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Transcript
Cindy Lee
1041791
Biochemistry 499
Affinity Chromatography: Homemade Microcystin-Sepharose
Cindy Lee, Marcia Craig
May 1, 2006
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Summary
Reversible phosphorylation of proteins is known to be a method of regulation of
many cellular pathways. The enzymes involved in the reactions in reversible
phosphorylation are protein kinases, which catalyze phosphorylation, and protein
phosphatases, which catalyze dephosphorylation. Protein phosphatases must be tightly
regulated because they are relatively few in number in the cell and must counterbalance
the actions of many different protein kinases. It has been recognized that abnormal
phosphorylation can cause diseases, such as cancer. To assist in the study of the
serine/threonine protein phosphatase class of protein phosphatases, an affinity column
made from microcystin-LR, a toxin and inhibitor of protein phosphatase-1 and -2A,
linked to Sepharose can be used for their purification and determination of their
regulatory subunits. In this project, a microcystin-Sepharose affinity column was created
and shown to bind protein phosphatase-1 and -2A and demonstrated how it could be used
for the purification of these enzymes.
Introduction
Reversible phosphorylation of proteins is a very important form of regulation in
almost every aspect of cell life [1]. The enzymes that catalyze reactions involved in
reversible phosphorylation are protein kinases and protein phosphatases. Protein kinases
catalyze the phosphorylation of proteins and phosphatases catalyze the dephosphorylation
of proteins. One family of phosphatases are the serine/threonine phosphatases, which
includes protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A).
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Abnormal phosphorylation has been known as a cause or consequence of many
human diseases, such as cancer. This stresses the importance of regulation of the
enzymes involved in reversible phosphorylation, especially the protein phosphatases.
There are relatively few Ser/Thr phosphatases in the cell and one catalytic subunit must
counterbalance the activity of many different protein kinases. Thus protein phosphatases
are subjected to spatial and temporal regulation.
The catalytic subunit of protein phosphatases do not exist as free molecules in
vivo; they are usually complexed with other proteins. These proteins can target the
phosphatases to particular subcellular locations or modify their activity [2]. PP1c (the
catalytic subunit of PP1) is regulated by the association of regulatory proteins. Proteins
that target PP1c commonly contain a four amino acid motif, RVXF. The catalytic
subunit of PP2A is bound to two regulatory subunits: a constant regulatory subunit of
65kDa (PR65), and a variable regulatory subunit that can be one of many diverse proteins
that respond to cell signaling [3].
There are a number of drugs and toxins that target protein phosphatases. One
such toxin is microcystin. Microcystin is a cyclic heptapeptide produced by
cyanobacteria, or blue-green algae. Microcystin is a potent inhibitor of PP1 and PP2A
and a potent liver carcinogen [4]. This inhibition of protein phosphatases causing
diseases implicates the importance of maintaining regulation of the enzymes involved in
reversible phosphorylation.
Over 50 types of microcystin have been identified. The general structure of
microcystin is cyclo(D-Ala-X-D-MeAsp-Y-Adda-D-Glu-Mdha-), where X and Y are
variable amino acids (see Figure 1). Microcystins may vary in degrees of methylation,
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configuration of Adda, or amino acids in the X and Y positions [4]. One of the most
common microcystins is microcystin-LR, where L-leucine is in the X position and Larginine is in the Y position.
Microcystin-LR interacts with PP1 and PP2A in two stages. The initial stage is a
fast (within minutes) binding of the Adda, glutamic acid, and methylaspartic acid
residues to the acidic, C terminal and hydrophobic grooves of PP1. The active site of
PP1 is located at the bifurcation point of these grooves [5]. The second stage is a slow
(over hours) formation of a covalent bond via Michael Addition of the thiol group of
cysteine 273 of PP1 (cysteine 266 of PP2A) to the -unsaturated carbonyl of the Mdha
group of microcystin. Figure 2a shows microcystin-LR in the active site of PP1 and the
covalent bond with the cysteine 273 residue.
An affinity column has been developed to take advantage of the interaction
between microcystin-LR and PP1 [2]. The -unsaturated carbonyl of the Mdha group
is used to link microcystin-LR to a Sepharose matrix, which also prevents a covalent
bond formation with PP1, but does not interfere with toxin-enzyme interactions. This
makes it possible for PP1 to bind to the microcystin-LR on the Sepharose matrix, and
allows its elution. Another aspect of this microcystin-Sepharose column is that the
RVXF motif binding site of PP1 is exposed because it is separate from the active site (see
Figure 2b). As mentioned, many regulatory proteins of PP1 have a RVXF motif, thus the
column allows for the association of these regulatory proteins.
