Transcript Document

Daniel L. Allen and Colin S. Burns
Chemistry Department
East Carolina University
[email protected]
A Literature and Experiment Based Evaluation of
Synthesis Techniques for Prothymosin-α Derived Peptides
General Protein Information
Prothymosin-α (ProTa) is a protein made up of 110 amino acids and located primarily in
the nucleus of cells. The precise function of this protein is unclear, however evidence
strongly suggests it is associated with several cellular activities and, in particular, cell
proliferation. Prior research indicates that ProTa has the following attributes:
•Chromatin Remodeling Activity1
•Immunomodulatory Activity2
•Growth-Promoting Effect3
•Zinc-Binding Property4
•Enhancement of Estrogen Receptor Transcriptional Activity5
•Protection of Cellular Apoptosis6
•Oxidative Stress Defense Gene Regulation6
•Enhancement of T Lymphocyte Function7
Because of ProTa’s high level of expression in a wide variety of cell types and the fact
that it is relatively conserved (especially in vertebrates), as can be seen in the alignments
of Figure 1 to the right, it is expected to play an essential role in organisms. Recent
research has also revealed that this protein possesses significant anti-HIV activity and
efforts are underway to understand how it suppresses viral replication. The use of
synthetic peptides derived from the central region of ProTa has also been shown to be
effective at inhibiting HIV replication. These small ProTa derived peptides can also be
used as a means to test the effects of variations in sequence identity in order to increase
the desired functional response in cells (anti-HIV activity for example). There is further
evidence that ProTa may be phosphorylated in vivo, and we hypothesize that this
phosphorylation may be significant with regards to its anti-HIV activity. Since the
phosphorylation state of a protein can be used to control its activity (i.e. functionally
turn it on or off), we would also like to know if phosphorylation increases or decreases
the protein’s anti-HIV properties. The protein itself can undergo many known posttranslational modifications and these too are shown in Figure 1 to the right and include
the following:
•Initiator Methionine Removal8
•N-Acetylation9
•Phosphorylation of Serine Residues10, 11
•Phosphorylation of Threonine Residues12, 13
•Caspase Cleavage at Three Binding Sites14
In addition to its wide range of functions and post-translational modifications, ProTa has
also been found to exist at multiple sites in the human genome. Interestingly, the
duplication events did not produce repeated genes ordered sequentially, but rather
created copies of the gene throughout the genome as can be seen in Figure 2 to the right.
Similar patterns exist in other species which indicates a temporally spaced series of
duplication events, especially in vertebrates. For example, Homo sapiens (humans) have
19 copies15, Pan troglodytes (chimpanzee) 2216, Mus musculus (mouse) 2617, Bos taurus
(cow) 1318, Danio rerio (zebra fish) 219, and Gallus gallus (chicken) 120. As can be seen
by comparing the human karyogram in Figure 2 to the chimpanzee’s in Figure 3, a broad
level of evolutionary conservation exists with regard to ProTa gene location. This is
most apparent when viewing chromosomes 6, 7, 11, 13, 14, 17 and 20, which have gene
placements in identical locations. The pattern of placement throughout the genome
indicates that the gene may be part of a transposon and/or that the various duplicates
may be expressed in response to varied signals which correspond to the different
functions that ProTa possesses. Further research would be necessary to evaluate these
hypotheses.
