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Histidine Phosphorylation, or Tyrosine Nitration, Affect Thymidylate Synthase Properties

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  • ICN Polfa Rzeszów S.A.

Abstract and Figures

Thymidylate synthase (TS) may undergo phosphorylation as endogenous protein present in mammalian cells and as recombinant protein (corresponding to human, rat, mouse or Trichinella spiralis TS) expressed in bacterial cells. The phosphorylated, compared to non-phosphorylated, recombinant enzyme forms show decreased (at least by 3-fold) molecular activity, bind their cognate mRNA (tested only with the rat enzyme) and repress their own mRNA translation (tested with human, rat and mouse enzyme). Recombinant human, mouse, rat and Trichinella spiralis TSs, expressed in E. coli, undergo phosphorylation on histidine residues; so do probably L1210 and calf thymus endogenous TS proteins. Endogenous calf thymus and L1210 thymidylate synthase proteins undergo nitration in vivo. Chemical nitration of human, mouse and C. elegans recombinant TS proteins distinctly affects catalytic potency, by lowering the V max app value.
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Pteridines / Vol. 20 / Special issue
137
Pteridines
Vol. 20, Special issue, pp. 137-142
T. Frączyk et al. : Thymidylate synthase histidine phosphorylation or tyrosine nitration
Histidine Phosphorylation, or Tyrosine Nitration, Affect
Thymidylate Synthase Properties
Tomasz Frączyk1, Tomasz Ruman2, Dagmara Rut2, Elżbieta Dąbrowska-Maś2, Joanna Cieśla1,
Zbigniew Zieliński1, Katarzyna Sieczka1, Janusz Dębski3, Barbara Gołos1, Patrycja Wińska1,
Elżbieta Wałajtys-Rode2, David Shugar3, Wojciech Rode1,2§
1 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland
2 Rzeszów University of Technology, Faculty of Chemistry, Rzeszów, Poland
3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland
Abstract
Thymidylate synthase (TS) may undergo phosphorylation as endogenous protein present in mammalian
cells and as recombinant protein (corresponding to human, rat, mouse or Trichinella spiralis TS)
expressed in bacterial cells. The phosphorylated, compared to non-phosphorylated, recombinant
enzyme forms show decreased (at least by 3-fold) molecular activity, bind their cognate mRNA (tested
only with the rat enzyme) and repress their own mRNA translation (tested with human, rat and mouse
enzyme). Recombinant human, mouse, rat and Trichinella spiralis TSs, expressed in E. coli, undergo
phosphorylation on histidine residues; so do probably L1210 and calf thymus endogenous TS proteins.
Endogenous calf thymus and L1210 thymidylate synthase proteins undergo nitration in vivo. Chemical
nitration of human, mouse and C. elegans recombinant TS proteins distinctly affects catalytic potency,
by lowering the Vmax
app value.
Key words: Thymidylate synthase (EC 2.1.1.45)
Introduction
Thymidylate synthase (TS; EC 2.1.1.45), a
target in chemotherapy of a number of diseases,
including cancer (1), catalyzes the N5,10-
methylenetetrahydrofolate (meTHF)-assisted
C(5)-methylation of dUMP (2), required for
DNA synthesis. It is, consequently, of interest to
examine possible post-translational modications
of the enzyme in living cells.
Possible phosphorylation of TS in rat cells
was previously demonstrated (3). Furthermore,
differing properties, including sensitivity to
inactivation by FdUMP and its analogues, of
TSs from parental and FdUrd-resistant mouse
leukemia L1210 cells were considered to result,
not from mutation, but rather posttranslational
modication(s), including phosphorylation (4).
Post-translational modifications also include
nitration of protein tyrosine residues, which may
affect the function of a protein, and is associated
with more than 50 diseases, including cancer,
due to intensied NO biosynthesis (5).
To learn more about molecular aspects of
TS phosphorylation, and the possibility of TS
tyrosine nitration in cells, and to determine to
what extent enzyme properties might be affected
by chemical nitration of TS tyrosine, we are
studying the endogenous enzyme from parental
and FdUrd-resistant mouse leukemia L1210 cells,
and calf thymus, and those of mouse, rat, human,
Trichinella spiralis and Caenorhabditis elegans
§Correspondence : Dr. W. Rode at the Nencki Institute of
Experimental Biology; F: +48-22-822-5342; Email: rode@
nencki.gov.pl
Pteridines / Vol. 20 / Special issue
138
T. Frączyk et al. : Thymidylate synthase histidine phosphorylation or tyrosine nitration
recombinant TSs, expressed in bacterial cells.