A microcystin-Sepharose affinity column has the potential to purify PP1 and
PP2A as well as show the association of regulatory proteins to these protein
phosphatases. The purification of PP1 and identification of some of its regulatory
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subunits has been done before [2,6]. However, the purification of PP2A using this
method was problematic in the literature [6].
The goal of this project was to create a microcystin-Sepharose column for
purification of PP1 and PP2A and for determining their regulatory subunits. We can learn
more about cell regulation from studying these proteins. In these experiments, we were
successful creating a column for PP1 purification. Preliminary results showed that PP2A
may be eluted with another toxin, okadaic acid, which inhibits PP2A more potently than
microcystin-LR [4].
Experimental Procedures
Materials
Microcystin-LR was obtained by purification of blue-green algae from Little
Beaver Lake in 1992 by Hue Anh Luu. The microcystin was purified by HPLC. Dried
fractions from these purifications were dissolved in water and analyzed for microcystin
by PNPP inhibition assays of PP1, and pooled [7]. Pooled fractions were run on the
HPLC to determine the purity and quantitate the amount of microcystin LR. 2aminoethanethiol, or cysteamine, was from Sigma. N-hydroxysuccinimide-activated CHSepharose 4B was from Amersham Biosciences/GE. PP1 and PP2A were purified by
Hue Anh Luu and Andrea Fong.
Reverse Phase High-Performance Liquid Chromatography
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All HPLC runs were on a Beckman with a microbore Vydac C18 column. HPLC
columns were equilibrated with 0.1% TFA/water and eluted with linear AB gradient
(0.3%B/minute), where solvent A is 0.1% TFA/water and solvent B is 0.1%
TFA/acetonitrile, over 3 hours at a flow rate of 0.2 mL/minute. The wavelength for
detection was 206nm.
Preparation of Aminoethanethiol-Microcystin
The reaction protocol of Moorehead was followed using 1mg of microcystin-LR
in water. The product was purified with a Waters C18 Long Body Plus Sep-Pak
cartiridge. The Sep-Pak was solvated with 10 mL of 0.1%TFA in acetonitrile, then
equilibrated with 10 mL of 0.1% TFA in water. The sample was then loaded and washed
with 10 mL of 0.1% TFA in 10% acetonitrile. Aminoethanethiol-MCLR was eluted off
with 0.1%TFA in acetonitrile until no more eluted off as shown with PNPP inhibition
assays. Eluted fractions containing aminoethanethiol-MCLR were run on the HPLC to
determine the completion of the reaction. The eluted fractions were dried with a
SpeedVac.
Preparation of Microcystin-thioethaneamino-Sepharose
Aminoethanethiol-MCLR (1 mg) was dissolved in 50mM sodium bicarbonate, pH
9.2 and reacted with 3.5 mL of N-hydroxysuccinimide-activated CH-Sepharose 4B resin
end-over-end overnight at 4oC. The resin was then centrifuged and the supernatant was
removed. An aliquot of the supernatant was run on the HPLC to determine the
completeness of the reaction.
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The resin was washed five times alternately with 50mM Tris-HCl pH 8, 0.5M
NaCl and 50 mM sodium acetate pH 4, and 0.5M NaCl. The resin was stored in 20%
ethanol at 4oC.
Preparation of Tris-Sepharose
Activated CH Sepharose 4B was reacted with 50mM Tris-HCl, pH 7.5, end-overend overnight at 4oC. The resin was stored in 20% ethanol at 4oC.
Determination of the Binding Capacity of Microcystin-Sepharose
50L of microcystin-Sepharose resin and of the Tris-Sepharose resin (control)
were placed in Eppendorf tubes and washed with Buffer A (50mM Tris-HCl, 0,1mM
EDTA, 0.5mM MnCl2, 0.2% beta-mercaptoethanol, pH 7.5). PP1 was diluted with
Buffer A and incubated end-over-end with the resin for 1 hour. The resin was then
centrifuged and the supernatant removed. The incubation step was repeated with new
PP1 until the resin was saturated (no longer binds additional PP1). Aliquots of the
supernatant from each step were used in PNPP assays to determine the percentage of
binding by comparing the microcystin-Sepharose resin to the Tris-Sepharose control
resin.
Binding of PP1 to Microcystin-Sepharose
50L of microcystin-Sepharose resin and of the Tris-Sepharose resin (control)
were placed in Eppendorf tubes and washed with Buffer A. PP1 was diluted with Buffer
A and incubated end-over-end with the resin for 1 hour. The resin was washed four times
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with 0.3M NaCl in Buffer A. After the last wash, 5L of resin slurry was removed for
testing with SDS PAGE. PP1 was eluted with 3M NaSCN in Buffer A (incubated endover-end for 30 minutes at 4oC). An aliquot of the elution was used for an SDS PAGE
gel. The resin was then washed four times with 0.3M NaCl in Buffer A. After the last
wash, 5L of resin slurry was removed for an SDS PAGE gel.