Evaluation of Synthesis Techniques
Figure 1
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1
------M SDA
------MSDA
------MSDA
------MSDA
----------------SDA
------MSDA
------MSDT
------MSDT
------MSDT
------MADT
------MADT
-----MESVA
MSTRKRKQEA
11
AVDTSSEITT
AVDTSSEITT
AVDTSSEITT
AVDTSSEITT
---------AVDTSSEITT
AVDTSSEITT
SVDASVEKTT
AVDASVEKTP
AVDASVEKTT
KVDTSKEVSA
KVDK-KEISA
AHKDEVTHVE
EDEEELQALP
21
KDLKEK-KEV
KDLKEK-KEV
KDLKEK-KEV
KDLKEK-KEV
----EK-KEV
KDLKXX-KEV
KDLKEK-KEV
KDLKSKDKEL
KDLKAKEKEV
KDLKAKEKEV
KDLKEK--KQ
KDLKEK--KL
SKIEELKRKS
SDESEEEEEY
31
VEEAEN GRDA
VEEAENGRDV
VEEAENGREA
VEEAENGRDA
VEEAENGREA
VEEAENGREA
VEEAENGRDA
VEETENGKDK
VEEAENGKDK
VEEAENGKDK
VEEAENGKDA
VEEAENGKDA
VDAKDVADHE
QSSDAEGDDY
41
PANGNAENEE
PANGNA-NEE
PANGNA-NEE
PANGNAQNEE
PANGNA-NEE
PANGNA-NEE
PANGNAQNEE
PANGNAENEE
PTNGNAENEE
PTNGKAENEE
PANGNA--EN
PANGKKAKEA
AKNGN--DEP
DDSEEGSEEE
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NGEQEADNEV
NGEQEADNEV
NGEQEADNEV
NGEQEADNEV
NGEQEA---NGEQEADNEV
NGEQEADNEV
NGEDGADNENGEQE--NEG
NGEPEVDNEG
EENGDQENEV
EENGDVED-V
VVKKIRKEAG
EEEEYDEDEG
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EEDGDEDEEA
EEDGDEDEEA
EEDGDEDEEA
EEDGDEDEEA
---------EEDGDEDEEA
EEDGDEDEEA
DDEGDEDDEA
DD--DEDDEV
DD--DDDDEV
DDD--EDEEA
EDD-EDDDEV
DEDGTEDDAS
KAEDQEDDDE
91
ESATGKRAAE
ESATGKRAAE
EAATGKRAAE
EAPTGKRVAE
-APTGKRAAE
EAATGKRAAE
EAPTGKRVAE
DGATGKRAAE
EGATGKRAAE
EGVTGKRAAE
EGGTGKRAAE
EGRAGKRAAE
QDTKGTNGTS
DGAGEEYDEE
101
DD-----EDD
DD-----EDD
DD-----EDD
DD-----EDD
DD-----EDD
DD-----EDD
DD-----EDD
DD----DEDD
DDE---DDDD
DDE---DDDD
DD----DDDE
DDEDDDDDDE
KCC------DDEGTNDTAD
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D----VDTKK
D----VDTKK
D----VDTKK
D----VDTKK
D----VDTKK
D----VDTKK
D----VETKK
D----VDAKK
D----VETKK
DD---VEIKK
DD---VDPKK
ED---VGTKK
-----VCVIT
KSGSAQAAKK
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QK----TDED
QK----TDED
QK----TDED
QK----TEED
QK----TDED
QK----TDED
QKK---TDED
QK----TDDD
QK----TDED
QK----TDED
QK----TDVQK----TD-D
S--------GKGGDVPQED
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D-------D-------D-------D-------D-------D-------D-------D-------D-------D---------------D---------------DVSEVEDED
Species Key
Variations from Human Sequence
1. Homo sapiens (Human) (0 changes)9
2. Pongo abelii (Sumatran orangutan) (2 changes)21
3. Bos taurus (Bovine) (3 changes)22
4. Mus Musculus (Mouse) (4 changes)23
5. Capra hircus (Goat) (4 changes)24
6. Sus scrofa (Pig) (5 changes)25
7. Rattus norvegicus (Rat) (7 changes)26
8. Rana esculenta (Edible frog) (25 changes)27
9. Xenopus tropicalis (Western clawed frog) (26 changes)28
10. Xenopus laevis (African clawed frog) (32 changes)29
11. Danio rerio (Zebrafish) (38 changes)30
12. Salmo salar (Atlantic salmon) (46 changes)31
13. Schistosoma mansoni (Blood fluke) (83 changes)32
14. Penicillium marneffei (strain ATCC 18224 / CBS 334.59 / QM 7333) (117
changes)33
61
DEEEEEGGEE
DEEEEEGGEE
DEEEEEGGEE
DEEEEEGGEE
---------DEEEEEGGEE
DEEEEEGGEE
-EEEEVD--E
DEEEEVD--E
DEEDEVD--E
DEEDDDV-AE
DEEEDVG-EE
DTDEDEKAHS
GEESKENAPA
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EEEEEEGDGE
EEEEEEGDGE
EEEEEEGDGE
EEEEEEGDGE
---------EEEEEEGDGE
EEEEEEGDGE
EDEEDEGEGD
EDEEDEVEGD
EDEEDEVEGE
EDEEDDGEGD
EDEEEDVEGD
PESRSAVAEL
PKKRKTAHDD
The goal of the experimental portion of this project was to develop useful techniques for
synthesizing small ProTa derived peptides to be used to evaluate the protein’s anti-HIV
activity. Specifically, three such peptides were created which addressed the issues
associated with the creation of phosphorylated peptides in vitro as well as glutamimide
formation as an unwanted side reaction during synthesis.
