Molecules in focus
Prothymosin a
Fernando Segade a ,Jaime Go
mez-Ma rquez b,*a
Department of Cell Biology and Physiology,Washington University School of Medicine,Box 8228,660South Euclid Ave.,St.Louis,
启航考研班怎么样MO 63110,USA
b
Departamento de BioquõÂm ica y BiologõÂa Molecular,Facultad de BiologõÂa ,Universidad de Santiago de Compostela,Campus Sur,
15706Santiago,Spain
Received 29March 1999;accepted 9July 1999
Abstract
Prothymosin a (ProT a )is a highly acidic protein widely distributed in mammalian cells.Since its discovery in 1984,the biological role of this protein has been controversial.Initially,ProT a was considered a thymic factor with a hormonal-like role in the maturation of T-lymphocytes.However,molecular and cellular analys led to conclude that ProT a is a nuclear protein required in proliferation events while failing to show a clear immunological e ect.The involvement of ProT a in changes in the compaction state of chromatin has been recently elucidated with the demonstration that this protein induces the unfolding of chromatin ®bres in a process that ems to be mediated by the interaction of ProT a with histone H1.This ®nding opens up new perspectives in the study of the dynamics of the genetic material in mammalian cells.Furthermore,the relationship between ProT a and apoptosis as well as with proliferation makes this protein an attractive target in the arch for modulators of cell death and tumour growth.#1999Published by Elvier Science Ltd.All rights rerved.
Keywords:Prothymosin a ;Histone H1;Chromatin decondensation;Cell proliferation
1.Introduction
Prothymosin a (ProT a )was ®rst isolated from rat thymus [1]as the precursor of thymosin a 1(T a 1),a small peptide (28amino acids)once con-sidered as a `thymic hormone'(reviewed in [2,3]).
According to the prevailing model at the time,
T a 1was a thymus-speci®c peptide with a critical involvement in the maturation and development of the immune system promoting the intrathymic maturation of T cells.By similarity to other short active peptides,it was hypothesid that T a 1and certain structurally related peptides,were not primary translation products but instead derived from a proteolytically procesd precursor.In the arch for the T a 1precursor,ProT a was successfully puri®ed from thymus only when strongly denaturing conditions that
The International Journal of Biochemistry &Cell Biology 31(1999)1243±1248
1357-2725/99/$-e front matter #1999Published by Elvier Science Ltd.All rights rerved.PII:S 1357-2725(99)00094-
1
/locate/ijbcb
*Corresponding author.Tel.:+34-981-563-100line 13310;fax:+34-981-596-904.
汉英在线翻译E-mail address:bnjgm@usc.es (J.Go mez-Ma rquez)Abbreviations:ProT a :prothymosin a ;H1:histone H1;T a 1:thymosin a 1.
prevented extensive proteolytic degradation were ud[1].Analysis of the ProT a quence indi-cated that T a1comprid the®rst28residues of ProT a.Since the extraction conditions that led to the isolation of ProT a did not yield T a1,it was suggested that ProT a was the only peptide pre-nt in cells.Accordingly,T a1and related pep-tides would be just artefacts generated by cleavage of ProT a during the extraction pro-cedures and not the result of speci®c processing events[3].However,whether or not T a1and re-lated peptides are natural products,arising from the intracellular turnover of ProT a,still remains to be de®nitively answered.
Several developments,including the detection of ProT a protein and mRNA in lymphoid and non-lymphoid mammalian tissues[4,5],the fact that ProT a does not contain signal peptide and that its mRNA is translated on free polyribo-somes,cast strong doubts on the concept of ProT a as a cretory peptide.The extremely acidic character of ProT a,its wide distribution and abundance and t
he prence of a putative nuclear localisation signal,led us to propo an intranuclear site of action for ProT a[6].This hy-pothesis was subquently con®rmed by veral groups[7,8]and,currently,the ascription of extracellular functions to ProT a in the immune system has been mostly abandoned.Nevertheless, some authors still consider ProT a and its related peptides as`thymic hormones'or biological re-spon modi®ers and u them in veral preclini-cal and clinical studies.While ProT a has exhibited some e ects in certain pharmacological or immunological assays,what is prently known about the biology of ProT a rules out that such`biological activities'are related to the phys-iological function of ProT a.gway
2.Structure
ProT a is a small protein(109±113amino acid residues),almost identical in all mammalian species where it has been isolated and shows v-eral atypical features[3].The protein lacks hydrophobic regions that could act as cretory signals,is devoid of histidine,sulphur and aro-matic residues.ProT a is highly hydrophilic and acidic(p I3.55)as expected from the abundance of aspartic and glutamic acids(50%of the total number of residues).Most of the acidic residues are clustered in the central region in what re-mbles a putative histone binding domain.Two small stretches of basic residues are located at both ends of the quence.The motif TKKQK at the C-terminus is a potent signal for th
e nuclear localisation of ProT a,whereas a very similar quence,KKEKK,located at the N-terminus apparently does not posss nuclear import ac-tivity.In physiological conditions ProT a is a monomeric protein that adopts a random coil conformation without any regular condary structure[9],what perhaps explains the poor immunogenicity of this molecule.