Material and Methods
Pro-Q® Diamond Phosphoprotein Gel Stain
and SYPRO® Ruby Protein Gel Stain were from
Molecular Probes. Inhibitase was from Eppendorf
(Hamburg, Germany), restriction endonucleases
were from Invitrogen and Rabbit Reticulocyte
Lysate System was from Promega (Madison, WI).
TS preparation
The endogenous enzyme proteins from parental
and FdUrd-resistant mouse leukemia L1210
cells (4), and calf thymus (6), were purified as
previously described. Ceanorhabditis elegans (8)
and mouse (9) coding regions were cloned into
pPIGDM4+stop vector and expressed as HisTag-
free proteins in BL21(DE3) or thymidylate
synthase-deficient TX61- (a kind gift from Dr.
W. S. Dallas) E. coli strain, respectively. Human
(10) or rat (11) TS coding regions were subcloned
into pET28a vector and expressed as HisTag-
containing proteins in E. coli BL21(DE3) strain.
Trichinella spiralis TS coding region (12) was
subcloned into pQE2 vector and expressed as
HisTag-containing protein in JM109 E. coli
strain. HisTag containing proteins were purified
on NiNTA His-Bind resin (Novagen) according
to manuf acturer protocol , and HisTag-free
proteins were purified as previously described
(11). Phosphatase inhibitors (50 mM NaF, 5 mM
Na-pyrophosphate, 0.2 mM EGTA, 0.2 mM
EDTA and 2 mM Na3VO4) were present in the
purification buffers. TS activity was measured
and kinetic parameters of the enzyme-catalyzed
reaction were determined as previously described
(13).
TS phosphorylation analysis with the Pro-Q®
Diamond Phosphoprotein Gel Stain
The assay for the presence of protein phosphate
groups was performed as previously described (4).
Separation of TS preparations into
phosphorylated and non-phosphorylated
fractions
This was done according to Wolschin et al. (14)
using metal oxide/hydroxide afnity chromatography
on Al(OH)3 beads.
Preparation and labelling of mRNA
This was done as previously described (7).
RNA electrophoretic mobility shift assay
RNA-protein binding experiments were
performed using an electrophoretic mobility shift
assay as earlier described (7, 15).
In vitro translation
Rabbit Reticulocyte Lysate System was used as
previously described (7).
Mass spectrometry analysis. This was presented
earlier (4).
31P NMR spectroscopy. Spectra were recorded
at Bruker Avance spectrometer operating in
quadrature mode at 500.13 MHz for 1H and
202.46 MHz for 31P nuclei. All spectra were
recorded at 277K both with and without proton
decoupling. The sample tube diameter was 5mm
and spectra were obtained at pH value of 7.5.
Each sample (740 μL) resulted from mixing
440μL of either 3.3 μM phosphorylated TS or
13.4 μM unphosphorylated TS fraction in 0.2M
Tris-HCl pH 7.5 or 7.8, 20% saccharose and
20mM β-mercaptoethanol with 300 μL of D2O.
Inorganic phosphate (Pi) was used as an internal
standard, with its resonance at 2.14-2.16 ppm (pH
7.8), 2.00-2.05 ppm (pH 7.5), 1.60-1.70 ppm (at
pH 5,0) or 0,0 ppm (at pH 1-1.5). Additionally,
the spectra were referenced using an external
standard (85% H3PO4). All protein samples were
analyzed by two dimensional NMR spectroscopy
using both 31P{1H} heteronuclear correlation
(HETCOR) and gradient-enhanced 1H-31P
heteronuclear single-quantum correlation (HSQC)
experiments. The HSQC experiments were
conducted with optimization for the long range
couplings using different 3JPH values (1-10Hz).
31P NMR-monitored amino acid
phosphorylation
To a solution of 10mg of a given amino acid
(histidine, arginine or lysine) in 0.2 M Tris-HCl
buffer (pH 7.8, 200 μl) potassium phosphoramidate
was added (at the molar ratio 7.5:1 of potassium
phosphoramidate to amino acid). After the reaction
mixture was shaken for 24 h at 277 K, 31P NMR
was monitored in a sample containing 200μL of
the reaction mixture and 300 μL of D2O.
