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The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin β4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Huntington disease (HD), a fatal neurodegenerative disorder, is caused by a lengthening of the polyglutamine tract in the huntingtin (Htt) protein. Despite considerable effort, thus far there is no cure or treatment available for the disorder. Using the approach of tandem affinity purification we recently discovered that prothymosin-α (ProTα), a small highly acidic protein, interacts with mutant Htt (mHtt). This was confirmed by co-immunoprecipitation and a glutathione S-transferase (GST) pull-down assay. Overexpression of ProTα remarkably reduced mHtt-induced cytotoxicity in both non-neuronal and neuronal cell models expressing N-terminal mHtt fragments, whereas knockdown of ProTα expression in the cells enhanced mHtt-caused cell death. Deletion of the central acidic domain of ProTα abolished not only its interaction with mHtt but also its protective effect on mHtt-caused cytotoxicity. Additionally, overexpression of ProTα inhibited caspase-3 activation but enhanced aggregation of mHtt. Furthermore, when added to cultured cells expressing mHtt, the purified recombinant ProTα protein not only entered the cells but it also significantly suppressed the mHtt-caused cytotoxicity. Taken together, these data suggest that ProTα might be a novel therapeutic target for treating HD and other polyglutamine expansion disorders.

Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for Cul3 Rbx1-mediated degradation of Nrf2. A majority of the INrf2/Cul3 Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal, and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that Cul3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-alpha both bind to the DGR region of INrf2. However, it is prothymosin-alpha and not Nrf2 that mediates nuclear import of INrf2/Cul3 Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/Cul3 Rbx1 complex. The INrf2/Cul3 Rbx1 complex inside the nucleus exchanges prothymosin-alpha with Nrf2, resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-alpha-mediated nuclear import of INrf2/Cul3 Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression.

Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tbeta4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tbeta4 and mediates its intracellular activity.

Our previous studies showed that thymosin beta4 (Tbeta4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with alpha1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tbeta4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-alpha1-acid glycoprotein (AGP) without Tbeta4 treatment. The AGP.PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tbeta4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.

Macromolecular translocation inhibitor II (MTI-II), which was first identified as an in vitro inhibitor of binding between the highly purified glucocorticoid receptor (GR) and isolated nuclei, is an 11.5-kDa Zn(2+)-binding protein that is also known as ZnBP or parathymosin. MTI-II is a small nuclear acidic protein that is highly conserved in rats, cows, and humans and widely distributed in mammalian tissues, yet its physiological function is unknown. To elucidate its in vivo function in relation to GR, we transiently transfected mammalian cells with an expression plasmid encoding MTI-II. Unexpectedly, we found that the expression of MTI-II enhances the transcriptional activity of GR. The magnitude of the transcriptional enhancement induced by MTI-II is comparable with that induced by the steroid receptor coactivator SRC-1. In contrast, MTI-II had little effect on the transcriptional activity of estrogen receptor. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, GR coprecipitates with MTI-II, and, vice versa, MTI-II coprecipitates with GR. The expression of various deletion mutants of MTI-II revealed that the central acidic domain is essential for the enhancement of GR-dependent transcription. Microscopic analysis of MTI-II fused to green fluorescent protein and GR fused to red fluorescent protein in living HeLa cells showed that MTI-II colocalizes with GR in discrete subnuclear domains in a hormone-dependent manner. Coexpression of MTI-II with the coactivator SRC-1 or p300 further enhances GR-dependent transcription. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, p300 and CREB-binding protein are coprecipitated with MTI-II. Furthermore, the knockdown of endogenous MTI-II by RNAi reduces the transcriptional activity of GR in cells. Moreover, expression of MTI-II enhances the glucocorticoid-dependent transcription of the endogenous glucocorticoid-inducible enzyme in cells. Taken together, these results indicate that MTI-II enhances GR-dependent transcription via a direct interaction with GR in vivo. Thus, MTI-II is a new member of the GR-coactivator complex.

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1 in vitro and in vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using an in vitro chromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.