ProT a is a very unusual phosphoprotein. Although the quence contains three motifs that remble the connsus for phosphorylation by cain kina II and veral rine and threonine residues are phosphorylated in vitro by cain kina II,not more than2%of puri®ed calf ProT a contain phosphate on the aminoterminal rine,the only residue found phosphorylated in vivo.A ries of studies established that native ProT a is highly modi®ed by phosphorylation and contains stoichiometric amounts of glutamyl phosphate[10].The apparent abnce of phos-phate in puri®ed ProT a is due to the extreme sus-ceptibility of the glutamyl phosphates to hydrolisis and the rapid transfer of the phosphate moeity to nearby rine residues to give stable phosphorine.
In humans,the gene for ProT a,PTMA, belongs to a multigene family of six members [11].Five members are procesd pudogenes and the sixth member of the family is the func-tional ProT a gene which,either using a canonical or a cryptic splice site,gives ri to two mRNAs who translation produces two ProT a variants di ering in one internal glutamic acid residue. The transcripti生活大爆炸第九季
on unit of PTMA shows a typical exon/intron structure and is split into®ve exons that span about3kb on chromosome2.Exon1 contains the5'untranslated region and the in-itiation methionine within a region that conforms to the Kozak connsus,whereas exon5includes
F.Segade,J.GoÂmez-MaÂrquez/The International Journal of Biochemistry&Cell Biology31(1999)1243±1248 1244
the stop codon and the entire 3'untranslated region.A polyadenylation signal is also prent in exon 5.Figure 1summaris the main charac-teristics of the ProT a gene and protein.
3.Synthesis and degradation
ProT a is widely expresd in mammalian cells and tissues [4,5].It is found in the nucleus of every mammalian cell and is continuously prent in all stages of the cell cycle [3,12].The ex-pression of the ProT a gene was found to be cor-related with cell proliferation in a wide variety of cells providing strong evidence for the involve-ment of ProT a in cell growth.The correlation between proliferation and ProT a gene expression was ®rst reported in NIH3T3®broblasts that were induced to proliferate by rum stimulation [4]and,subquently,in lymphocytes [5].Many other systems responded to growth stimulation by increasing the levels of ProT a transcript:con-canavalin/IL2-stimulated lymphoc
ytes,thymo-cytes and hepatocytes during liver regeneration [12].Moreover,the transcription of the ProT a gene has been found regulated by the c-myc gene [13],a key player in the control of proliferation.The conception that ProT a is directly required for proliferation was ®rst obrved in synchro-nid human myeloma cells;the cells were
unable to divide when cultured in the prence of ProT a antin oligonucleotides until the dis-appeared from the medium [14].A later report showed that the do±respon e ect exerted by ProT a antin oligonucleotides in human HL-60cells,produced an inhibition of cell prolifer-ation at low concentrations and an induction of apoptosis at higher concentrations [15].This ®nd-ing suggests that the actions of ProT a are critical not only for proliferation but also for the integ-rity of the mammalian cell.Not unexpectedly,the overexpression of ProT a was able to acceler-ate cell proliferation,shortening the duration of the G1pha [16,17]and retard di erentiation of HL-60cells [17].
Little is known about the degradation pathway of ProT a inside the cell.Nonetheless,the identi®-cation of veral peptides (T a 1and related pep-tides)that di er only in their length,and who entire quences are prent at the N-terminus of ProT a ,makes conceivable that the peptides could be normal intermediaries originated during the catabolism of ProT a .4.Biological function
Until a short time ago,remained the biological function of ProT a elusive and confusing.Since its discovery,veral physiological roles have been propod [3].Although some authors still support a role related to immunity,the molecular analys carried out on ProT a both at protein and gene levels have ®nally provided indisputable evidence that favour only an intracellular func-tion inside the nucleus.Nevertheless,the intra-nuclear function of ProT a has also been controversial and activities in nucleosome asm-bly/disasmbly,in the regulation of cell cycle,in the nucleocytoplasmic transport and in the pro-duction,processing or export of RNA,were advanced at one time or another [3,10].
