The Thymosins

7 The Thymosins Prothymosin , Parathymosin, and -Thymosins: Structure and Function

Ewald Hannappel and Thomas Huff Institute for Biochemistry/Faculty of Medicine, University of Erlangen-Nu¨rnberg, 91054 Erlangen, Germany

I. Introduction II. Polypeptide b1 III. a-Thymosins and Prothymosin  A. Structure of Prothymosin B. Prothymosin and Cell Proliferation C. Prothymosin and Zn2+ D. Bipartite Nuclear Localization Signal and Caspase 3 Cleavage Site E. Phosphorylation of Prothymosin F. Intracellular Partners of Prothymosin G. Prothymosin and Small RNA H. Extracellular Thymosin a1 and Prothymosin IV. Parathymosin A. Zn2+-Binding Protein B. Bipartite Nuclear Localization Signal C. Phosphorylation of Parathymosin D. Parathymosin and Glucocorticoid Action V. -Thymosins A. Purification of b-Thymosins B. Amino Acid Sequences and Phylogenetic Distribution of b-Thymosins C. b-Thymosins and G-Actin Vitamins and Hormones Volume 66

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Copyright 2003, Elsevier Science (USA). All rights reserved. 0083-6729/03 $35.00

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D. b-Thymosins E. b-Thymosins F. b-Thymosins G. Thymosin b4 VI. Conclusions References

and F-Actin and Cancer in Angiogenesis and Wound Healing and AcSDKP

The studies on thymosins were initiated in 1965, when the group of A. White searched for thymic factors responsible for the physiological functions of thymus. To restore thymic functions in thymic-deprived or immunodeprived animals, as well as in humans with primary immunodeficiency diseases and in immunosuppressed patients, a standardized extract from bovine thymus gland called thymosin fraction 5 was prepared. Thymosin fraction 5 indeed improved immune response. It turned out that thymosin fraction 5 consists of a mixture of small polypeptides. Later on, several of these peptides (polypeptide 1, thymosin 1, prothymosin , parathymosin, and thymosin 4) were isolated and tested for their biological activity. The research of many groups has indicated that none of the isolated peptides is really a thymic hormone; nevertheless, they are biologically important peptides with diverse intracellular and extracellular functions. Studies on these functions are still in progress. The current status of knowledge of structure and functions of the thymosins is discussed in this review. ß 2003, Elsevier Science (USA).

I. INTRODUCTION The thymus is the site of maturation of T lymphocytes. This maturation requires the thymic microenvironment. Even today, the sequence of this maturation within the thymus is not completely understood. In 1970 it was well accepted that a vital part of the process by which the thymus works occurs via humoral factors (Goldstein and White, 1970). Various cell-free extracts were prepared from thymus and tested for their ability to counteract effects of neonatal thymectomy in rodents. Because little was known at the time about the complex processes involved in the maturation of T lymphocytes, testing the biological activities of extracts was not reliable. Klein and co-workers (1965) studied the enhancement of incorporation of [3H] thymidine into DNA of mouse nodes by thymic extracts. In 1966 the same group presented a standardized fractionation of calf thymus and coined the term ‘‘thymosin’’ for the isolated lymphocytopoietic factor (Goldstein et al., 1966). Thymosin was thought to be a single 12.6-kDa polypeptide (Goldstein et al., 1972). The starting material was thymosin

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fraction 5, which was prepared by a standardized five-step procedure: (1) extraction of thymus in isotonic NaCl solution, (2) heat denaturation from 0
to 80 C within 25 min, (3) acetone precipitation, (4) ammonium sulfate precipitation, and (5) ultrafiltration. However, at least two of these steps are disputable in terms of changing the peptide pattern of the extract by proteolysis: proteolysis will occur during the thawing of frozen calf thymus in the cold as well as during the heat denaturation step. Later it turned out that thymosin fraction 5 or fraction 5A (Goldstein and Low, 1985) prepared by a different ammonium sulfate fractionation contained not only one biologically interesting peptide but consisted of a mixture of small polypeptides ranging from approximately 1 to 15 kDa. On an isoelectric focusing gel, 30–40 polypeptide components or fragments were identified. The large number of peptides present in thymosin fraction 5 may indeed represent fragments of larger polypeptides generated during preparation of thymosin fraction 5A and not genuine thymic peptides. Because of the large number of peptides detected a nomenclature for the family of polypeptides present in thymosin fraction 5 was suggested (Goldstein et al., 1977). The thymosins were divided into three main groups according to their isoelectric points: -thymosins, pI below 5.0; thymosins, pI between 5.0 and 7.0; and -thymosins, pI above 7.0. Numerical subscripts were included simply to denote the chronological order of isolation. The decision concerning which peptides to isolate first was crucial. Depending on the point of view, the wrong and right peptides were chosen. Thymosin fraction 5 contains several peptides that are present in higher amounts and these peptides were selected for further purification (Low et al., 1979). Probably the wrong peptides were chosen with respect to thymic hormones, because the nonlymphoid (stromal) cells situated in the thymus providing nursing to immature T cells (van Ewijk, 1991) are outnumbered by T cells to die (Smith et al., 1989; MacDonald and Lees, 1990). However, it turned out that biologically important peptides were isolated with respect to general cell function. The first two peptides isolated from thymosin fraction 5 were 1 and 1 (Low and Goldstein, 1979). Whereas the former was classified as a thymosin on the basis of its activity in certain assays, the latter was termed polypeptide 1. Polypeptide 1 did not show biological activity in the bioassay systems used, indicating that it might not be an important molecule for T cell maturation.

II. POLYPEPTIDE 1 Polypeptide 1 consists of 74 amino acid residues. At least four independent groups sequenced the polypeptide 1 completely or partially. It was isolated from bovine thymus as a polypeptide that has lymphocyte-

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differentiating properties and was designated as ubiquitous immunopoietic polypeptide, UBIP (Goldstein et al., 1975). It was supposed to induce the differentiation of T and B cells via -adrenergic receptors and adenylate cyclase activation. However, the effect on T and B cells was caused by endotoxin present in the UBIP preparations (Low et al., 1979). Because UBIP was present in various animal cells, yeast, bacteria, and plants (Goldstein et al., 1975; Schlesinger and Goldstein, 1975) as well as insect eggs (Gavilanes et al., 1982) UBIP was renamed ubiquitin(1–74). Ubiquitin(1–74), which is extended at the C terminus by a dipeptide (Gly-Gly), is connected with histone 2A via an isopeptide bond in the chromosomal protein A24, forming uH2A (Goldknopf and Busch, 1977). Today, polypeptide 1 is well known as part of ubiquitin, or ATPdependent proteolysis factor 1 (APF-1) (Ciechanover et al., 1980). Ubiquitin plays a key role in a variety of cellular processes, such as ATP-dependent degradation of cellular proteins (Glickman and Ciechanover, 2002); regulation of transcription (Conaway et al., 2002); spermiogenesis by tagging histones 2A, 2B, and 3 (Jason et al., 2002); apoptosis (Jesenberger and Jentsch, 2002); and ribosome biogenesis (Finley et al., 1989). Ubiquitin is a globular protein but the last four C-terminal residues (–73LRGG76) extend from the globular structure and are accessible (Vijay-Kumar et al., 1987). The bond between arginine and glycine is cleaved during the preparation of thymosin fraction 5, generating polypeptide 1. In the ATP-dependent proteolysis system, the C-terminal glycine residue is used for ubiquitinylation of target proteins via an isopeptide bond.