Cellular ATP depletion in diverse cell types results in the net conversion of monomeric G-actin to polymeric F-actin and is an important aspect of cellular injury in tissue ischemia. We propose that this conversion results from altering the ratio of ATP-G-actin and ADP-G-actin, causing a net decrease in the concentration of thymosinactin complexes as a consequence of the differential affinity of thymosin beta4 for ATP- and ADP-G-actin. To test this hypothesis we examined the effect of ATP depletion induced by antimycin A and substrate depletion on actin polymerization, the nucleotide state of the monomer pool, and the association of actin monomers with thymosin and profilin in the kidney epithelial cell line LLC-PK1. ATP depletion for 30 min increased F-actin content to 145% of the levels under physiological conditions, accompanied by a corresponding decrease in G-actin content. Cytochalasin D treatment did not reduce F-actin formation during ATP depletion, indicating that it was predominantly not because of barbed end monomer addition. ATP-G-actin levels decreased rapidly during depletion, but there was no change in the concentration of ADP-G-actin monomers. The decrease in ATP-G-actin levels could be accounted for by dissociation of the thymosin-G-actin binary complex, resulting in a rise in the concentration of free thymosin beta4 from 4 to 11 microm. Increased detection of profilin-actin complexes during depletion indicated that profilin may participate in catalyzing nucleotide exchange during depletion. This mechanism provides a biochemical basis for the accumulation of F-actin aggregates in ischemic cells.

We propose phage display combined with enzyme-linked immunosorbent assay as a tool for the systematic analysis of protein-protein interactions by investigating the binding behavior of variants to a partner protein. Via enzyme-linked immunosorbent assay we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity. Hence this method enables us to calculate the relative affinities of a large number of mutants. As model systems, we investigated actin-binding motifs conserved in a number of proteins binding monomeric or filamentous actin. The hexapeptide motifs LKKTET, present in thymosin beta4, and LKKEKG, present in the villin headpiece, were mutated, and the variants were analyzed. Study of the positional tolerance allows postulating that the motifs, although similar in primary structures adopt different conformations when bound to actin. In addition, our data show that the second and the fourth amino acid of the thymosin beta4 motif and the first three residues of the villin headpiece motif are most important for actin binding. The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.

Thymosin alpha(1) (T alpha(1)) and thymosin T alpha(11) (T alpha(11)) are polypeptides with immunoregulatory properties first isolated from thymic extracts, corresponding to the first 28 and 35 amino acid residues, respectively, of prothymosin alpha (ProT alpha), a protein involved in chromatin remodeling. It has been widely supposed that these polypeptides are not natural products of the in vivo processing of ProT alpha, since neither was found in extracts in which proteolysis was prevented. Here we show that a lysosomal asparaginyl endopeptidase is able to process ProT alpha to generate T alpha(1) and T alpha(11). In view of its catalytic properties and structural and immunological analyses, this protease was identified as mammalian legumain. It selectively cleaves some of the asparaginyl-glycine residues in the ProT alpha sequence; specifically, Asn(28)-Gly(29) and Asn(35)-Gly(36) residues are cleaved with similar efficiency in vitro to generate T alpha(1) and T alpha(11), respectively. By contrast T alpha(1) is the main product detected in vivo, free in the cytosol, at concentrations similar to that of ProT alpha. The data here reported demonstrate that T alpha(1) is not an artifact but rather is naturally present in diverse mammalian tissues and raise the possibility that it has a functional role.

Actobindin is an actin-binding protein from amoeba, which consists of two beta-thymosin repeats and has been shown to inhibit actin polymerization by sequestering G-actin and by stabilizing actin dimers. Here we show that actobindin has the same biochemical properties as the Drosophila or Caenorhabditis elegans homologous protein that consists of three beta-thymosin repeats. These proteins define a new family of actin-binding proteins. They bind G-actin in a 1:1 complex with thermodynamic and kinetic parameters similar to beta-thymosins. Like beta-thymosins, they slow down nucleotide exchange on G-actin and make a ternary complex with G-actin and Latrunculin A. On the other hand, they behave as functional homologs of profilin because their complex with MgATP-G-actin, unlike beta-thymosin-actin, participates in filament barbed end growth, like profilin-actin complex. Therefore these proteins play an active role in actin-based motility processes. In addition, proteins of the actobindin family interact with the pointed end of actin filaments and inhibit pointed end growth, maybe via the interaction of the beta-thymosin repeats with two terminal subunits.

Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin beta(4), and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin beta(4) (residues 6-22) confirm the presence of an extensive binding surface between actin and thymosin beta(4), and explain why thymosin beta(4) and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin beta(4) peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin beta(4). Intracellular concentrations of thymosin beta(4) and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin beta(4)-actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin beta(4) can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.

We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin beta(4) and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.

Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 micrometer and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin beta(4) to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin beta(4), then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin beta(4) is a surprising result that suggests that significant allosteric changes affect the thymosin beta(4) binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2(1)2(1)2(1) and diffraction to 3.10 A. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.

Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Domínguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.

Human and monkey prothymosin alpha contain activated carbonyl groups on glutamic acid residues. Three lines of evidence indicate the existence of unusual phosphates. 1) Prothymosin alpha continued to be metabolically labeled with [32P]orthophosphoric acid despite a mutation at Ser1, the sole site of phosphate in purified bovine prothymosin alpha (Sburlati, A. R., De La Rosa, A., Batey, D. W., Kurys, G. L., Manrow, R. E., Pannell, L. K., Martin, B. M., Sheeley, D. M., and Berger, S. L. (1993) Biochemistry 32, 4587-4596). 2) Immediately upon cell lysis, the pH stability curves of metabolically labeled native [32P]prothymosin alpha or a [32P]histidine-tagged variant resembled the pH stability curve of acetyl phosphate. 3) After a brief incubation at pH 7, these curves changed from a pattern diagnostic for an acyl phosphate to that characteristic of a serine or threonine phosphate, an observation consistent with transfer of phosphate in vitro. Our data indicate that most of prothymosin alpha's phosphates are subject instantaneously to hydrolysis, based on the observation that greater than 90% of the phosphate initially found at pH 7 disappeared at the extremes of pH. Rapid loss of phosphate was not affected by the presence of phosphatase inhibitors including 50 mM sodium fluoride, 1 mM okadaic acid, and 0.5 mM calyculin A. The amount of phosphate missing could not be ascertained, but the trifling amount recovered on Ser or Thr depended heavily on conditions favoring the transient survival of labile phosphate. Further analysis using COS cells lysed in the presence of sodium borohydride showed that: 1) phosphate recovered on prothymosin alpha decreased 8-fold when lysates were treated with borohydride; 2) the reagent caused 4-8 glutamic acid residues/molecule to vanish; 3) using [3H]NaBH4, label was introduced into proline, a product derived from reductive cleavage of phosphoglutamate; and 4) [3H]proline was localized almost exclusively to a peptide with pronounced homology to the histone binding site of nucleoplasmin, a chromatin remodeling protein found in Xenopus laevis. Our data demonstrate that prothymosin alpha is energy-rich by virtue of stoichiometric amounts of glutamyl phosphate.

Prothymosin alpha is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin alpha contains stoichiometric amounts of phosphate on the glutamyl groups of the protein and that in vitro the phosphate undergoes rapid hydrolysis or transfer to a nearby serine residue. Here an assay for the presence of acyl phosphates in vivo has been developed by measuring stable phosphoserine and phosphothreonine in vitro. The assay was used to determine the half-life of the acyl phosphates on prothymosin alpha in vivo by pulse-labeling HeLa cells with [32P]orthophosphate and chasing using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of [32P]ATP by expansion of the pool; and incubation with inorganic phosphate. Regardless of the method, the phosphate turned over with a half-life of 75-90 min. The ability of cells to phosphorylate old prothymosin alpha molecules was established by demonstrating equivalent labeling of the protein with [32P]orthophosphate in the presence and absence of cycloheximide. The half-life of the acyl phosphates was also studied in resting and growing NIH3T3 cells, with measured values of 30-35 and 70 min, respectively. Our data suggest that the "activity" of prothymosin alpha involves the turnover of its acyl phosphates and that it participates in a function common to all nucleated mammalian cells regardless of whether they are quiescent or undergoing rapid proliferation. This is the first measurement of the stability of protein-bound acyl phosphates in vivo.