The first two peptides dealt with the difficulty of creating peptides with bulky
phosphorylated residues. A ten residue amino acid chain corresponding to residues 8190 in the human ProTa sequence (EAESATGKRA) was first created with a
phosphoserine at a 0.1 mmol scale. All reagents were in 0.4 mmol excess except for the
phosphoserine which was used in 0.3 mmol excess to reduce wasted reagent. The
difficulty of the phosphoserine coupling was circumvented by allowing the coupling
times of the residue and the subsequent residue to be increased to 2 hours (from the
usual 1 hour). The resultant peptide was purified via HPLC and then analyzed by mass
spectrometry. It was found to be highly successful with o a small percentage of the
product containing deletions. What’s more, the vast majority of the deletions that did
occur were in residues other than the phosphoserine meaning that the conditions and
extended coupling time used for that residue were adequate.
Post-Translational Modifications to Human Sequence
7 Initiator methionine removed8
8-36 Thymosin α1
8-139 Prothymosin α
8 N-acetylation9, Phosphoserine for approximately 2% of prothymosin during
entire cell cycle10, 11
14, 19, 20 Phosphothreonine created in the cytosol by prothymosin α
phosphorylating kinase which is composed of two protein subunits of
mass 64 and 60 kDa12
127 Phosphothreonine13
47 Deletion in prothymosin α isoform 2
101-117 3 caspase binding sites (DDED, DEDD, DDVD)14
96-97, 119-122 Proposed nuclear localization site14
This figure shows the various post-translational modifications of Human ProTa as well as the level of conservation of sequence between
various species (see keys above). Note that the numbers associated with the modifications listed correspond to their position in the
graphic and not the number of the amino acid(s) affected in the human sequence.
Figure 2
This figure shows a human karyogram with the various locations
of ProTa replicates marked by arrows. The most highly expressed
replicate is marked by a rectangle.15
The second phosphorylated peptide synthesized utilized the same sequence and
conditions except that a phosphothreonine was used and not a phosphoserine. In the case
of this peptide, both the conditions used and results obtained were the same meaning
that extended coupling times again produced proper incorporation of the phosphorylated
residue.
Figure 3
This figure shows a chimpanzee karyogram with the various
locations of ProTa replicates marked by arrows. The most highly
expressed replicate is marked by a rectangle.16
Conclusion
The experimental goals of this project centered on developing high-fidelity synthesis
techniques for both the highly charged phosphorylated as well as other glutamimide
prone ProTa derived peptides. These were both elucidated with a high success rate
attributable to altered coupling times and conditions during the synthesis process. The
literature evaluation focused on mapping the points of post-translational modifications,
comparing the human sequence to various phylogenetic lineages, assessing and
describing the various functions attributed to the peptide as well as where these
functions are expressed, and assessing the expression of the protein throughout the cell
cycle. As research continues, elucidation of the evolution of the protein and the
ramifications this has towards the overall functions will be explored. Additional testing
of the ProTa derived peptides for anti-HIV activity will undoubtedly continue.
The final peptide synthesized was derived from residues 66-75 of human ProTa
(EEEGDGEEED) and was created to model techniques to evade cyclization and
glutamimide formation during synthesis of larger peptide sequences. It utilized a benzyl
ester protecting group (Obzl) on the glutamate residues to prevent both synthetic as
well as analytical problems attributable to negative charges. Additionally, this peptide
used a nonconventional dimethoxybenzyl protected glycine at position 71 as a way to
prevent the previously mentioned glutamimide formation. As before, a 0.4 mmol surplus
of reagents was used for all residues except for the Fmoc-(Dmb)Gly-OH where a 0.3
mmol excess was used. The products were again purified and analyzed and found to
contain a much smaller percentage of cyclized peptides compared to the non-Dmb
protected product.
References
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1112. Pages: 201-209. 2007. Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Santiago de Compostela, Santiago, Galicia, Spain.
2 “Novel Function of Prothymosin Alpha as a Potent Inhibitor of Human Immunodeficiency Virus Type 1 Gene Expression in Primary Macrophages.” Mosoian, Arevik, et. al. J. Virol. 2006 80: 9200-9206.
3 “Prothymosin α promotes cell proliferation in NIH3T3 cells.” Wu C.-L.; et. al. LifeSciences Volume 61, Number 21, 17 October 1997 , pp. 2091-2101(11).
4 “TI: Divalent metal cation binding properties of human prothymosin α.” Nina V. Chichkova, et. al. European Journal of Biochemistry. Vol: 267.15. Pages 4745-4752. 2000. Belozersky Institute of Physico-Chemical Biology
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5“Prothymosin Alpha Selectively Enhances Estrogen Receptor Transcriptional Activity by Interacting with a Repressor of Estrogen Receptor Activity.” Martini, Paolo G. V et. al. Mol. Cell. Biol. 2000 20: 6224-6232.