III. -THYMOSINS AND PROTHYMOSIN  Thymosin 1 is a small peptide (28 amino acid residues) isolated from thymosin fraction 5. Its isoelectric point is 4.2 and the molecular mass is 3108 Da (Low and Goldstein, 1985). The N-terminal serine residue is acetylated, and the peptide contains no aromatic amino acid residue (Fig. 1). Later, two peptides related to thymosin 1 were isolated from calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, was designated des-(25–28)-thymosin 1. The other, named thymosin 11, contained seven additional amino acid residues at the COOH terminus. (Caldarella et al., 1983). However, none of these peptides were detectable in larger amounts when strongly denaturing conditions were used during extraction of calf thymus (Hannappel et al., 1982b). When rat thymus was pulverized in a mortar chilled in dry ice and added thereafter to boiling water a peptide of 111 amino acids was isolated that contained the entire sequence of thymosin 1 at its N terminus (Haritos et al., 1984a). Therefore, the new peptide was designated prothymosin  (Fig. 1). If

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FIGURE 1. Amino acid sequences of thymosin 1, thymosin 11, and prothymosins from various species: Homo sapiens (human, Q15249), Bos taurus (bovine, P01252), Rattus norvegicus (rat, P06302), Mus musculus (mouse, P26350), Rana esculenta (frog, Q90ZK2), and Brachydanio rerio (zebrafish, Q8QGP0). The numbers in parentheses are the accession codes for the SWISSPROT and TrEMBL databases. The numbering is according to human prothymosin. The bipartite nuclear localization signal is outlined; the caspase 3 cleavage sites (–DxxD–) located inside the bipartite NLS are shown in boldface; additional potential caspase 3 sites of non mammalian prothymosins are shown in italic. Invariant residues are indicated by asterisks and highly conserved residues are indicated by colons.

thymosin 1 is produced solely by artificial proteolysis during preparation of thymosin fraction 5 or whether this proteolysis resembles the natural processing of prothymosin  to thymosin 1 in certain tissues is still not settled (Freire et al., 1985; Franco et al., 1992; Frillingos et al., 1992; Sarandeses et al., 2003). Prothymosin  and its mRNA have been detected almost ubiquitously in a wide variety of tissues (Haritos et al., 1984b; Clinton et al., 1989a). In a cDNA library constructed from human spleen mRNA, a clone was isolated that contained a 503-base pair insert including the entire coding sequence of prothymosin. The presence of an initiator codon immediately preceding the codon for the N-terminal serine residue, and a terminator codon immediately following the codon for the Cterminal Asp-109, suggested that prothymosin is synthesized without formation of a larger precursor. Analysis of the 50 sequence preceding the initiator methionine codon excluded the potential presence of a hydrophobic signal peptide (Goodall et al., 1986). According to a survey of human cDNA libraries the prothymosin gene was among the most abundantly expressed genes together with the 90-kDa heat shock protein, myosin light chain, and ribosomal proteins (Adams et al., 1995). The complete amino acid sequences of human (Eschenfeldt and Berger, 1986; Pan et al., 1986; Gomez-Marquez et al., 1989), calf (Panneerselvam et al., 1988b), rat (Haritos et al., 1985a; Frangou-Lazaridis et al., 1988) and mouse (Schmidt

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and Werner, 1991) prothymosin  have been established. These prothymosins vary slightly in length (human and calf, 109; mouse, 110; and rat, 111 amino acid residues) and are different in a few amino acid residues (Fig. 1). Even the presence of a putative prothymosin homolog in yeast (Makarova et al., 1989) and Escherichia coli (Vartapetian et al., 1992) has been reported. This supposed high evolutionary conservation of prothymosin was an argument in favor of a fundamental role for the peptide in eukaryotic and prokaryotic cells. Later, the presence of prothymosin or a prothymosin gene in animals other than mammals was shown to be highly unlikely (Trumbore et al., 1998). More recently, however, cDNA clones encoding prothymosins have been identified in Rana esculenta (Aniello et al., 2002; De Rienzo et al., 2002) and zebrafish (TrEMBL accession code, Q8QGP0). The amino acid sequences are highly conserved (70%). In addition, the bipartite nuclear localization site separated by multiple caspase 3 cleavage sites is conserved in the C-terminal region of all prothymosins (Fig. 1). The cDNA of frog prothymosin is expressed ubiquitously. Because the prothymosin expression varies during the spermatogenetic cycle concomitantly with germ cell maturation, prothymosin might contribute to the efficiency of spermatogenesis in species with seasonal breading (Aniello et al., 2002).

A. STRUCTURE OF PROTHYMOSIN

Because structure and function of molecules are related, the peculiar chemical features of prothymosin must be considered. All prothymosins are devoid of cysteine, methionine, and aromatic amino acid residues. Thus, they do not absorb at 280 nm. About half the amino acids residues are acidic amino acids. The ratio between glutamic acid to aspartic acid drops from about 2 in mammalian prothymosin to about 1 in frog and zebrafish. Eight lysine residues are conserved in all prothymosins; four are clustered in the N-terminal region (residues 14, 17, 19, and 201) and the others are found in the C-terminal region (residues 87, 101, 102, and 104). A lysine residue in frog and zebrafish replaces the arginine residue at position 30 in mammalian prothymosins, whereas the second arginine residue at position 88 is conserved. Mammalian prothymosins contain five times more acidic than basic amino acid residues, have isoelectric points below 3.5, and belong to the most acidic proteins known in nature. Because of this amino acid composition, prothymosins are soluble in boiling buffer or diluted perchloric acid and partition to the aqueous phase of a phenol extraction. These properties have been successfully used for purification of prothymosins

1

Numbering according to human prothymosin .

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(Haritos et al., 1985b; Vartapetian et al., 1988; Sburlati et al., 1990; Watts et al., 1990; Evstafieva et al., 1995). Because of the large number of acidic amino acid residues prothymosin is highly negatively charged and adopts a random coil-like conformation with no regular secondary structure (Gast et al., 1995), permitting interactions with cationic structures. However, at low pH (3) and rather low concentrations (5 M) compared with intracellular concentrations (up to 0.3 pg/cell, 100 M 2) (Haritos et al., 1984b; Franco et al., 1992; Sburlati et al., 1993), the natively unfolded prothymosin is capable of forming regular elongated fibrils (Pavlov et al., 2002). The low pH presumably decreases the repulsive forces caused by the high negative charge of the protein at neutral pH and thereby induces its partial folding. Whether this change in structure might occur in vivo under certain physiological or pathophysiological conditions is currently unknown. B. PROTHYMOSIN AND CELL PROLIFERATION

Prothymosin expression is elevated in proliferating tissues including colon cancer (Mori et al., 1993) and hepatocellular carcinoma (Wu et al., 1997) and is correlated with c-Myc mRNA expression (Vareli et al., 1995). Prothymosin was found in proliferating lymphoma and transformed 3T3 cells but not in resting cells (Eschenfeldt and Berger, 1986). Antisense RNA or synthetic antisense DNA oligomers of prothymosin are able to inhibit cell division in myeloma cells (Sburlati et al., 1991). Prothymosin gene transcription is directly regulated by activated c-Myc via an E-box element (CACGTG) localized in the first intron of the gene (Eilers et al., 1991). In estrogen receptor-positive breast cancer cells the prothymosin mRNA was rapidly increased by estrogen accompanied by a 6-fold increase in prothymosin content. Prothymosin was found to selectively enhance estrogen receptor (ER) transcriptional activity by sequestering repressor of ER activity, rendering the estrogen–ER complex accessible to coactivators such as SRC-1 (Martini et al., 2000). It is noteworthy that prothymosin, a protein enhancing estrogen–ER transcriptional effectiveness, is itself upregulated by estrogen. The estrogen effect on prothymosin expression in breast cancer cells is mediated via two upstream half-palindromic TGACC motifs in the prothymosin promotor (Garnier et al., 1997; Martini and Katzenellenbogen, 2001). Despite the fact that overexpression of prothymosin promotes tumor formation, mice were injected with murine bladder cancer cells (MBT-2) in conjugation with replication-defective retroviruses encoding prothymosin. These mice exhibited smaller tumor mass, lower tumor incidence, and higher 2 Assuming an even distribution of the peptide in a volume of 270 fl corresponding to the intracellular volume of human mononuclear leukocytes.