Prothymosin alpha (ProTalpha) is an acidic protein involved in cell proliferation. Its phosphorylation status is correlated with proliferative activity. Here we report the isolation and characterization of a ProTalpha-phosphorylating kinase (ProTalphaK) from mouse splenocytes that seems to be responsible for the in vivo phosphorylation of ProTalpha and that differs from other protein kinases reported to date. This enzyme, mainly located in the cytosol, has an molecular mass of 180 kDa and appears to be made up of two proteins of 64 and 60 kDa. Its activity was markedly enhanced by mitogenic activation of cells. The ProTalpha residues phosphorylated by the enzyme in vitro are a Thr at position 7 and another Thr at positions 12 or 13, both located within casein kinase 2 (CK-2) consensus motifs; these are the same residues as are phosphorylated in vivo. The new enzyme shows a number of clear structural and catalytic differences from CK-2. It phosphorylates histones H2B and H3, although with weaker activity than ProTalpha. An enzyme with the same characteristics was also found in other murine tissues and cell lines.

Thymosin beta 4 is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of Tbeta 4 in actin assembly in the high range of concentrations (up to 300 micron) at which Tbeta 4 is found in highly motile blood cells. Tbeta 4 behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 micron). As the concentration of Tbeta 4 increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The Tbeta 4-actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the copolymerization of actin and Tbeta 4-actin complex, the critical concentration is the sum of free G-actin and Tbeta 4-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by Tbeta 4-G-actin complex. The incorporation of Tbeta 4-actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, Tbeta 4 is not a simple passive actin-sequestering agent, and at high concentrations the ability of Tbeta 4-actin to copolymerize with actin reduces the sequestering activity of G-actin-binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing beta-thymosins.

beta-Thymosins are the currently favored candidates for maintaining the large actin monomer (G-actin) pool in living cells. To determine if beta-thymosin behaves like a simple G-actin buffering agent in the complex environment of a cell, we overexpressed thymosin beta10 (Tbeta 10) in NIH3T3 cells and determined the effect on the monomer/polymer equilibrium. Tbeta 10 is the predominant beta-thymosin isoform in the NIH3T3 cell line, and it is present in approximately equal molar ratio to profilin and cofilin/actin depolymerizing factor, two other well characterized actin monomer binding proteins. Clonal cell lines that overexpressed three times more Tbeta 10 had 23-33% more polymerized actin than control cells, and the filaments appeared thicker after staining with fluorescent phalloidin. There was no change in total actin, profilin, and cofilin/actin depolymerizing factor content. The overexpressing cells were more motile; they spread faster and had higher chemotactic and wound healing activity. Assuming that there is no compensatory inactivation of the other classes of monomer binding proteins, our paradoxical observation can be accounted for quantitatively by a parallel in vitro study (Carlier, M.-F., Didry, D., Erk, I., Lepault, J., Van Troys, L., Vanderkekove, J., Perelroizen, I., Yin, H. L., Doi, Y., and Pantaloni, D., (1996) J. Biol. Chem. 271, 9231-9239). beta-Thymosin at levels comparable with that found in the overexpressing cells binds actin filaments and decreases the critical concentration (C(c)) for actin polymerization. This reduces the monomer buffering ability of beta-thymosin, so that above a certain threshold an incremental increase in thymosin does not lead to a corresponding increase in G-actin. Furthermore, the decrease in C(c) reduces the buffering capacity of the other actin monomer binding proteins. As a consequence, an increase in beta-thymosin does not necessarily result in a proportionate increase in actin monomer content in a complex environment containing other actin monomer binding proteins. The outcome depends on the level of beta-thymosin expression relative to the composition of the other actin monomer binding protein. Our results suggest that beta-thymosins are not simple actin buffering proteins and that their biphasic action may have physiological significance.