TS tyrosine in vitro nitration
The reaction was performed at 4ºC in the
presence of 20 μM dUMP (stabilization in the
absence of 2-mercaptoethanol) in a reaction
mixture containing 200 mM Na/K phosphate
buffer pH 7.5, equimolar concentration of
Pteridines / Vol. 20 / Special issue
139
T. Frączyk et al. : Thymidylate synthase histidine phosphorylation or tyrosine nitration
NaHCO3 and H2O2 (5-70 mM), NaNO2 at
concentration by 5% exceeding the latter (5.25-
73.5 mM) and the enzyme (5 μM dimer). To
start the reaction, H2O2 was added, the sample
mixed 30 sec and next incubated 5 min.
While nitrotyrosine content was determined
spectrophotometrically (16), to the remaining
reaction mixture 2-mercaptoethanol (20 mM) was
added, followed by either protein precipitation
with 10% (w/v) TCA or sample dilution (≥300-
fold) with 50 mM Na/K phosphate buffer pH
7.5, containing 0.1% Triton X-100 and 10 mM
2-mercaptoethanol. The diluted preparation
preserved TS activity for at least 2h, allowing
to study enzyme properties. To the control
reaction mixture TS was added after mixing and
incubating the remaining components, in order to
inactivate the produced peroxynitrite.
Immunoblotting
Previously described method was used (4).
Results and Discussion
TS preparations highly puried in the presence
of phosphatase inhibitors, including endogenous
TS forms from L1210 parental and FdUrd-
resistant cells (4), and calf thymus (Fig. 1), as
well as mouse, rat, human and Trichinella spiralis
recombinant TSs expressed in bacterial cells (not
shown), as analyzed with the Pro-Q® Diamond
Phosphoprotein Gel Stain following SDS-PAGE,
contained phosphorylated forms present in a low
proportion, except for the calf thymus TS where
their apparent content was distinctly higher
(Fig. 1). However, MS analysis of the bands
revealed no phosphorylated amino acid residues.
Furthermore, MS analysis of IEF fractions
of TS preparations from parental and FdUrd-
resistant mouse leukemia L1210 cells, whose
differing sensitivity to inactivation by FdUMP
and its analogues was previously found not due
to mutations (4), demonstrated phosphorylation
of Ser10 and Ser16 only in the resistant enzyme,
although the Pro-Q® Diamond Phosphoprotein
Gel Stain indicated also phosphorylation of
parental TS.
Enrichment of the phosphorylated fraction, by
separation from that non-phosphorylated, of each
of the four recombinant TS preparations using
metal oxide/hydroxide affinity chromatography
on Al(OH)3 beads (6), yielding always
1% of the total purified TS protein, allowed
to demonstrate that TS phosphorylation is
responsible for a 3-20-fold lower Vmax
app (Fig.
2), with unaltered Km
app (not shown) for either
substrate or cofactor. Moreover, only the
phosphorylated fractions showed ability to repress
in vitro translation of TS cognate (Figs. 3A-B), as
well as luciferase (not shown), mRNA, and with
rat recombinant TS (other enzyme forms were
not tested) only phosphorylated form was able to
bind the cognate mRNA (Fig. 3C). Surprisingly,
while MS analyses did not reveal the presence of
phosphorylated residues in any of the fractions
investigated, 31P NMR spectroscopy demonstrated
Fig. 1: Phosphorylation of calf thymus endogenous TS
(lanes marked 2 in gels A and B; lanes marked 1 contain
PeppermintStickTM phosphoprotein molecular weight
standards providing a mixture of phosphorylated and
non-phosphorylated proteins ), determined following
PAGE under denaturing (SDS-PAGE) conditions. Gel
was stained first for phosphoprotein (Pro-Q
Diamond
Phosphoprotein Gel Stain; A) and later for protein
(SYPRO
Ruby Protein Gel Stain; B).
Fig. 2: Vmax
app values of phosphorylated (phos) and
non-phosphorylated (non-phos) fractions of human
(hTS), mouse (mTS), rat (rTS) and T. spiralis (T.sp.TS)
recombinant TS preparations. Each value is expressed
as mean ± ½ 95% CI for 3-5 experiments. Statistical
signicance: *p<0,001, **p<0,01.
Pteridines / Vol. 20 / Special issue
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T. Frączyk et al. : Thymidylate synthase histidine phosphorylation or tyrosine nitration
Fig. 3: The effect of protein phosphorylation on non-catalytic properties of thymidylate synthase.