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survival rate, as well as higher antitumor cytotoxic activities compared with those injected with control viruses. This effect was not observed in severe combined immunodeficiency mice, suggesting that prothymosin exerted its effect through its immunomodulatory activities. However, as to be expected, tumor cell growth in monolayer culture and colony formation in soft agar was enhanced in the prothymosin gene-modified MBT-2 clones. The growth-promoting effect was circumvented by removing the nuclear localization signal of prothymosin from the construct (Shiau et al., 2001). C. PROTHYMOSIN AND ZN2+

Mice of the RF/J strain are defective in some aspects of cellular immunity, as evidenced by their susceptibility to infections with Candida albicans. When the mice were fed a high-zinc diet and treated daily with 160 ng of prothymosin, an increase occurred in resistance to infections with Candida (Salvin et al., 1987). In the presence of Zn2+ or small unilamellar vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidic acid (10:1), thymosin 1 adopts a partially structured conformation. A turn is present between residues 5 and 8, whereas the region between residues 17 and 24 adopts an -helical conformation (Grottesi et al., 1998). Binding of divalent metal cation by proteins is dependent on negatively charged amino acid side chains. Therefore the divalent cation-binding properties of prothymosin were evaluated (Chichkova et al., 2000). Prothymosin could bind up to 3 Zn2+ ions in the presence of 100 mM NaCl (KD of 0.23 mM) specifically and up to 13 Zn2+ ions in the absence of NaCl (KD of 0.04 mM) unspecifically. Zinc ions significantly enhanced the binding of prothymosin to HIV-1 Rev but not to histone H1, two putative binding partners (Papamarcaki and Tsolas, 1994; Kubota et al., 1995). Binding of zinc to prothymosin (reported: KD of 1 mM) induces compaction and considerable rearrangement of protein structure into a compact, partially folded, premolten globulin-like conformation (Uversky et al., 2000). No interactions with Mg2+, Mn2+, Cu2+, Ni2+, or Co2+, and a weak interaction with Ca2+, were observed (Chichkova et al., 2000; Uversky et al., 2000). D. BIPARTITE NUCLEAR LOCALIZATION SIGNAL AND CASPASE 3 CLEAVAGE SITE

In 1988 it was first postulated by sequence comparison with nuclear localization signals (NLSs) of other proteins ‘‘that prothymosin is located inside the cell nucleus and that its activity might be to organize some protein complexes’’ (Gomez-Marquez and Segade, 1988). It was recognized that the

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sequence – 101KKQK104 – might serve as NLS. The nuclear localization of prothymosin was confirmed by several groups (Watts et al., 1989; Clinton et al., 1991; Manrow et al., 1991; Castro and Barcia, 1996) but questioned by others (Tsitsiloni et al., 1989). To localize prothymosin in cells wild-type and mutant human prothymosins were expressed as green fluorescent protein (GFP) fusion proteins in human cells. This study led to the identification of a bipartite NLS (–87KR-X12-KKQK104–). Wild-type prothymosin appeared to be exclusively nuclear and excluded from the nucleoli. Mutations (K87R or K101R) in both parts of the putative bipartite NLS resulted in a marked (but not complete) redistribution to the cytoplasm (Rubtsov and Vartapetian, 1996; Rubtsov et al., 1997). This bipartite NLS is conserved in all described prothymosins, including those from frog and zebrafish (Fig. 1). The bipartite NLS is separated by 12 (mammalian), 13 (frog), or 14 (zebrafish) mainly acidic amino acid residues that display several overlapping amino acids motifs, –DxxD–, conforming to caspase 3 recognition sites. When HeLa cells undergo apoptosis, DNA becomes fragmented, caspases are activated, and prothymosin is cleaved at sites near the C terminus. Consequently, the larger part of the bipartite NLS is removed and the truncated peptide forfeits known functions such as nuclear localization. Caspase 3 attacks prothymosin directly. The truncated peptide is deficient in phosphate. Because phosphorylation of prothymosin is supposed to occur in the nucleus this reflects loss of nuclear compartmentalization. Multiple caspase 3 recognition sites are conserved in all prothymosins between amino acid residues 90 and 100. Prothymosins from human, bovine, mouse, and frog possess the motif three times (DDVD, DEDD, and DDVD), whereas the last motif is lost in rat (DDVE). In zebrafish, five potential caspase 3 recognition sites can be identified in this region (DDDDDEDDVD). In addition, three more putative caspase 3 recognition sites are located between positions 63 and 82 of frog and zebrafish prothymosins (Fig. 1). These sites are lost in the mammalian prothymosins. Prothymosin appears to be a carefully designed substrate for caspase 3 (Enkemann et al., 2000; Evstafieva et al., 2000). Most recently it has been reported that prothymosin inhibits activation of caspase 3 by blocking apoptosome formation (Jiang et al., 2003). Because prothymosin itself is a substrate of caspase 3, the inhibition of apoptosome formation might be abolished by cleavage of prothymosin.

E. PHOSPHORYLATION OF PROTHYMOSIN

Prothymosin is phosphorylated in vivo, but there is still considerable controversy about the amino acid residue(s) phosphorylated. It was noticed that prothymosin contains three motifs that resemble consensus sequences for phosphorylation of serine or threonine by casein kinase-2 (Barcia et al.,

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1992). In mitogenically stimulated murine splenic lymphocytes, prothymosin and thymosin 1 were phosphorylated at Thr-7 and Thr-12/13 and not at serine residues, pointing to a kinase different from casein kinase-2 (Barcia et al., 1993). In 2000, this prothymosin kinase was characterized. Despite the fact that the phosphorylation sites of prothymosin are Thr-7 and Thr-12/13 the identified kinase phosphorylates neither thymosin 1 nor thymosin 11. The kinase is located in the cytosol throughout the cell cycle and its activity increases during the S phase and decreases at entry into the G2 phase. Prothymosin kinase is activated by phosphorylation in a mitogen-initiated pathway that is dependent on protein kinase C; however, protein kinase C does not phosphorylate prothymosin kinase directly (Perez-Estevez et al., 2000). It has also been reported that serine or glutamate residues of prothymosin can be phosphorylated. The phosphorylated serine residue was identified as the N-terminally acetylated amino acid residue. Only about 2% of prothymosin was determined to be phosphorylated throughout the cell cycle under steady state conditions. However, prothymosin is plentiful during rapid growth (0.3 pg/cell, 0.02% of total protein). Thus the number of phosphorylated prothymosin molecules may be about 300,000 per cell (Sburlati et al., 1993). This nevertheless low degree of phosphorylation of prothymosin and, even more, its constancy during the cell cycle were the reasons to scrutinize the source of the phosphate residue found at the acetyl serine residue of prothymosin. On the basis of the careful observation that greater than 90% of the phosphate found initially in prothymosin disappeared rapidly, it was recognized that phosphate groups had been linked to prothymosin in an energy-rich bond. The initial sites of phosphorylation are glutamate residues. The formed glutamyl phosphates are extremely labile and are hydrolyzed (90%) or transferred to serine or threonine to form stable esters in vivo (Trumbore et al., 1997). The free energy of hydrolysis of the mixed acid anhydride glutamyl phosphate is higher than that of ATP, and each prothymosin bears several phosphates simultaneously. Therefore, it might be that the glutamyl phosphate bonds in prothymosin are able to supply energy for processes in the nucleus (Trumbore et al., 1997). Because the phosphate residue could also be transferred back to ADP for energetic reasons, glutamyl phosphorylated prothymosin could serve as an energy buffer in the nucleus. This would be reminiscent of the creatine phosphate–creatine system of fast-fatigable muscle fibers. The high-energy status as well as the unusual phosphorylation of glutamate residues of prothymosin raise questions concerning how this reaction is catalyzed. Until now, no glutamyl kinase of prothymosin has been identified. Cell cycle-specific differences in the half-life of glutamyl phosphorylated prothymosin were observed in NIH 3T3 cells: 35 min in G0, 83–89 min

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during arrest in or progression through the S phase, and 174 min during M-phase arrest (Wang et al., 1997; Tao et al., 1999). As indicated by the long half-life of prothymosin phosphate during the M phase, the glutamyl phosphate of prothymosin is not required for mitosis. The half-life of prothymosin phosphate was about 150 min in quiescent (G0) and rapidly growing cells in the presence of actinomycin D, a potent inhibitor of transcription. Thus, it was proposed that prothymosin glutamyl phosphates fuel an energy-requiring step in the production, processing, or export of RNA (Tao et al., 1999). The short half-life of prothymosin glutamyl phosphates observed in quiescent NIH 3T3 cells can be explained by the lower prothymosin content and the lower number of phosphate groups per prothymosin when compared with rapidly growing cells. Resting NIH 3T3 cells synthesize RNA at about one-ninth the rate of rapidly growing cells. Concomitantly, the amount of prothymosin decreases to one-ninth and the number of phosphates incorporated in prothymosin drops from 4–8 to 2–4 in resting cells (Tao et al., 1999). F. INTRACELLULAR PARTNERS OF PROTHYMOSIN