Binding sites of actin and thymosin beta 4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys10, Cys374, or the sulfur atom of the ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S), the actin derivatives were reacted with synthetic thymosin beta 4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate cross-linking as followed by UV spectroscopy was found for Cys374 of actin and Cys6 of thymosin beta 4, indicating that the N terminus of thymosin beta 4 is in close proximity (< or = 9.2 A) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin beta 4 analogs when the cross-linkers were anchored at Cys10 of actin. A second contact site was identified by cross-linking of Cys17 and Cys28 in thymosin beta 4 with the ATP gamma S derivative bound to actin, indicating that the hexamotif of thymosin beta 4 (positions 17-22) is in close proximity (< or = 9.2 A) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin beta 4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.

The 11.5-kDa zinc-binding protein (ZnBP, parathymosin-alpha), a potent inactivator of 1-phosphofructokinase, is found only in the cytoplasm of most tissues despite the presence of the putative nuclear localization signal PKRQKT. Recent reports on nuclear uptake of ZnBP could not exclude the participation of unspecific diffusion. We show here that wild-type ZnBP overexpressed in COS cells accumulates exclusively in the nucleus but that ZnBP with a mutated or deleted PKRQKT motif appears both in the nucleus and in the cytoplasm. In contrast, fusion proteins between ZnBP and parts of the endoplasmic reticulum protein calreticulin required the intact PKRQKT motif for nuclear import. The motif RKR, located nine amino acids upstream of the PKRQKT motif, is also involved in the active nuclear import of ZnBP. In contrast to rat hepatocytes and kidney cells in situ, which have ZnBP almost exclusively in the cytosol, we find ZnBP in Reuber H35 hepatoma cells and normal rat kidney cells only in the nuclei. Freshly isolated rat hepatocytes translocate their ZnBP to the nucleus in < 24 h during standard cell culture conditions.

Prothymosin alpha is a widely distributed polypeptide whose function, though unknown, seems to be related to cell proliferation. In vitro, it is a substrate for casein kinase-2. In this work, extracts of mitogenically stimulated murine splenic lymphocytes labeled with [32P] orthophosphate were found to contain [32P]prothymosin alpha. Phosphorylation activity was highly dependent on mitogenic activation with concanavalin A plus interleukin-2. While cells remained viable, phosphorylation increased with stimulation time in the presence of [32P]orthophosphate. Structural analysis showed that prothymosin alpha was phosphorylated at Thr residues located among its first 14 amino acids, whereas its in vitro phosphorylation by casein kinase-2 affects both Ser and Thr residues in this fragment, apparently in similar proportions. Thus, casein kinase-2 seems not to be responsible for the phosphorylation of prothymosin alpha in vivo. Prothymosin alpha was also found to be phosphorylated in proliferating murine thymocytes and HeLa cells; the phosphorylation sites were the same as in splenic lymphocytes, but the rate of phosphorylation was about 5 times lower. In thymocytes and subconfluent HeLa cells, the [32P]prothymosin alpha concentrations of the cytosolic and nuclear fractions were similar; in splenic lymphocytes, [32P]prothymosin alpha was found mostly in cytosol.

The beta-thymosins are a family of related peptides. Recently, thymosin beta 4 was identified as a significant actin monomer sequestering protein in cells. To determine if other beta-thymosins also bind actin, and how they may participate in the regulation of actin polymerization, we expressed thymosin beta 4 and its major homolog, thymosin beta 10, in bacteria and characterized their interactions with actin. Equilibrium sedimentation studies showed that thymosin beta 4 behaved as a monomeric protein in solution. Both beta-thymosins bound skeletal muscle actin and inhibited actin polymerization with similar Kd values (between 0.7-1 microM). They were not inhibited by polyphosphoinositides. Kinetic measurements showed that at high ratios of beta-thymosin to actin, beta-thymosin decreased the rate of barbed end filament growth. However, in spite of a close agreement between the kinetic and steady state Kd values, the rate of barbed end filament growth was slightly, but reproducibly, larger than expected, and this deviation was particularly noticeable at lower ratios of beta-thymosin to actin. We conclude that unlike profilin, beta-thymosins are primarily actin monomer sequestering proteins, although some aspects of their interactions with actin are still not completely understood.