(A and B) The effect of human, rat and mouse thymidylate synthases on the in vitro translation of their respective
mRNAs.
Rabbit reticulocyte lysate was incubated with 40 nM mRNA and increasing concentrations of the unphosphorylated
(A) or phosphorylated (B) enzyme forms obtained after protein separation on metal oxide/hydroxide affinity
chromatography. The concentration of phosphate buffer was 10.6 mM in all samples. The control contained all
reagents except for the protein. The in vitro translation products were separated on 14% polyacrylamide gel and
autoradiographed. The plots resemble the density of TS bands expressed as % of control. Each point is the average of
two to four experiments.
(C) The effect of phosphorylation on binding of rat thymidylate synthase to its own mRNA.
32P-labelled rTS mRNA (0.66 nM, 105 cpm) was incubated alone or with increasing concentrations of rHisTag-TS
before or after separation of the protein into non-phosphorylated and phosphorylated enzyme forms on metaloxide
affinity chromatography. The complexes of mRNA-protein were treated with RNase T1 and heparin and subjected
to non-denaturing polyacrylamide gel chromatography followed by autoradiography. The plot resembles the density
of bands expressed as % of control, presented as means of ± SD (bars). Each point is the average density from three
separate experiments.
A
B
C
Fig. 4: 31P NMR spectrum of the enriched phosphorylated fraction of human recombinant TS with marked positions of
the resonances of phosphorylated standards. The insert presents the corresponding spectrum of the non-phosphorylated
TS fraction.
Pteridines / Vol. 20 / Special issue
141
T. Frączyk et al. : Thymidylate synthase histidine phosphorylation or tyrosine nitration
clearly the presence of phosphorylated residues
only in the phosphorylated enzyme fractions.
Further analyses of the 31P NMR spectra,
including their time-dependent changes following
acidification (not shown), and comparison with
those of synthetic phosphoramidate derivatives
of basic amino acids (Lys, Arg and His), and
commercially available phospho-amino acids
(Fig. 4), revealed the presence of phosphorus in
a phosphoramidate (acid-labile) bond, pointing
to modification of histidine residue(s). Results
of an MS analysis of peptides enriched from the
recombinant mouse TS preparation trypsin digest
using TiO2 beads (Phos-Trap, PerkinElmer),
the enrichment resulting presumably from
phosphohistidine binding by the beads, pointed to
His298 being the most probable phosphorylation
site. Which protein kinases are responsible for the
phosphorylation of TS, remains to be established.
Highly purified TS preparations isolated from
calf thymus, and L1210 parental and FdUrd-
resistant cells were found to be nitrated (Fig.
5), based on a specific reaction with anti-
nitrotyrosine antibodies (Sigma-Aldrich,
NO409), suggesting the enzyme to undergo this
modication endogenously in normal and tumor
tissues.
Each human, mouse and Ceanorhabditis
elegans recombinant TS preparation, incubated
in vitro in the presence of NaHCO3, NaNO2
and H2O2, underwent tyrosine nitration (Fig.
5), leading to a Vmax
app 2-fold lower following
nitration of 1 (with human or C. elegans TS) or 2
(with mouse TS) tyrosine residues per monomer.
Enzyme interactions with dUMP, meTHF or
5-uoro-dUMP were not distinctly inuenced.
In co nclus i on, co valen t modi f icati o n s,
phosphorylation and tyrosine nitration, may
inuence catalytic (both modications) and non-
catalytic (tested only with phosphorylation)
properties of TS. The enzyme may undergo
phosphorylation as endogenous protein present
in mammalian cells and as recombinant
protein overexpressed in bacterial cells. The
phosphorylated, compared to non-phosphorylated,
forms show decreased (at least by 3-fold)
molecular activity, bind their cognate mRNA
(tested only with the rat enzyme) and repress
their own mRNA translation (tested with human,
rat and mouse enzyme). Recombinant human,
mouse, rat and Trichinella spiralis TSs, expressed
in E. coli, undergo phosphorylation on histidine
residues; so do probably L1210 and calf thymus
endogenous TS proteins. Endogenous calf thymus
and L1210 thymidylate synthase proteins undergo
nitration in vivo. Chemical nitration of human,
mouse and C. elegans recombinant TS proteins
distinctly affected catalytic potency.
Fig. 5: Nitrotyrosine detection by specific antibodies
in chemically nitrated mammalian recombinant TS and
endogenous enzyme preparations purified from tumour
and normal tissues and separated by SDS-PAGE (without
2-mercaptoethanol).
