttranslational
modification (nPTM) argpyrimidine (Apy)
was incorporated into Hsp27 through
protein semisynthesis, and the impact
was investigated at the molecular level.
Apy was found to reduce Hsp27 chaper-
one activity and oligomerization, without
affecting folding. This suggests that
nPTMs are able to alter protein function.
M. Matveenko, E. Cichero, P. Fossa,
C. F. W. Becker*
&&&& — &&&&
Impaired Chaperone Activity of Human
Heat Shock Protein Hsp27 Site-
Specifically Modified with Argpyrimidine
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!