Studies have shown that prothymosin is a nuclear protein involved in cell proliferation and associated with the nucleosome linker histone H1 (Papamarcaki and Tsolas, 1994). It was recognized that prothymosin binds to nucleosome core histones, in particular, H3 and H4 (Diaz-Jullien et al., 1996), indicating that prothymosin might be involved in chromatin remodeling (Gomez-Marquez and Rodriguez, 1998; Karetsou et al., 1998). Affinity chromatography on prothymosin–Sepharose columns was used to identify proteins in subcellular extracts of transformed human lymphocytes that interact with prothymosin directly or indirectly. The most abundant prothymosin-binding proteins were histones H2A, H2B, H3, and H4. Of the nuclear transport proteins, karyopherin 1, Rch-1, Ran, and RCC1 were detected at high concentrations; NTF2, nucleoporin p62, and Hsp70 were detected at low concentrations; whereas transportin, CAS, and Ran BP1 were not detected. Of the cell cycle control proteins, PCNA, Cdk2, and cyclin A were detected at high concentrations; cdc2, Cdk4, and cyclin B were detected at low concentrations; and cyclin D1, D3, Cip1, and Kip1 were not detected. In agreement with the involvement of the karyopherin 1–Rch1 complex in the nuclear import of NLS-bearing proteins, prothymosin might be transported by this heterodimer into the nucleus. In the nucleus, prothymosin may interact with proteins involved in DNA metabolism and cell cycle control (Freire et al., 2001). Prothymosin interacts with Rev proteins (HTLV-I Rex and HIV-1 Rev), as has been shown by affinity chromatography experiments (Kubota et al., 1995). Rev proteins may possibly be involved in the export of prothymosin from the nucleus. Prothymosin lacks a leucine-rich nuclear export signal

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(NES) and Rev proteins may function as piggybacks for nuclear export of prothymosin. It has been demonstrated that the Epstein–Barr virus nuclear antigen (EBNA3C) interacts with prothymosin in regulating histone acetylation (Cotter and Robertson, 2000). Amino acid residues between positions 366 and 400 of EBNA3C are responsible for the interaction with prothymosin. Moreover, prothymosin interacts with two domains (CH1 and CH3/HAT) of histone acetyltransferase p300 in EBV-infected cells. These two domains of p300 are also responsible for the interaction with the N and C terminus of EBNA3C (residues 1–207 and 620–992), suggesting that these interactions are important for transcriptional regulation and gene expression of the transformed cells (Subramanian et al., 2002). Prothymosin may remove histone H1 from the nucleosome and be involved in the recruitment of other general transcription factors. Therefore prothymosin seems to be involved in chromatin decondensation and acetylation of core histones as it interacts with the histone acetyl transferase p300 at two domains, interacting additionally with other transcriptional factors including RNA polymerase II. Another histone acetyltransferase has been identified as interacting with prothymosin. Prothymosin interacts physically with the CREB-binding protein (CBP), which is a versatile transcription coactivator. The site of interaction was mapped within the N-terminal domain of CBP (residues 1–771) and a region of prothymosin composed of two polyglutamate stretches (Karetsou et al., 2002).

G. PROTHYMOSIN AND SMALL RNA

Prothymosin has been identified as a 13-kDa protein that is present in RNA–protein complexes in human, bovine, and yeast cells (Makarova et al., 1989). As described earlier, the presence of prothymosin in yeast has not been confirmed (Trumbore et al., 1998). Prothymosin is covalently attached to small, unidentified cytoplasmic RNA in mammalian cells. In E. coli cells overexpressing recombinant rat prothymosin, several bacterial tRNAs were identified to be linked via their 50 terminus (Vartapetian et al., 1997). The points of attachment were mapped by mutational analysis to several sites on prothymosin (positions 14–20, 89–98, and 102–106). The attachment of tRNA to prothymosin occurs via a stable bond resistant to denaturing conditions as well as proteolytic degradation of prothymosin. tRNA attachment seems to occur within a cluster of lysine residues located at both termini of prothymosin. Modification of prothymosin by tRNA between positions 89 and 98 might interfere with the function of the bipartite NLS as well as with the caspase 3 cleavage sites. However, tRNA linking to prothymosin appears to be relatively inefficient, at least in E. coli.

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The biological meaning of attaching tRNAs to prothymosin is unclear at present (Lukashev et al., 1999).

H. EXTRACELLULAR THYMOSIN 1 AND PROTHYMOSIN

Prothymosin was called ‘‘thymic hormone’’ because it was detected in blood serum (60 pM) (Panneerselvam et al., 1987) together with its cleavage product thymosin 1 (80–400 pM) (Naylor et al., 1992; Weller et al., 1992; Molinero et al., 2000). Extracellular thymosin 1 stimulates endothelial cell migration, angiogenesis, and wound healing (Malinda et al., 1998). The stimulation of angiogenesis has also been shown for prothymosin (Koutrafouri et al., 2001). Thymosin 1 is able to increase interleukin 2 receptors on mitogenstimulated human lymphocytes (Sztein et al., 1986; Sztein and Serrate, 1989) and promotes secretion of Th1 cytokines such as interferon (IFN- ) (Serrate et al., 1987). Combinations of thymosin 1 and nucleoside analogs (famciclovir) or interferon  are still under investigation for treating patients with chronic hepatitis B and C virus infections (Billich, 2002; Lau et al., 2002). In hepatitis B e antigen (HBeAg)-positive Chinese patients, the clearance of HBeAg is significantly greater in those treated with a 6-month course of thymosin 1 (Chien et al., 1998); however, the effect is less pronounced in white patients (Mutchnick et al., 1999). Thymosin 1, together with low doses of interferon or interleukin 2, is highly effective in restoring several immune responses depressed by tumor growth and cytostatic drugs and increases the antitumor effect of chemotherapy while markedly reducing the general toxicity of treatment (reviewed in Bodey et al., 2000; Garaci et al., 2000). It has been reported that thymosin 1 can positively modulate hematopoietic functions of murine bone marrow cells and restore myelopoiesis in tumor-bearing mice (Paul and Sodhi, 2002). Mammary carcinomas were observed 3 months after injection of nitrosomethylurea into rats. Daily administration of thymosin 1 (10 g) reduced carcinoma incidence and prolonged survival time (Moody et al., 2002). All these effects attributed to thymosin 1 require that extracellular thymosin 1 be able to influence the intracellular metabolism of target cells. The mechanism of action might either be by modulating cytokine receptors via interaction of thymosin 1 with negatively charged membranes (Grottesi et al., 1998) or by a specific receptor. Specific receptors with low (15 nM) and high (250 pM) affinity for prothymosin have been identified on the surface of human peripheral blood mononuclear cells (Cordero et al., 1994). Three binding partners (31, 29, and 19 kDa) have been purified by affinity chromatography from membranes of phytohemagglutinin (PHA)-activated lymphoblasts. The putative receptor for prothymosin was localized in a

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caplike structure at one of the poles of blood mononuclear cells (Pineiro et al., 2001).

IV. PARATHYMOSIN Compared with prothymosin much less is known about parathymosin. Research has focused mainly on prothymosin and neglected parathymosin. Because of many similarities, prothymosin and parathymosin are thought to build a protein family with similar functions. However, there are differences in their biological function that strongly recommend treating these two peptides as separate entities. Parathymosin was the first thymosin that was not isolated completely or in a fragmented form from thymosin fraction 5. During the isolation of prothymosin an unknown peptide was purified as a by-product (Haritos et al., 1985b). Because of its structural homology to prothymosin in size and amino acid composition (Fig. 2), it was named parathymosin  (Haritos et al., 1985c). The amino acid sequence of rat parathymosin was determined (Komiyama et al., 1986). Parathymosin as prothymosin is present in various nonlymphoid tissues of rat in high concentrations (217 g/g liver; 78 g/g thymus). Tissues high in parathymosin tend to be low in prothymosin (110 g/g liver; 916 g/g thymus) (Clinton et al., 1989a). Parathymosin has been quantified in different rat tissues by an enzyme-linked immunosorbent assay (ELISA). All tissues except erythrocytes contain parathymosin (Brand et al., 1991). Early on in parathymosin research, it was speculated that

FIGURE 2. Amino acid sequences of parathymosins from various species: Homo sapiens (human, P20962), Bos taurus (bovine, P08814), Rattus norvegicus (rat, P04550), and Mus musculus (mouse, Q9D0J8). The numbers in parentheses are the accession codes for the SWISSPROT and TrEMBL databases. The bipartite nuclear localization signal is outlined; potential caspase 3 sites are shown in italic. The question mark points to a potential mistake in the amino acid sequence of bovine parathymosin, because all other parathymosins possess a lysine reside at position 79 (Trompeter et al., 1996). The nucleic acid sequence of bovine parathymosin has not been determined. Invariant residues are indicated by asterisks, and highly conserved residues are indicated by colons.