Proteins were stained (C) or, following transfer to
PVDF membrane, underwent first reaction with anti-
nitroY antibodies (A), followed by treatment with
anti-TS antibodies (B). Additional negative controls
included treatment with anti-nitroY antibodies either
in the presence of 10 mM nitrotyrosine or following
reduction of nitrotyrosine to aminotyrosine with 100 mM
Na2S2O4 at pH 9,0 (not shown). Nitrated BSA (positive
control; lane 1), mouse recombinant TS nitrated with 8
mM (0.8 mol nitroY/mol TS subunit; lane 2), 12 mM
(1.6 mol nitroY/mol TS subunit; lane 3) or 12 mM
inactivated (negative control, 0 mol nitroY/mol TS; lane
4) peroxynitrite, and endogenous TS purified from calf
thymus (lane 5), and L1210 parental (lane 6) and FdUrd-
resistant (lane 7) cells.
A
B
C
Pteridines / Vol. 20 / Special issue
142 T. Frączyk et al. : Thymidylate synthase histidine phosphorylation or tyrosine nitration
Acknowledgments
Supported by the Ministry of Science and
Higher Education (grant number N401 0612 33
and N401 2334 34).
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... In view of a previous report on possible phosphorylation of the enzyme in cultured rat cells (Samsonoff et al., 1997 ), such a modification was sought by conventional mass spectrometry of isoelectric focusing fractions of native proteins of the two enzyme forms. Surprisingly, phosphorylation of Ser10 and Ser16 was found, but only in the resistant cell enzyme (Frączyk et al., 2009), although staining of proteins separated by SDS/PAGE with the Pro-Q® Diamond Phosphoprotein Gel Stain showed phosphorylation of the enzyme forms from both cell lines (Cieśla et al., 2006). One possibile explanation of this apparent U n c o r r e c t e d P a p e r i n P r e s s discrepancy is that at least the enzyme from the parental cells is phosphorylated on basic amino acids, which escaped detection by the former procedure. ...
... A similar problem appeared during studies of four recombinant enzyme preparations (human, rat, mouse and Trichinella spiralis thymidylate synthases), whose phosphorylation could be demonstrated by Pro-Q® Diamond staining , but not by conventional MS analysis. However, 31 P NMR spectra showed the presence of phosphohistidine residues in phosphorylated fraction enriched from each of the recombinant enzyme preparations (Frączyk et al. 2009). Interestingly, the phosphorylation of the enzyme's His residue(s) affected not only its catalytic (by lowering the V max app values), but also non-catalytic (by affecting repression of the enzyme's own mRNA in vitro translation ) properties (Frączyk et al., 2009). ...
... However, 31 P NMR spectra showed the presence of phosphohistidine residues in phosphorylated fraction enriched from each of the recombinant enzyme preparations (Frączyk et al. 2009). Interestingly, the phosphorylation of the enzyme's His residue(s) affected not only its catalytic (by lowering the V max app values), but also non-catalytic (by affecting repression of the enzyme's own mRNA in vitro translation ) properties (Frączyk et al., 2009). The possible function(s) of the phosphorylation of thymidylate synthase is of obvious interest but, as with many other phosphorylated proteins (Lienhard, 2008), further studies are needed to learn more about it. ...
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... Considering that CX-4945 at 3 μM affected the expression of DHFR at the protein but not mRNA level, the possible regulatory role of CK2-mediated phosphorylation in translation/degradation of DHFR protein should be taken into account. Recently, it was demonstrated that phosphorylation of TYMS affected its catalytic and non-catalytic properties, including translation and binding of its own mRNA (39)(40)(41). CK2-mediated phosphorylation of DHFR might have a similar effect on enzyme properties. It should also be noted that phosphorylated TYMS bound and inhibited translation not only of its own mRNA, but also of other mRNAs encoding DHFR, SHMT, thymidine kinase and deoxycitidylate deaminase proteins (41). ...