Binding of PP2A to Microcystin-Sepharose
The same procedure as the PP1 binding experiment was used with the exception
of the elution step. Okadaic acid, in excess, was used to elute the PP2A from the resin.
Results
Quantitation of Microcystin by HPLC
A standard of microcystin-LR was run on the HPLC to determine the retention
time (Figure 3a). The retention time for microcystin-LR was 121.04 minutes. Pooled
fractions of microcystin that were run on the HPLC showed a microcystin-LR peak at
121.27 minutes, but there were also impurities (Figure 3b). The total microcystin-LR in
the pooled fractions was calculated to be ~10 mg based on comparison of peak heights in
the chromatograms.
Preparation of Aminoethanethiol-Microcrystin
An aliquot of the reaction product of microcystin treated with aminoethanethiol
that was purified on the Sep-Pak cartridge was run on the HPLC (Figure 3c). There was
a shift in the retention time of microcystin-LR from 121.04 minutes before the reaction to
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113.89/115.02 minutes after the reaction. There was no residual peak at 121.27 minutes,
thus the reaction is assumed to be ~95% complete.
Preparation of Microcystin-thioethaneamino-Sepharose
An aliquot of the supernatant after centrifuging the reacted aminoethanethiolmicrocystin-LR and NHS-activated CH Sepharose 4B was run on the HPLC (Figure 3d).
The missing peak at 113.89/115.02 minutes indicates the absence of aminoethanethiolmicrocystin in solution. This was expected if aminoethanethiol-microcystin was linked
to the Sepharose that was removed from solution by centrifugation. The absence of the
peak at 113.89/115.02 minutes suggests the reaction went to ~95% completion.
Binding Capacity of Microcystin-Sepharose
Activity assays of PNPP were carried out with aliquots of the supernatant after
incubation of the resin with PP1. The percentage of activity compared to the control was
used to calculate the quantity of PP1 that bound to the resin. After four additions of PP1,
59 g of PP1 bound to 50 L of resin, which was calculated to contain approximately
14.3 g of microcystin. However, the saturation point was not reached, thus 59g is a
lower limit of the binding capacity. (Unfortunately, there were time constraints.) 1 mg
of microcystin was calculated to bind 4.2mg of PP1.
PP1 Binding Experiments
Figure 4 shows the SDS PAGE gel of the experiment. Comparison between the
control Tris-Sepharose resin and the microcystin-Sepharose resin shows that PP1 bound
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specifically to the microcystin-Sepharose resin. The markers are in lane 1. Lane 2 was
the PP1 that was incubated with the resin. Lanes 3 and 4 were the resins after incubation
with PP1 and then washed with 0.3M NaCl. Lanes 5 and 6 were the resins after elution
with 3M NaSCN and lanes 7 and 8 were the elutions. The faint band in lane 6, the
microcystin-Sepharose resin after elution, indicates there was residual PP1 that did not
elute from the resin. The elution in lane 8 confirms that PP1 was eluted with 3M
NaSCN.
PP2A Binding Experiments
Figure 5 shows the SDS PAGE gel of the experiment. Comparison between the
control Tris-Sepharose resin and the microcystin-Sepharose resin shows that PP2A bound
specifically to the microcystin-Sepharose resin. The markers are in lane 1. Lane 2 was
the PP2A that was incubated with the resin. Lanes 3 and 4 were the resins after
incubation with PP2A and then washed with 0.3M NaCl. Lanes 5 and 6 were the resins
after elution with excess okadaic acid and lanes 7 and 8 were the elutions. The band in
lane 6, the microcystin-Sepharose resin after elution, indicates there was residual PP2A
that was not eluted by okadaic acid. There is a very faint band in lane 8, which represents
elution of PP2A by okadaic acid.
Discussion
This project was successful in creating a microcystin-Sepharose for purifying PP1
and PP2A. Based on the quantitation using HPLC, there are approximately 10mg of
microcystin-LR in the pooled fractions that can be use for creating microcystin-
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Sepharose resin. From the binding capacity experiments, 10mg of microcystin-LR can
bind 42 mg of PP1, which would make large-scale purification of PP1 possible.
The reactions involved in creating the resin, microcystin-LR with
aminoethanethiol and the product of that reaction with NHS-activated Sepharose, were
high yielding, ~95%. Though there were impurities in the pooled fractions (figure 3b)
and appeared to react with aminoethanethiol (figure 3c shows that all peaks were shifted
to the left), they did not react with the NHS-activated Sepharose (figure 3d). This
showed that purification of microcystin-LR before any reactions was unnecessary
because reactions with NHS-activated Sepharose are specific for microcystin.