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parathymosin might modulate the action of prothymosin in protecting sensitive strains of mice against opportunistic infection with Candida albicans. The amino acid sequences of human (Clinton et al., 1989b) and bovine (Panneerselvam et al., 1988a) parathymosin are highly homologous to that of rat prothymosin. Mouse parathymosin has not been characterized at the peptide level; however, its cDNA was identified by annotating 21,076 cDNAs analyzed by the RIKEN Mouse Gene Encyclopaedia Project (Kawai et al., 2001). All parathymosins sequenced are acetylated at the N-terminal serine and consist of 101 amino acid residues (Fig. 2). The isoelectric point of rat parathymosin is 4.15, slightly less acidic compared with a pl of 3.55 in the case of rat prothymosin (Haritos et al., 1985c). The translated part of the rat parathymosin gene is interrupted by one large intron (2589 bp) and three small introns (191, 150, and 167 bp). The presence of an initiator codon immediately preceding the codon for the N-terminal serine residue and a terminator codon immediately following the codon for the C-terminal Ala-101 suggested that prothymosin is synthesized without formation of a larger precursor (Trompeter and So¨ ling, 1992). A. ZN2+-BINDING PROTEIN

An acidic zinc-binding protein (ZnBP) of 11.5 kDa, purified from rat liver, inhibits the glycolytic enzyme phosphofructokinase-1. The reversible inhibition results from a dissociation of the tetrameric enzyme into its inactive protomers and is dependent on the presence of 1–20 M Zn2+, whereas at higher concentrations of zinc (100 M) inhibition was completely abolished (Brand and So¨ ling, 1986). In 1989 the cDNA sequencing of ZnBP revealed its identity with parathymosin (Trompeter et al., 1989). The protein has four binding sites for zinc (KD  6 M). Two of them were called specific because zinc binding is still observed in the presence of high salt (0.75 mM Mg2+, 100 mM NaCl). Four clusters of acidic amino acid residues responsible for zinc binding and inactivation of phosphofructokinase were identified between positions 35 and 78 of parathymosin. By further proteolytic cleavage the two specific zinc-binding sites were located to amino acid residues between positions 51 and 72, whereas the region between positions 35 and 50 is necessary for binding and inactivation of phosphofructokinase (Brand et al., 1988). The zinc-binding sites of parathymosin are different from zinc finger motifs. It has been shown by parathymosin–Sepharose affinity chromatography that the interaction of parathymosin with many enzymes of carbohydrate metabolism is zinc specific. From liver cytosol the following enzymes were retained: hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, phosphofructokinase-1, aldolase, glycerol-3-phosphate dehydrogenase, glyceral-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, and the phosphorylated form of pyruvate kinase L. Thus a possible role of

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parathymosin in supramolecular organization of carbohydrate metabolism was proposed (Brand and Heinickel, 1991). B. BIPARTITE NUCLEAR LOCALIZATION SIGNAL

Injection of parathymosin into Xenopus oocytes led to a nuclear uptake of the peptide (Watts et al., 1990), the motif –79KR-X10-KRQK94– comparable to a similar motif of prothymosin (–87KR-X12-KKQT104–), has been identified as a bipartite NLS (Trompeter et al., 1996). The bipartite NLS of prothymosin is separated by 12 amino acid residues, which generate several functional caspase 3 cleavage sites, whereas that of parathymosin is separated by 10 amino acid residues displaying only one potential caspase 3 cleavage site. Yet it has not been studied whether parathymosin can be cleaved by caspases. Immunocytochemical localization of parathymosin revealed a cell-type specific distribution between cytosol and nucleus despite the bipartite NLS. In most cells, the cytoplasm was stained exclusively. In contrast, in duodenal and jejunal crypt cells immunostaining was nuclear, whereas the more mature cells at the top of the villi contained most of the antigen in the cytoplasm. Immunostaining of nuclei was also observed in pancreatic duct cells. Duodenal and jejunal mucosae have a high proliferation rate compared with other somatic tissue; the cells at the top of mucosal villi are terminally differentiated. Thus these data indicated that parathymosin is redistributed between cytoplasm and nucleus depending on the proliferation/differentiation process of the tissue (Brand et al., 1991). Nuclear parathymosin is excluded by nucleoli and correlates with early replication sites, as has been shown by indirect immunofluorescence labeling and confocal scanning laser microscopy (Vareli et al., 2000). Despite the data just presented parathymosin was isolated as a zinc-binding protein inhibiting the cytosolic phosphofructokinase, binding to several enzymes of carbohydrate metabolism, and shown to be located in the cytosol in many rat tissues. The puzzle was solved when it was recognized that the translocation of parathymosin from the cytosol to the nucleus was negatively correlated with cell density. Thinly seeded hepatocytes keep their parathymosin in the nucleus, whereas at high cell density parathymosin is retained in the cytoplasm. Thus at high cell density the bipartite NLS must be inactivated, possibly by covering the NLS with a cytosolic protein. Freshly prepared rat liver cytosol indeed contains a protein with an apparent molecular mass of >250 kDa, which is able to conceal the NLS of parathymosin. No protein inhibiting nuclear import of parathymosin could be isolated from permanent cell lines, in accordance with the fact that in these proliferating cells parathymosin was always observed in the nucleus (Trompeter et al., 1999). The following scenario might be conceivable. Resting cells synthesize the protein inhibiting nuclear import of parathymosin, and thus parathymosin is retained in the

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cytosol and inhibits phosphofructokinase partially. Cells meet their ATP demand mainly by oxidative glucose breakdown. However, when cells in primary culture or tumor cells proliferate, their demand for ATP increases and they metabolize glucose anaerobically to lactate (Brand and Hermfisse, 1997). The concentration of the protein inhibiting nuclear import of parathymosin decreases and parathymosin is translocated into the nucleus (Trompeter et al., 1999). There it binds to histone A1 (Kondili et al., 1996) and supports replication of DNA (Vareli et al., 2000). The inhibition of the glycolytic key enzyme phosphofructokinase is abolished, leading to a stimulation of anaerobic glycolysis. C. PHOSPHORYLATION OF PARATHYMOSIN

No reports exist on potential phosphorylation or any other posttranslational modifications of parathymosin except the acetylation of its N terminus. This is surprising because of the sequence homology to prothymosin. Only a doublet detected by Western blot with specific antibodies against parathymosin might indicate the presence of a rather stable posttranslational protein modification (Brand et al., 1991), which might be a phosphorylated form of parathymosin. D. PARATHYMOSIN AND GLUCOCORTICOID ACTION

Whereas prothymosin was found to enhance estrogen receptor transcriptional activity by acting as an inhibitor of an anticoactivator (Martini et al., 2000), parathymosin inhibits activated glucocorticoid-receptor binding to DNA containing glucocorticoid-response elements. Inhibition of glucocorticoid–receptor binding to nuclei is mediated by the acidic domain(s) of parathymosin located between residues 43 and 79. This inhibitory activity of parathymosin may not require zinc. Glucocorticoids inhibit proliferation and promote differentiation of cells. Thus, in proliferating cells, parathymosin would accelerate proliferation by inhibiting glucocorticoid action (Okamoto and Isohashi, 2000).

V. -THYMOSINS The first -thymosin isolated from thymosin fraction 5 was termed thymosin 4 (Low and Goldstein, 1982). This polypeptide has been reported to have an effect on thymus-dependent maturation of lymphoid stem cells by inducing the terminal deoxynucleotidyltransferase (Low et al., 1981). It also inhibits migration of macrophages (Thurman et al., 1984) and exerts effects on hypothalamus and pituitary (Rebar et al., 1981). Shortly after the characterization of thymosin 4, two highly homologous peptides named 8

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FIGURE 3. Amino acid sequences of -thymosins from various species: 4 (P01253); 4Ala (P34032); 4Xen (Xenopus laevis, P18758); 9 (P21752); 9Met (P21753); 10 (P13472); 11 (Oncorhynchus mykiss, P26351); 12 (Oncorhynchus mykiss, P26352); 12perch (Lateolabrax japonicus, P33248); 13(Mihelic and Voelter, 1994); 14 (Stoeva et al., 1997); 15 (P97563); scallop (Argopectan irradians; Safer and Chowrashi, 1997); zebrafish (Q9W7M8); and sea urchin (Arbacia punctulata; Safer and Chowrashi, 1997). Other -thymosins have been identified only by nucleic acid sequencing: 4Y (O14604); 4nb (Q99406); 15mouse (Q9D2R9); carp-A (Cyprinus carpio, Q91955); carp-B (Q9I954); 10torp (Torpedo marmorata, Q9I980); quail (Coturnix coturnix japonica, Q9DET5); sycon (Sycon raphanus, Q9GUA6); strongyl (Strongylocentrotus purpuratus, O76538); and gilli (Gillichthys mirabilis, Q9DFJ9). The numbers in parentheses are the accession codes for the SWISS-PROT and TrEMBL databases. Invariant residues are indicated by asterisks, and highly conserved residues are represented by colons.

and 9 were purified by isoelectric focusing (Hannappel et al., 1982a). When thymosin fraction 5 was used as the starting material for purification, thymosin 8 was isolated. However, purification starting with fresh-frozen calf thymus by a procedure that minimizes proteolysis yielded thymosin 9 (Hannappel et al., 1982a). Thymosin 9 is identical to 8 except for the presence of an additional dipeptide (AK) at the C terminus (Fig. 3). This indicates that artificial proteolysis might occur during preparation of thymosin fraction 5.