6 "New Functions of a Well-Known Protein: Prothymosin α Is Involved in Protecting Cells from Apoptosis and Oxidative Stress." Evstafieva, A. G., et al. Molecular Biology 39.5 (Sep. 2005): 631-645. Biomedical Reference
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7 “Enhancement of Human T Lymphocyte Function by Prothymosin α: Incremed Production of Interleukin-2 and Expression of Interleukin-2 Receptors in Normal Human Peripheral Blood t Lymphocytes.” Constantin N.
Baxevanis, et. al. Immunopharmacology and Immunotoxicology, 1990, Vol. 12, No. 4, Pages 595-617.
8Identification of N-terminal acetylation of recombinant human prothymosin alpha in Escherichia coli. Wu J et. al. Biophys Acta. 2006 Aug;1760(8):1241-7. Epub 2006 Apr 19. [PubMed: 16737780]
9"The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA." Eschenfeldt W.H., Berger S.L. Proc. Natl. Acad. Sci. U.S.A. 83:9403-9407(1986) [PubMed:
3467312]
10Phosphorylation of human and bovine prothymosin alpha in vivo. Sburlati A.R., et. al. Biochemistry 32:4587-4596(1993) [PubMed: 8485135]
11Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Olsen J.V., et. al. Cell 127:635-648(2006) [PubMed: 17081983]
12A 180-kDa protein kinase seems to be responsible for the phosphorylation of prothymosin alpha observed in proliferating cells. Pérez-Estévez A, et. al. J Biol Chem. 1997 Apr 18;272(16):10506-13 [PubMed: 9099694]
13A quantitative atlas of mitotic phosphorylation. Dephoure N., et. al. Natl. Acad. Sci. U.S.A. 105:10762-10767(2008) [PubMed: 18669648]
14The thymosins. Prothymosin alpha, parathymosin, and beta-thymosins: structure and function. Hannappel E, Huff T. Vitam Horm. 2003;66:257-96. Review. [PubMed: 12852257]
15http://www.ensembl.org/Multi/blastview/BLA_9I9mYNTM6
16http://www.ensembl.org/Multi/blastview/BLA_fnr5Fq8S9
17http://www.ensembl.org/Multi/blastview/BLA_NRZ5NXXQM
18http://www.ensembl.org/Multi/blastview/BLA_dhOq7d6bV
19http://www.ensembl.org/Multi/blastview/BLA_c6kDCmCZY
20http://www.ensembl.org/Multi/blastview/BLA_nIrLon2Jk
21The German cDNA consortium. Submitted (NOV-2004) to the EMBL/GenBank/DDBJ databases
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23"Nucleotide sequence of the murine prothymosin alpha cDNA and its deduced primary and secondary protein structure." Schmidt G., et. al. Biophys. Acta 1088:442-444(1991) [PubMed: 2015308]
24"Isolation and partial sequence of goat spleen prothymosin alpha." Frillingos S., et. al. Cell. Biochem. 108:85-94(1991) [PubMed: 1770947]
25"Evaluation and characterization of a porcine small intestine cDNA library: analysis of 839 clones." Winteroe A.K., et. al. Genome 7:509-517(1996) [PubMed: 8672129]
26"Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs." Frangou-Lazaridis M., et. al. Biochem. Biophys. 263:305-310(1988) [PubMed: 3377505]
27"First evidence of prothymosin in a non-mammalian vertebrate and its involvement in the spermatogenesis of the frog Rana esculenta." Aniello F., et. al. 110:213-217(2002) [PubMed: 11744386]
28NIH - Xenopus Gene Collection (XGC) project Submitted (FEB-2005) to the EMBL/GenBank/DDBJ databases
29NIH - Xenopus Gene Collection (XGC) project Submitted (JUN-2003) to the EMBL/GenBank/DDBJ databases
30"A zebrafish prothymosin alpha-like protein selectively enhances proliferation of embryonic hematopoietic progenitor cells." Marty S.D., et. al. Submitted (APR-2001) to the EMBL/GenBank/DDBJ databases
31"Salmo salar full-length cDNAs." cGRASP (B.F. Koop & W.S. Davidson) Leong J., et. al. Submitted (JAN-2009) to the EMBL/GenBank/DDBJ
32"The genome of the blood fluke Schistosoma mansoni." Berriman M., et. al. Submitted (APR-2009) to the EMBL/GenBank/DDBJ databases
33"Genome sequence of Penicillium marneffei strain ATCC 18224." Fedorova N.D., et. al. Submitted (OCT-2007) to the EMBL/GenBank/DDBJ databases
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