There are veral pieces of evidence that put together suggest an involvement of ProT a in chromatin organisation:its high concentration in the nucleus,its acidic nature which rembles some chromatin/chromosomal proteins,its struc-tural similarity to nucleoplasmin (a
chromatin
Fig.1.Structure of the human PTMA gene,showing the ®ve
exons (boxed,with the protein coding regions in black)and the ProT a protein,indicating the N-terminal basic domain,the localisation of T a 1,the central acidic region and the nuclear localisation si
gnal (NLS)domain.The asterisk indi-cates the position of the additional Glu residue in the alterna-tively spliced variant.
F.Segade,J.Go Âmez-Ma Ârquez /The International Journal of Biochemistry &Cell Biology 31(1999)1243±12481245
remodelling protein in Xenopus )and its ability to bind the linker histone H1(H1)in vitro.Recently,two di erent groups have focud their rearch on the activity of ProT a in relation to chromatin.One of them concluded that this pro-tein participates in chromatin decondensation through its interaction with H1[18].The authors supported such conclusion bad on the following facts:the accessibility of the microcco-cal nuclea to chromatin was greatly incread in HL-60cells overexpressing ProT a and the size of the nuclea-generated fragments corre-sponded to tho lacking H1.Therefore it is likely that ProT a acts by removing H1from chromatin inducing its decondensation.Simultaneously,another authors reported the immunoisolation of H1-ProT a complexes from NIH 3T3cell extracts [19]providing strong evi-dence for the existence of an interaction between both proteins in vivo.Furthermore,they carried out a detailed analysis of the interactions ProT a -H1and its e ect on the association of H1with chromatin.The conclusion was that ProT a may modulate the interaction of H1with chromatin by contacts between the acidic region of P
roT a and H1.Bad on the and previous ®ndings,in Fig.2we show a hypothetical model to explain the action of ProT a in the changes of the high-order structure of chromatin.therefore的用法
The elucidation of the function of ProT a opens new and exciting possibilities to under-stand the structure and dynamics of chromatin.The prence of ProT a along the cell cycle together with its ability to interact with H1suggests that ProT a may participate,directly or indirectly,in esntial process that require prior decondensation of the chromatin ®bres such as transcription and/or DNA replication.Moreover,it is tempting to speculate that ProT a might be one of the master proteins that participate also in the unpacking of chromosomes that takes place after mitosis to begin the cell
cycle.
Fig.2.Schematic reprentation of the propod role of ProT a in the decondensation of chromatin through its interaction with his-tone H1.According to this model,the interaction of ProT a with H1provokes the unfolding of the 30-nm chromatin ®bres facilitat-ing the occurrence of biochemical process that need chromatin decondensation to take place.It does not escape to us that other
proteins must be involved in the remodelling of chromatin shown in the ®gure.
F.Segade,J.Go Âmez-Ma
extrovertÂrquez /The International Journal of Biochemistry &Cell Biology 31(1999)1243±12481246jeso
5.Possible medical applications
The myc proto-oncogenes encode for transcrip-tion factors that regulate the expression of v-eral genes,among them the gene for ProT a[13].
A clear correlation has been found between the expression of c-myc and its target genes in veral types of human tumours[3,20].Thus,it has been reported an association between ProT a levels and a
n increa in risk of death from breast cancer [21].It is then likely that the levels of ProT a in speci®c types of tumours might be employed as a prognostic factor.On the other hand,given that the elimination of ProT a from the cell is capable of inducing apoptosis[15]there exists the tanta-lising possibility of stopping or at least delaying the growth of tumour cells through the manipu-lation of ProT a levels.Finally,a-thymosins have been ud in the past as biological respon modi®ers in an attempt to potentiate the immune respon in patients undergoing cancer therapy. Although from a physiological point of view, such pharmacological e ects should be con-sidered as xenobiotic or artefactual,the potential therapeutic u of ProT a and/or its derivatives cannot be dismisd.
Acknowledgements
We are grateful to Tom Broekelmann for his help with Fig.1.We also thank to Dr.Francisco Boa n for the critical reading of the manuscript and for his help in the elaboration of Fig.2.