A. PURIFICATION OF -THYMOSINS

To isolate -thymosins, various purification schemes have been developed in our laboratory with particular emphasis to avoid proteolysis

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during the isolation procedure (Hannappel et al., 1982b; Hannappel, 1986; Huff et al., 1997b). The procedures for purification of -thymosins consist basically of four steps: (1) extraction of cells or tissue and simultaneous denaturation of proteases, (2) concentration and desalting by solid-phase extraction, (3) separation of the peptides according to their isoelectric points (isoelectric focusing or chromatofocusing), and (4) separation according to hydrophobicity [reversed-phase high-performance liquid chromatography (HPLC)]. B. AMINO ACID SEQUENCES AND PHYLOGENETIC DISTRIBUTION OF -THYMOSINS

Currently, 14 other -thymosins (Fig. 3) from various vertebrates and invertebrates have been described (Huff et al., 2001). They form a family of highly conserved polar 5-kDa peptides consisting of 40–44 amino acids. Members of this family have been found in species ranging from mammals to echinoderms but not in yeast or in prokaryotes. The presence of an initiator codon immediately preceding the codon for the N-terminal serine or alanine residue, and a terminator codon immediately following the codon for the C-terminal amino acid residue, suggest that -thymosins are synthesized without formation of a larger precursor. The N-terminal residue is always acetylated. Except for one phenylalanine present at position 12 of all -thymosins and a tyrosine at position 40 of 15, -thymosins do not contain aromatic amino acid residues. Thus, they almost do not absorb at 280 nm and can only be detected by absorption below 220 nm. Because many substances absorb below 220 nm, we recommend that -thymosin be separated by reversed-phase HPLC and that the separated peptides be detected by postcolumn derivatization with fluorescamine (Hannappel, 1986; Huff et al., 1997b). According to searches in expressed sequence tag (EST) and cDNA databases, other species may also contain members of the -thymosin family (Fig. 3). In addition, in Drosophila melanogaster and Caenorhabditis elegans, two larger gene products have been identified as containing three repeats of a -thymosin-like sequence (Fig. 4). The Drosophila protein (ciboulot, Cib) binds to G-actin (see later) but, unlike conventional -thymosins, it participates in actin polymerization and is functionally more similar to profilin (Boquet et al., 2000). -Thymosins are largely unstructured in water, comparable to prothymosin and parathymosin. However, addition of trifluoroethanol promotes the formation of -helical structures involving residues 4–16 and 30–40 (Zarbock et al., 1990; Czisch et al., 1993; Feinberg et al., 1996). In most mammalian tissues, at least two -thymosins are expressed (Fig. 3). Tumor tissue might contain additional -thymosins (Bao et al., 1996, 1998; Gold et al., 1997). Thymosin 4 is usually the main peptide,

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FIGURE 4. Comparison of the three -thymosin-like sequences of Cib protein of Drosophila melanogaster (O97428) and a hypothetical 17.0-kDa protein of Caenorhabditis elegans (O17389). The percentages given are calculated as the sum of invariant (*) and highly conserved (:) residues divided by the number of amino acids of the corresponding segment times 100.

representing about 70–80% of the total -thymosins in normal tissue of adult rats (450 g/g spleen, 11 g/g muscle) (Hannappel, 1986). It is present in high concentrations (up to 400 M) in rodent tissue, tumor cells, and cell lines from various mammalian species (Hannappel et al., 1982c; Xu et al., 1982). EBV-transformed human cell lines contain up to 1 pg of thymosin 4 per cell, and 1% of total protein synthesis is dedicated to the synthesis of thymosin 4 (Hannappel and Leibold, 1985). High concentrations of thymosin 4 have been detected in whole blood (12–19.5 mg/liter), mononuclear leukocytes (183–380 fg/cell), polymorphonuclear leukocytes (269–564 fg/cell), and human platelets (6.9–31.7 fg/cell), whereas serum contained less than 1% of the thymosin 4 present in whole blood. Incidentally, this is still equal to a concentration of about 20 nmol/liter of serum. No -thymosins have been detected in erythrocytes (Hannappel and van Kampen, 1987). The high intracellular concentration and ubiquitous distribution of -thymosins prompted us early to speculate that thymosin 4 is not a thymic hormone but fulfills some general function in cells, for example, as part of the cytoskeletal system.

C. -THYMOSINS AND G-ACTIN

In spite of data pointing to a general function of -thymosins inside of cells, the concept of thymosin 4 as a thymic hormone lasted until the early

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FIGURE 5. Schematic representation of G-actin sequestering by thymosin 4. For clarity thymosin 4 has been moved to the right by the length of the arrow. Thymosin 4 forms a 1:1 complex with G-actin. The ATP molecule bound to G-actin is depicted in the center of G-actin formed by its four subdomains (I–IV). The C terminus (C) and the N terminus (N) of G-actin reside in subdomain I. Thymosin 4 interacts with G-actin in an extended form like a clip covering G-actin domains III, I, and II and inhibits G-actin polymerization by steric hindrance (Safer et al., 1997). 1990s, when Safer and co-workers established that the previously isolated 5-kDa actin-sequestering peptide (Fx) is identical to thymosin 4 (Safer et al., 1990, 1991), Thymosin 4 sequesters G-actin in a 1:1 complex and inhibits salt-induced polymerization (Fig. 5). All other -thymosins studied also possess G-actin-sequestering activity in various in vitro systems (Hannappel and Wartenberg, 1993; Heintz et al., 1993; Yu et al., 1993; Jean et al., 1994; Huff et al., 1995). -Thymosins have a 50- to 100-fold higher affinity for MgATP–actin than for MgADP–actin (Carlier et al., 1993; Jean et al., 1994). The dissociation constant of the thymosin 4–ATP– G-actin complex is in the range of 0.5 to 2.5 M. Both potential helices of thymosin 4 seem to be important for complex stability. On the basis of chemical and enzymatic cross-linking a structure was proposed for the thymosin 4–G-actin complex. Thymosin 4 interacts in an extended form with subdomains III, I, and II of G-actin and inhibits polymerization by steric hindrance (Fig. 5) (Safer et al., 1997). The stability of the 4–G-actin complex (Huff et al., 2001) can be altered by changes in the amino acid sequence of thymosin 4. This is most impressive in the case of thymosin 4Ala, in which only the first amino acid residue of thymosin 4 is changed

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from serine to alanine, which results in a 3- to 5-fold higher affinity for G-actin (Huff et al., 1995). On the other hand, oxidation of the methionine residue at position 6 ( 4-sulfoxide) increases the dissociation constant of the complex by about 20-fold (Jean et al., 1994; Huff et al., 1995; Huff and Hannappel, 1997). Concomitantly, a 20-fold molar excess of thymosin 4-sulfoxide is necessary to inhibit polymerization of G-actin. Whether the oxidation of intracellular thymosin 4 to its sulfoxide is a mechanism to modulate affinity for G-actin is questionable. The herbicide paraquat (1,10 dimethyl-4,40 -dipyridylium dichloride) causes damage to human lung, presumably by oxidative stress. In an in vitro situation incubation with paraquat destabilizes the complex of thymosin 4 with G-actin in a timeand concentration-dependent manner as indicated by an about 50-fold increase in the dissociation constant. However, even in the presence of high paraquat concentrations, HPLC analysis revealed no oxidation of thymosin 4. Thus, paraquat might act directly on G-actin (Huff et al., 1998). Comparable to the oxidation of thymosin 4, truncation of the first 6 or 12 amino acid residues ( 47–43 or 413–43) increases the dissociation constant by about 20-fold. However, peptides 47–43 and 413–43 are no longer able to inhibit polymerization of G-actin despite a level of binding to G-actin comparable to thymosin 4-sulfoxide. Truncation of the first 23 amino acid residues completely abolishes the interaction with G-actin (Fig. 5) (Huff et al., 1995). G-actin covalently cross-linked to thymosin 47–43 can be polymerized by high salt to fibers even in the absence of F-actin-stabilizing phalloidin (Ballweber et al., 2002). Changes in the C-terminal structure of thymosin 4 also modulate the interaction with G-actin. Truncation of the last 26 amino acids ( 41–16) wipes out the interaction with G-actin completely. After removing the putative C-terminal helix of thymosin 4, the peptide 41–30 still inhibits saltinduced actin polymerization, although a 25-fold molar excess over G-actin was needed (Fig. 5) (Vancompernolle et al., 1992). The effect of C-terminal truncation is only minute when the last two amino acid residues of thymosin 10 were removed (Huff et al., 1997a). Chimeras of thymosin 4 and 15 were generated because thymosin 15 identified in rat prostatic carcinoma (Bao et al., 1996) binds G-actin with a 2.4-fold higher affinity than does thymosin 4. Replacement of the 10 C-terminal amino acids residues of thymosin 15 with those of thymosin 4 reduced the actin-binding affinity of the chimera relative to thymosin 15. Complementary replacement of the thymosin 4 C-terminal amino acid residues with those of thymosin 15 led to increased G-actin binding (Eadie et al., 2000). The central sequence motif 17LKKTETQEK25 of thymosin 4 seems to be important for the interaction with G-actin. This sequence motif is highly homologous to the well-known actin-binding sequence of actobindin (Safer et al., 1991) and is flanked on both sides by a potential helical region.