The binding capacity experiments showed that at least 59 g of PP1 bound to 50
L of resin that contains ~14.3 g of microcystin-LR. Unfortunately, the maximum
capacity of the resin was not determined due to time constraints. Work can be continued
to determine the saturation point. This is important to establish because it will allow
more efficient purification of PP1 and more efficient binding of regulatory proteins to
PP1.
The binding experiments with PP1 verified that it could be purified using
microcystin-Sepharose resin, as shown in the literature [2]. Comparing lanes 3 and 4 of
figure 4, PP1 bound to the microcystin-Sepharose resin but not the control (TrisSepharose) resin indicating that PP1 bound specifically and not non-specifically. Also,
PP1 used in this experiment was not completely pure as there are visible faint bands at 15
and 75 kDa (lane 2, figure 4), but these proteins did not bind to the microcystinSepharose resin. This shows this resin can be used for purification.
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PP1 was successfully eluted with 3M NaSCN as shown with the band in lane 8
(figure 4), though some PP1 remained on the resin (lane 6, figure 4). This implies the
resin may only be used a finite number of times. However, the residual PP1 may be
eluted with more 3M NaSCN or with denaturation methods, such as SDS [6].
Using microcystin-Sepharose for purification of PP2A has been problematic in
the past [6]. PP2A binds to the resin so strongly that it could only come off when boiled
in SDS. In this experiment, we used another toxin, okadaic acid, to compete with
microcystin-Sepharose for the binding site of PP2A. Okadaic acid is a more potent
inhibitor of PP2A and unlike microcystin, it does not form a covalent bond with the
phophatase.
The binding experiments with PP2A showed that PP2A, like PP1, binds
specifically to the microcystin-Sepharose because it did not bind to the control resin. The
elution (lane 8, figure 5) showed a very faint band suggesting PP2A was successfully
eluted with okadaic acid. The band from the resin after elution (lane 6, figure 5) was also
lighter than the band from before elution. If PP2A can be eluted from the resin,
microcystin-Sepharose would be a good method for purification because the PP2A used
for binding had contaminating proteins (lane 2, figure 5), which did not bind to the resin
(lane 4, figure 5).
The microcystin-Sepharose affinity column may be a method of obtaining pure
PP2A for crystallography and thus structure determination. The structure of PP2A has
not been determined, unlike that of PP1. Current methods of purifying PP2A may not
suffice to produce high enough purity for crystals to form. From the SDS PAGE gel
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(lane 2, figure 5), it is obvious the PP2A had many contaminating proteins, which would
hinder crystal growth.
Future work with the microcystin-Sepharose resin can be done to determine the
association of proteins with PP1 and PP2A. In this case, it would be important to saturate
the resin with the enzyme to maximize the efficiency of protein binding. Though both
phosphatases bind to the resin, PP1 can be eluted with a different method than PP2A (3M
NaSCN) to ensure only PP2A is present on the resin. Thus, the resin can be used to
determine the regulatory subunits of PP1 and PP2A and would give a great deal of
information about cell regulation involving reversible phosphorylation.
A microcystin-Sepharose affinity column was successfully produced and shown
how it can purify PP1 and PP2A. It also has the potential for determining the regulatory
subunits of these protein phosphatases and providing insight into cell regulation, which
may also lead to new drug targets for diseases.
Acknowledgements
I would like to thank Dr. Charles F.B. Holmes for his supervision and the opportunity to
work in the Holmes lab and all members of the Holmes lab for providing assistance and
materials. I would like to especially thank Marcia Craig for all her supervision and
technical assistance.
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References
[1] Cohen, P. (2002) Nature Cell Biology 4, E127-E130
[2] Moorhead, G., MacKintosh, R.W., Morrice, N., Gallagher, T., MacKintosh, C. (1994)
FEBS Letters 356, 46-50
[3] Zolnierowicz, S., Van Hoof, C., Andjelkovic, N., Cron, P., Stevens, I., Merleved, W.,
Goris, J., Hemmings, B.A. (1996) Biochem. J. 317, 187-194
[4] Craig, M., Holmes, C.F.B. (2000) Freshwater Hepatotoxins Microcystin and
Nodularin; Mechanism of Toxicity and Effects on Health. Seafood and Freshwater
Toxins: Pharmacology,Physiology and Detection, Luis Botana, ed. Marcel Dekker,
New York, Basel
[5] Goldberg, J., Huang, H., Kwan, Y., Greengard, P., Nairn, A., Kuriyan, J. (1995)
Nature 376, 745-753
[6] Tran, H., Ulke, A., Morrice, N., Johannes, C., Moorhead, G.B.G. (2004) Molecular &
Cellular Proteomics 3, 257-265
[7] An, J-S., Carmicheal, W.W. (1994) Toxicon 32, 1495-1507
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