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Background/aim: Recently, we demonstrated the ability of inhibitors of protein kinase 2 (casein kinase II; CK2) to enhance the efficacy of 5-fluorouracil, a thymidylate synthase (TYMS)-directed drug for anticancer treatment. The present study aimed to investigate the antileukemic effect of simultaneous inhibition of dihydrofolate reductase (DHFR), another enzyme involved in the thymidylate biosynthesis cycle, and CK2 in CCRF-CEM acute lymphoblastic leukemia cells. Materials and methods: The influence of combined treatment on apoptosis and cell-cycle progression, as well as the endocellular level of DHFR protein and inhibition of CK2 were determined using flow cytometry and western blot analysis, respectively. Real-time quantitative polymerase chain reaction was used to examine the influence of silmitasertib (CX-4945), a selective inhibitor of CK2 on the expression of DHFR and TYMS genes. Results: The synergistic effect was correlated with the increase of annexin V-binding cell fraction, caspase 3/7 activation and a significant reduce in the activity of CK2. An increase of DHFR protein level was observed in CCRF-CEM cells after CX-4945 treatment, with the mRNA level remaining relatively constant. Conclusion: The obtained results demonstrate a possibility to improve methotrexate-based anti-leukemia therapy by simultaneous inhibition of CK2. The effect of CK2 inhibition on DHFR expression suggests the important regulatory role of CK2-mediated phosphorylation of DHFR inside cells.
... 9). And C. elegans TS Thr 126 , also homologous to human Ser 124 , underwent phosphorylation in vitro (Table 4).[44]). Lower panel: corresponding spectrum of the non-phosphorylated TS fraction (m-TS).Fig. ...
... Higher eukaryotes are known to contain kinases and phosphatases that control the reversible phosphorylation of histidine residues, yet primary sequence analysis indicates that these organisms do not possess homologues of the 'two-component' systems described above [8]. Evidence acquired by functional characterization of individual proteins suggests the involvement of phosphohistidine in mammalian cell signalling, both by virtue of functioning as a catalytic intermediate in phosphotransfer reactions [5,[9][10][11][12][13][14] and as a reversibly regulated moiety that regulates protein structure and thus function [15][16][17][18][19][20][21][22][23][24][25], akin to the more widely characterized phosphorylation-induced regulation. Specifically, HHK (histone H4 histidine kinase) activity has been reported in a wide range of cell types and is often associated with cell growth and development. ...
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A significant number of proteins in both eukaryotes and prokaryotes are known to be post-translationally modified by the addition of phosphate, serving as a means of rapidly regulating protein function. Phosphorylation of the amino acids serine, threonine and tyrosine are the focus of the vast majority of studies aimed at elucidating the extent and roles of such modification, yet other amino acids, including histidine and aspartate, are also phosphorylated. Although histidine phosphorylation is known to play extensive roles in signalling in eukaryotes, plants and fungi, roles for phosphohistidine are poorly defined in higher eukaryotes. Characterization of histidine phosphorylation aimed at elucidating such information is problematic due to the acid-labile nature of the phosphoramidate bond, essential for many of its biological functions. Although MS-based strategies have proven extremely useful in the analysis of other types of phosphorylated peptides, the chromatographic procedures essential for such approaches promote rapid hydrolysis of phosphohistidine-containing peptides. Phosphate transfer to non-biologically relevant aspartate residues during MS analysis further complicates the scenario.
... It should be added that recombinant human, mouse, rat and T. spiralis TSs, expressed in E. coli, may also undergo phosphorylation on histidine residues, the modification strongly influencing catalytic and non-catalytic properties of the enzyme. L1210 and calf thymus endogenous TS proteins also seem to undergo acid-labile phosphorylation[51]. ...
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Thymidylate synthase (TS) was found to be a substrate for both catalytic subunits of human CK2, with phosphorylation by CK2alpha and CK2alpha' characterized by similar K(m) values, 4.6microM and 4.2microM, respectively, but different efficiencies, the apparent turnover number with CK2alpha being 10-fold higher. With both catalytic subunits, phosphorylation of human TS, like calmodulin and BID, was strongly inhibited in the presence of the regulatory subunit CK2beta, the holoenzyme being activated by polylysine. Phosphorylation of recombinant human, rat, mouse and Trichinella spiralis TSs proteins was compared, with the human enzyme being apparently a much better substrate than the others. Following hydrolysis and TLC, phosphoserine was detected in human and rat, and phosphotyrosine in T. spiralis, TS, used as substrates for CK2alpha. MALDI-TOF MS analysis led to identification of phosphorylated Ser(124) in human TS, within a sequence LGFS(124)TREEGD, atypical for a CK2 substrate recognition site. The phosphorylation site is located in a region considered important for the catalytic mechanism or regulation of human TS, corresponding to the loop 107-128. Following phosphorylation by CK2alpha, resulting in incorporation of 0.4mol of phosphate per mol of dimeric TS, human TS exhibits unaltered K(m) values for dUMP and N(5,10)-methylenetetrahydrofolate, but a 50% lower turnover number, pointing to a strong influence of Ser(124) phosphorylation on its catalytic efficiency.