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Already the first six amino acid residues of thymosin 4 are indispensable for inhibition of salt-induced actin polymerization. Even minute changes in the amino acid sequences ( 4 vs. 4Ala, 4 vs. 4-sulfoxide, or 10 vs. 101–41) might be reflected in the actin-binding affinity and be important for the biological function(s). D. -THYMOSINS AND F-ACTIN

Thymosin 4 is supposed to be the main G-actin-sequestering peptide in mammals. However, it has been demonstrated that thymosin 4 is able to interact with filamentous actin (Carlier et al., 1996; Sun et al., 1996). The ability of thymosin 4 to depolymerize F-actin decreased with increasing concentrations of thymosin 4. At concentrations >100 M, thymosin 4 stabilized F-actin and was incorporated into filamentous actin at low molar ratios (Carlier et al., 1996). The notion that thymosin 4 is not just a simple G-actin-sequestering peptide was supported by the observation that the number and thickness of actin filaments increased in NIH 3T3 cells overexpressing thymosin 10 (Sun et al., 1996). In the presence of phalloidin, chemically cross-linked thymosin 4  actin can be incorporated into F-actin. As expected, chemically cross-linked thymosin 47–43  actin can polymerize even in the absence of phalloidin. Data suggest that thymosin 4 interacting with actin adopts a structural fold similar to that observed in the presence of trifluoroethanol (Ballweber et al., 2002). In a complementary manner, the structure of G-actin is changed by thymosin 4 binding (De La Cruz et al., 2000). E. -THYMOSINS AND CANCER

G-actin-sequestering -thymosins might be involved in cancerogenesis and metastatic potential of tumors because -thymosins could supply a pool of G-actin when cells need filaments (Fig. 6). It is not clear how -thymosins might influence metastasis but it is likely to relate to the need for cells to migrate (Ridley, 2000). Expression of thymosins 4, 10, and 15 appears to be involved in the manifestation of the malignant phenotype of human tumor cells. In general, increased levels of -thymosins seem to correspond to tumor malignancy. In the human breast cancer cell line MCF-7 the expression of thymosins 4 and 10 is differentially regulated (Otto et al., 2002). Several points should be taken into account when discussing the role of -thymosins in cancerogenesis and the metastatic potential of tumors: first, in most studies, only one -thymosin has been investigated, although most cells express several -thymosins, which may or may not share the same biological function(s). The expression of -thymosins is regulated differentially. However, neither the molecular basis of the gene expression nor the need of cells for different -thymosins is understood (Huff et al.,

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FIGURE 6. Functions attributed to -thymosins. The expression of -thymosins (&, ) seems to be involved in differentiation and dedifferentiation and in the organization of actin cytoskeleton (depicted as lines inside of cells). Thymosin 4 released from cells can exert additional effects. It could be oxidized to its sulfoxide or processed to AcSDKP. AcSDKP is degraded by angiotensin 1-converting enzyme (ACE). There is also evidence that -thymosins can act directly on cells, resulting in a change in gene expression or chemotaxis of certain cells. Neither receptors (?) on cells nor the signal transduction pathway(s) (?) have been characterized. -Thymosins can be cross-linked to proteins (fibrin, etc.) by transglutaminases (blood coagulation factor XIIIa).

2001). Second, often only the change in the mRNA is determined, which does not necessarily give information about the intracellular concentration of the -thymosin (Scho¨ bitz et al., 1990, 1991a, b). Third, if the cancerogenic function of -thymosins is related to their G-actin-sequestering ability, it might be helpful to monitor changes in G-and F-actin. Thymosin 10 overexpression, for example, seems to be a general event in a wide variety of human carcinomas (human colon carcinonas, germ cell carcinomas of different histological types, breast carcinomas, ovarian carcinomas, uterine carcinomas, colon carcinomas, and esophageal carcinoma cell lines) (Califano et al., 1998). With the advent of DNA array methodology, there is an increasing number of reports indicating that -thymosin expression is correlated with pathological alterations of blood vessels and cancer (Tung et al., 2001; Sardi et al., 2002). By comparing

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melanoma cells with low and high metastatic potential on DNA arrays, the mRNAs for thymosins 4 and 10 were expressed at higher levels in highly metastatic cells (Clark et al., 2000). Thymosin 15 was described as a novel regulator of tumor cell motility in Dunning rat prostatic carcinoma correlated with metastatic potential (Bao et al., 1996). In a clinical pilot study on human prostate cancer, it has been shown that thymosin 15 might be a useful marker to identify high-risk patients (Chakravatri et al., 2000). This -thymosin is also proposed as a marker for other carcinomas (Gold et al., 1997; Bao et al., 1998). To explore the molecular mechanism of transcriptional regulation of thymosin 15, the rat thymosin 15 gene was isolated and characterized (Bao and Zetter, 2000). Thymosin 15 binds G-actin with a 2.4-fold higher affinity than does thymosin 4 (Eadie et al., 2000), which might be responsible for the metastatic potential of those cells. F. -THYMOSINS IN ANGIOGENESIS AND WOUND HEALING

-Thymosins may be involved in angiogenesis (Grant et al., 1995; Malinda et al., 1997), wound healing (Frohm et al., 1996; Malinda et al., 1999), and apoptosis (Iguchi et al., 1999; Niu and Nachmias, 2000). The mRNA for thymosin 4 increased 5-fold in endothelial cells growing on Matrigel compared with cells growing on plastic (Fig. 6). Endothelial cells transfected with thymosin 4 showed an increased rate of attachment and spreading, as well as an accelerated rate of tube formation on Matrigel. Because an antisense oligonucleotide to thymosin 4 inhibited these effects, thymosin 4 might be involved early in the differentiation of endothelial cells and vessel formation (Grant et al., 1995). Hepatocyte growth factor, exerting motogenic effects on various target cells, induces the expression of thymosin 4 in human umbilical vein endothelial cells (HUVECs) (Oh et al., 2002). Thymosin 4 exhibits chemoattractant activity for HUVECs and keratinocytes (Malinda et al., 1997, 1999). The angiogenic effects of various ‘‘thymosin’’ peptides have been studied in the chick chorioallantoic membrane model. Thymosin 4, prothymosin, and thymosin 1 were found to enhance angiogenesis, whereas parathymosin, thymosin 9, and thymosin 10 were inhibitory (Koutrafouri et al., 2001). Because transglutaminases also participate in all these cellular reactions, we investigated whether thymosin 4 might be participating in transglutaminase-catalyzed reactions. Thymosin 4 serves as a specific glutaminly substrate for guinea pig transglutaminase, using dansylcadaverine as aminyl substrate. Thymosin 4 can be labeled rapidly at two residues (Gln-23 and Gln-36) whereas the third glutamine residue at position 39 reacts slowly (Huff et al., 1999). Gln-23 and Gln-36 are conserved in most or all -thymosins (Fig. 3). Despite the presence of nine lysine residues, thymosin