References
[1]A.A.Haritos,G.J.Goodall, B.L.Horecker,
Prothymosin a:isolation and properties of the major immunoreactive form of thymosin a1in rat thymu
s, Proc.Natl.Acad.Sci.USA81(1984)1008±1011. [2]T.L.K.Low, A.L.Goldstein,Thymic hormones:an
overview,Methods Enzymol.116(1985)213±215. [3]M.R.Smith,Prothymosin a:in arch of a function,
Leuk.Lymphoma18(1995)209±214.
[4]W.H.Eschenfeldt,S.L.Berger,The human prothymosin
a gene is polymorphic and induced upon growth stimu-
lation:evidence using a cloned cDNA,Proc.Natl.
Acad.Sci.USA83(1986)9403±9407.
[5]J.Go mez-Ma rquez,F.Segade,M.Dosil,J.G.Pichel,
X.R.Bustelo,M.Freire,The expression of prothymosin
a gene in T lymphocytes and leukemic lymphoid cells is
tied to lymphocyte proliferation,J.Biol.Chem.264 (1989)8451±8454.
[6]J.Go mez-Ma rquez,F.Segade,Hypothesis:prothymosin
a is a nuclear protein,FEBS Lett.226(1988)217±219.
[7]J.D.Watts,P.D.Cary, D.Crane-Robinson,
Prothymosin a is a nuclear protein,FEBS Lett.245 (1989)17±20.
[8]R.E.Manrow, A.R.Sburlati,J.A.Hanover,S.L.
Berger,Nuclear targeting of prothymosin a,J.Biol.
Chem.266(1991)3916±3924.
[9]K.Gast,H.Damaschum,K.Eckert,K.Schulze-
Forster,R.H.Maurer,M.Mu ller-Frohne,D.Zirwer,J.
Czarnecki,G.Damaschum,Prothymosin a:a biologi-cally active protein with random coil conformation, Biochemistry34(1995)13211±13218.
[10]L.Tao,R.-H.Wang,S.A.Enkemann,M.W.Trumbore,
S.L.Berger,Metabolic regulation of protein-bound glu-tamyl phosphates:Insights into the function of Prothymosin a,J.Cell.Physiol.178(1999)154±163. [11]R.W.Manrow, A.Leone,M.S.Krug,W.H.
Eschenfeldt,S.L.Berger,The human prothymosin a gene family contains veral procesd pudogenes lacking deleterious lesions,Genomics13(1992)319±331.
[12]X.R.Bustelo,A.Otero,J.Go mez-Ma rquez,M.Freire,
Expression of the rat prothymosin a gene during T-lym-phocyte proliferation and liver regeneration,J.Biol.
Chem.266(1991)1443±1447.蛆虫的意思
[13]M.Eilers,S.Schirm,J.M.Bishop,The myc protein acti-
vates transcription of a-prothymosin gene,EMBO J.10 (1991)133±141.
[14]A.R.Sburlati,R.E.Manrow,S.L.Berger,Prothymosin
a antin oligomers inhibit myeloma cell division,
Proc.Natl.Acad.Sci.USA88(1991)253±257. [15]P.Rodrõguez,J.E.Vin uela,L.Alvarez-Ferna ndez,J.
Go mez-Ma rquez,Prothymosin a antin oligonucleo-tides induce apoptosis in HL-60cells,Cell Death Di er.六级通过率
6(1999)3±5.
[16]C.-L.Wu,A.-L.Shiau,C.-S.Lin,Prothymosin a pro-
motes cell proliferation in NIH3T3cells,Life Sci.61 (1997)2091±2101.
[17]P.Rodrõguez,J.E.Vin uela,L.Alvarez-Ferna ndez,M.
Buceta,A.Vidal,F.Dominguez,J.Go mez-Ma rquez, Overexpression of prothymosin a accelerates prolifer-ation and retards di erentiation in HL-60cells, Biochem.J.331(1998)753±761.
[18]P.Rodriguez,J.Go mez-Ma rquez,Prothymosin a is a
chromatin-remodelling protein in mammalian cells, Biochem.J.333(1998)1±3.
[19]Z.Karetsou,R.Sandaltzopoulos,M.Frangou-
出国留学出国Lazaridis, C.-Y.Lai,O.Tsolas,P.B.Becker,T.
Papamarcaki,Prothymosin a modulates the interaction of histone H1with chromatin,Nucleic Acids Res.26 (1998)3111±3118.
F.Segade,J.GoÂmez-MaÂrquez/The International Journal of Biochemistry&Cell Biology31(1999)1243±12481247