Article
Protein phosphorylation is an important post-translational modification that is an integral part of cellular function. The O-phosphorylated amino-acid residues, such as phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr), have dominated the literature while the acid labile N-linked phosphorylated amino acids, such as phosphohistidine (pHis), have largely been historically overlooked because of the acidic conditions routinely used in amino-acid detection and analysis. This review highlights some misinterpretations that have arisen in the existing literature, pinpoints outstanding questions and potential future directions to clarify the role of pHis in mammalian signalling systems. Particular emphasis is placed on pHis isomerization and the hybrid functionality for both pHis and pTyr of the proposed τ-pHis analogue bearing the triazole residue.
Article
Crystal structure is presented of the binary complex between potassium phosphoramidate-phosphorylated recombinant C. elegans thymidylate synthase and dUMP. On each monomer a single phosphoserine residue (Ser127) was identified, instead of expected phosphohistidine. As 31P NMR studies of both the phosphorylated protein and of potassium phosphoramidate potential to phosphorylate different amino acids, point to histidine as the only possible site of the modification, thermodynamically favored intermolecular phosphotransfer from histidine to serine is suggested.
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Crystal structures were solved of the binary complexes Trichinella spiralis and Caenorhabditis elegans thymidylate synthases with deoxyuridine monophosphate (dUMP), with crystals obtained by the vapor diffusion method in hanging drops. For the T. spiralis thymidylate synthase-dUMP complex, the diffraction data were collected at the BESSY Synchrotron to 1.9 Å resolution. The crystal belongs to the space group P1 with two dimers in the asymmetric unit (ASU). For the C. elegans TS-dUMP complex crystal, the diffraction data were collected at the BESSY Synchrotron to 2.48 Å resolution, and the crystal belongs to the space group P 32 2 1, with two monomers (one dimer) in the ASU. Structural comparisons were made of both structures and each of them with the corresponding mouse thymidylate synthase complex.
Article
This year (2012) marks the 50th anniversary of the discovery of protein histidine phosphorylation. Phosphorylation of histidine (pHis) is now widely recognized as being critical to signaling processes in prokaryotes and lower eukaryotes. However, the modification is also becoming more widely reported in mammalian cellular processes and implicated in certain human disease states such as cancer and inflammation. Nonetheless, much remains to be understood about the role and extent of the modification in mammalian cell biology. Studying the functional role of pHis in signaling, either in vitro or in vivo, has proven devilishly hard, largely due to the chemical instability of the modification. As a consequence, we are currently handicapped by a chronic lack of chemical and biochemical tools with which to study histidine phosphorylation. Here, we discuss the challenges associated with studying the chemical biology of pHis and review recent progress that offers some hope that long-awaited biochemical reagents for studying this elusive posttranslational modification (PTM) might soon be available.
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Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA. Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels. The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system. The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA. This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA. The inclusion of dUMP, 5-fluoro-dUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein. Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human beta-actin, and yeast tRNA. These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.
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Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5, 10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50-100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [32P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme's contribution to the cell cycle may be more complex than believed previously.
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Inflammation in asthma, sepsis, transplant rejection, and many neurodegenerative diseases associates an up-regulation of NO synthesis with increased protein nitration at tyrosine. Nitration can cause protein dysfunction and is implicated in pathogenesis, but few proteins that appear nitrated in vivo have been identified. To understand how this modification impacts physiology and disease, we used a proteomic approach toward targets of protein nitration in both in vivo and cell culture inflammatory disease models. This approach identified more than 40 nitrotyrosine-immunopositive proteins, including 30 not previously identified, that became modified as a consequence of the inflammatory response. These targets include proteins involved in oxidative stress, apoptosis, ATP production, and other metabolic functions. Our approach provides a means toward obtaining a comprehensive view of the nitroproteome and promises to broaden understanding of how NO regulates cellular processes.