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4 does not participate as an aminyl substrate in the transglutaminase reaction. Thymosin 4 can be cross-linked by tissue transglutaminase to some proteins (fibrinogen, fibrin, collagen, and actin) but not to others (plasmin, hexokinase, alcohol dehydrogenase etc). After activation of human platelets with thrombin, thymosin 4 is released and cross-linked to fibrin. Because factor XIIIa is coreleased from platelets, this transglutaminase might mediate cross-linking (Fig. 6). This provides a potential mechanism to ‘‘fix’’ thymosin 4 near sites of platelet activation or injury, permitting it to contribute in the extracellular space to biological processes associated with clotting and wound repair (Huff et al., 2002). Glucocorticoids upregulate the expression of antiinflammatory mediators, and thus the characterization of these molecules could give the therapeutic benefits of steroids without toxic side effects. Supernatants from monocytes and macrophages cultured in the presence of glucocorticoids contain thymosin 4-sulfoxide as an active component. Whereas thymosin 4 is not active, thymosin 4-sulfoxide promotes dispersive locomotion of neutrophils, lowers their adhesion to endothelial cells, and inhibits their chemotactic response to fMLP. Thymosin 4-sulfoxide is a potent inhibitor of carrageenin-induced edema in mouse paw. Thymosin 4 seems to act inside of cells in its methionyl form as the main G-actin-sequestering peptide, whereas outside of cells the oxidized, methionyl-sulfoxide form of thymosin 4 attenuates inflammatory response (Fig. 6) (Young et al., 1999). It has been noticed that thymosin 4 promotes wound healing and decreases inflammation in heptanol-damaged corneas (Sosne et al., 2001). Similar effects on wound healing and inflammation were observed following alkali injury of corneas from mice. Mouse corneas topically treated with 5 g of thymosin 4 twice daily demonstrated accelerated reepithelialization and decreased signs of inflammation when compared with controls. mRNA transcript levels were decreased for IL-1 , macrophage inflammatory proteins (MIP-1, MIP-1 , and MIP-2), and monocyte chemoattractant protein 1. Thus, thymosin 4 may provide a treatment for severe traumatic corneal injuries (Sosne et al., 2002). Thymosin 4 is also active in accelerating wound repair in full-thickness wounds in both db/db diabetic and aged mice (Philp et al., 2003). The FDA has just given the permission to begin phase 1 clinical trials with thymosin 4 in wound healing in the US. When we determine thymosin 4 by reversed-phase HPLC in cells or tissue, we can easily identify -thymosins containing methionine residues, because the oxidized, methionyl-sulfoxide-containing form elutes about 2 to 5 min earlier from the column compared with the nonoxidized form (eluting at about 50 min). Exposing methionine-containing -thymosins to neutral or alkaline pH causes oxidation of the methionine residue by oxygen. To avoid oxidation we keep the peptides at slightly acidic pH or add thiodiglycol. Because it has been demonstrated that thymosin 4 and

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thymosin 4-sulfoxide possess different biological properties in terms of G-actin sequestering inside of cells or modulation of inflammation outside of cells, it becomes more and more important to discriminate between the two forms of thymosin 4. These two forms can be distinguished only by reversed-phase HPLC or MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight mass spectrometry). This shift in retention time or molecular mass is used in our laboratory routinely to identify methioninecontaining -thymosins by oxidation with diluted H2O2.

G. THYMOSIN 4 AND ACSDKP

The tetrapeptide AcSDKP (Seraspenide, Goralatide) is a physiological regulator of hematopoiesis, blocking the transition from G0/G1 to S phase of hematopoietic stem cells (Monpezat and Frindel, 1989). It was originally purified from fetal calf bone marrow (Lenfant et al., 1989). AcSDKP represents the N-terminal sequence of thymosin 4 and can be generated by a single cleavage step employing a mammalian Asp-N-like protease (Fig. 6) (Grillon et al., 1990). The protease responsible for conversion of thymosin 4 to AcSDKP has not been isolated from bone marrow. We noticed that thymosin 4 might be a substrate for caspase 3 because of its N-terminal sequence (AcSDKPD-M . . . ), which is similar to the multiple caspase 3 cleavage sites (–DxxD–) of prothymosin. The generated pentapeptide (AcSDPKD) might then be converted by a carboxypeptidase to AcSDPK. However, we were unable to cleave thymosin 4 with caspase 3 in vitro (our unpublished results). Many indirect data indicate that AcSDKP might originate from thymosin 4; however, the final proof is still missing. Thymosin 4 itself is reported to inhibit normal bone marrow progenitor cell growth. Although the inhibitory effect of thymosin 4 is similar to that of AcSDKP, a truncated form devoid of AcSDKP was also active (Bonnet et al., 1996). AcSDKP concentrations have been determined in blood (2 nM) and testicular interstitial fluid (22 nM) by a highly specific enzyme immunoassay (EIA). The authors speculate that AcSDPK might play a role in the regulation of spermatogenesis (Stephan et al., 2000). The half-life of AcSDKP in human plasma is about 80 min. AcSDKP is hydrolyzed by angiotensin 1-converting enzyme (ACE) to AcSD and KP, which is cleaved rapidly in blood to lysine and proline (Rieger et al., 1993; Rousseau et al., 1995). Consequently, higher concentrations of AcSDKP are found in patients treated with ACE inhibitors (Azizi et al., 1997; Comte et al., 1997). Because ACE inhibitors can cause anemia in some patients, it is currently discussed whether the increased level of AcSDKP caused by ACE inhibitors might be responsible for partial resistance of patients to erythropoietin treatment (Le Meur et al., 2001) or not (Abu-Alfa and Perazella, 2002).

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VI. CONCLUSIONS The work on ‘‘thymosin’’ started in 1965. Since then more than 1800 scientific papers have been published. Thymosin fraction 5 was the starting material for the isolation of many biologically important peptides. However, none of them really kept the promise to be a thymic hormone. Polypeptide 1 turned out to be identical to ubiquitin truncated by a dipeptide at the C terminus. Ubiquitin is indispensable for ATP-dependent proteolysis in cells. Thymosin 1 is generated from a larger polypeptide named prothymosin . Despite its suggestive name it became evident that the main function of prothymosin  is not as the precursor of thymosin 1; rather, it has several biological activities of its own. Prothymosin might be involved inside the nucleus in several processes controlling transcription and estrogen receptor activity. More studies will be necessary to understand the molecular mechanism by which prothymosin remodels chromatin structure and modulates transcription. Outside cells, other functions have been attributed to thymosin 1 and prothymosin (stimulation of cell migration, angiogenesis, and wound healing). In addition, thymosin 1 is used in clinical trials for treatment of viral infections (hepatitis). Parathymosin has been isolated as a Zn2+-binding protein that inhibits the glycolytic enzyme phosphofructokinase. It interacts with several other enzymes of carbohydrate metabolism and has therefore been proposed to play a role in the supramolecular organization of this important metabolic pathway. Besides this, it translocates from cytosol to the nucleus, dependent on cell density. At high cell density the bipartite NLS of parathymosin is covered by a cytosolic protein and parathymosin binds to and inhibits phosphofructokinase. At low cell density, parathymosin is translocated to the nucleus, where it binds to histone A1 and supports replication of DNA. In contrast to prothymosin, which enhances estrogen receptor transcriptional activity, parathymosin inhibits glucocorticoid-receptor binding to DNA. -Thymosins and especially thymosin 4 are the main G-actinsequestering peptides in mammals, thus playing an essential role in regulation of the microfilamental system. However, it is not clear why often two -thymosins are expressed in mammalian cells. Their differential expression patterns may indicate that they possess different functions in cells and tissues under normal and pathological conditions. Another interesting part of future research on -thymosins will be to identify other intracellular components interacting with -thymosins. Increased levels of -thymosins seem to correspond to tumor malignancy. However, the underlying molecular mechanism is not completely understood. The activity in angiogenesis and wound healing of -thymosins will become an active field of research. At least for some of the described effects an extracellular function for -thymosins seems to be obvious. It will be a major goal to search for the

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molecular mechanisms [receptor(s), signal transduction, interaction with other biomolecules] mediating the effects attributed to extracellular thymosins. It is important to keep in mind that the thymosins are a group of biologically important peptides with similarities and dissimilarities. They constitute three families at minimum: the prothymosin, parathymosin, and -thymosin families.

ACKNOWLEDGMENTS Our work on the thymosins started during a postdoctoral fellowship (E.H.) from 1979 to 1981 at the former Roche Institute of Molecular Biology in the group of Prof. Dr. B. L. Horecker (Nutley, NJ). The authors’ work has been supported since then by the Deutsche Forschungsgemeinschaft (Grants Ha 1148 and Hu 865) and in part by the Deutsche Krebshilfe.

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