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N{sup 4}-Hydroxy-dCMP (N{sup 4}-OH-dCMP), N{sup 4}-methoxy-dCMP (N{sup 4}-OMe-dCMP), and their 5-fluoro congeners were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N{sup 4}-OH-dCMP was not a substrate and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based. K{sub i} values for N{sup 4}-OH-dCMP and its 5-fluoro analogue were in the range 10{sup {minus}7}-10{sup {minus}8} M, 2-3 orders of magnitude higher for the corresponding N{sup 4}-OMe analogues. The 5-methyl analogue of N{sup 4}-OHdCMP was 10{sup 4}-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N{sup 4}-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N{sup 4}-OH-dCMP, suggesting interaction of both N{sup 4}-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N{sup 4}-OH-dCMP increased its affinity from 2- to 20-fold formore » the enzyme from different sources. With the Ehrlich and tapeworm enzymes, N{sup 4}-OH-FdCMP and FdUMP were almost equally effective inhibitors.« less
Article
1.1. Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells.2.2. The enzyme is a dimer composed of 35,000 mol. wt monomers.3.3. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP.4.4. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34† C, reflecting a change of rate-limiting step in dTMP formation.5.5. MgATP at millimolar concentrations inhibits mouse thymus enzyme.
Article
N4-Hydroxy-dCMP (N4-OH-dCMP), N4-methoxy-dCMP (N4-OMe-dCMP), and their 5-fluoro congeners (syntheses of which are described) were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N4-OH-dCMP was not a substrate (no dihydrofolate produced; no tritium released with 5-3H-labeled molecule), and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based, with arrest of a step posterior to addition of cofactor and blocking abstraction of the C(5) hydrogen. Ki values for N4-OH-dCMP and its 5-fluoro analogue were in the range 10(-7) - 10(-8) M, 2-3 orders of magnitude higher for the corresponding N4-OMe analogues. The 5-methyl analogue of N4-OH-dCMP was 10(4)-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N4-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N4-OH-dCMP, suggesting interaction of both N4-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N4-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.
Article
Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA. The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein. The missing region was obtained by screening a rat genomic library. The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da. Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues. Six of these differences are in the first 21 amino acids from the amino-end. The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature. The three species differed completely at only four sites. Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS. The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein. Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed. The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool.
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This chapter discusses the detection and quantitation methods of nitrotyrosine residues in proteins. Nitrotyrosine is detected in human diseases associated with oxidative stress and is visualized using immunological techniques in atherosclerotic plaques of human coronary vessels, in lungs of infants with acute lung injury and sepsis, and in adult respiratory distress syndrome (ARDS). High-performance liquid chromatography (HPLC) analysis is used to detect nitrotyrosine in synovial fluid from patients with rheumatoid arthritis. Peroxynitrite can be synthesized from sodium nitrite and acidified hydrogen peroxide. Selective tyrosine nitration can be accomplished by titrating protein with tetranitromethane (TNM) at neutral or alkaline conditions (pH 7–8). TNM is a potent carcinogen, which must be handled carefully. The residual TNM and trinitromethane must be removed prior to nitrotyrosine quantitation. Nitrotyrosine is essentially nonfluorescent whereas aminotyrosine is highly fluorescent and has a characteristic emission spectrum. Thus, fluorescent detection of aminotyrosine can be used as an alternative to direct detection of nitrotyrosine. Quantitation of nitrotyrosine using the solid-phase immunoradiochemical method has the advantage of high sensitivity and does not require sample manipulation.
Article
A method is presented for expressing human thymidylate synthase (TS) to the extent of 25-30% of the protein in Escherichia coli. By this procedure, 200-400 mg of pure enzyme can be obtained from a 2-liter culture of cells. The key to the level of expression appears to be related to the conversion of purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product. Conversion of the penultimate proline to a leucine did not diminish expression, but while the isolated native enzyme contained only proline on its amino-terminal end, the mutated enzyme was found to contain methionine on its amino terminus. By contrast, the expression of the unmodified TS cDNA represented only about 0.1-0.2% of the total cellular protein. Unlike recombinant rat and human TSs, the respective enzymes purified to homogeneity from eukaryotic cells were blocked at the amino ends and possessed 2- to 4-fold lower specific activities. To determine at what level the impairment of expression occurred, an in vitro transcription, translation system was employed and the results showed that while transcription was unaffected, the translation of native TS mRNA was reduced by at least 20-fold relative to modified TS mRNA using a rabbit reticulocyte translation system. Thus, it appears that at least for the TS gene, expression is greatly influenced by the GC content of the 5' coding region of the gene in both prokaryote and eukaryote systems.