The primary structure of an elastase inhibitor located in the cytoplasm of leukocytes obtained from the equine species has been determined. By sequence comparison, the protein was found to be a member of the serpin family with strong identity to plasminogen activator inhibitor-2. In contrast to other serpins this protein contained no carbohydrate and had a blocked amino terminus. Preliminary evidence indicates that the inhibitor has the additional feature of being a thymosin beta 4-binding protein, since this polypeptide was always located in purified preparations of the protein. This suggests a physiological role for cytoplasmic elastase inhibitors in the thymosin beta 4-regulated rearrangement of the cytoskeleton of leukocytes.

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.

At least 50% of the actin in resting human platelets is unpolymerized, and the bulk of this unpolymerized actin is complexed with a recently identified acidic, heat-stable 5-kDa peptide, named "Fx." Purified Fx binds stoichiometrically to muscle G-actin, forming a complex identifiable by nondenaturing polyacrylamide gel electrophoresis. Formation of the complex inhibits salt-induced polymerization of G-actin. Here we report that Fx is indistinguishable from thymosin beta 4, an acidic, heat-stable 5-kDa peptide first isolated from calf thymus and thought to be a thymic hormone. The complete amino acid sequence of Fx was determined and was found to be identical with that of thymosin beta 4. Authentic thymosin beta 4 is functionally equivalent to Fx, forming a 1:1 complex with actin monomers and inhibiting polymerization. The widespread distribution and high intracellular concentration of thymosin beta 4 (Fx) strongly suggest that it plays a significant role in regulating actin polymerization in many cell types.

Prothymosin alpha is a highly acidic protein which lacks an amino-terminal signal peptide, yet was once thought to be a precursor for thymosin alpha 1, a putative peptide hormone secreted by the thymus. Here, two lines of evidence are presented that strongly implicate prothymosin alpha as a nuclear protein: 1) in COS cells transfected with the human prothymosin alpha gene copious amounts of prothymosin alpha were present in sealed nuclei obtained by treating these cells with cytochalasin B and enucleating them centrifugally. 2) Constructs in which human prothymosin alpha nucleic acid sequences were fused in-frame either near the amino terminus of the beta-galactosidase gene in pCH110 or at the carboxyl terminus, when expressed in COS cells, resulted in nuclear localization of the fusion protein; indirect immunofluorescence in situ was used as the assay. The basic cluster of amino acids at the carboxyl terminus of prothymosin alpha, TKKQKT, has been identified as part of the nuclear targeting signal, whereas the basic cluster of amino acids situated within the thymosin alpha 1 sequence at the amino terminus failed to effect nuclear transport.

Prothymosin alpha and thymosin alpha 1 are believed to be thymus-derived, hormone-like materials with immunomodulatory functions performed outside the cell. These functions are inconsistent with the existence of a full length cDNA clone that does not encode an amino-terminal signal peptide or several consecutive hydrophobic residues. A study of the prothymosin alpha mRNAs and genes was undertaken in search of evidence for secreted forms of the protein. Prothymosin alpha mRNA was localized exclusively on free, rather than membrane-bound, polysomes. Upon screening cosmid and plasmid libraries totaling 2 X 10(6) clones, a gene family consisting of six members was identified. Sequence information from the 5'-ends of all the genes indicated that none encodes an amino-terminal signal peptide. One of the genes, apparently by means of alternate splicing, gives rise to two prothymosin alpha mRNAs, one of which has an additional internal glutamic acid codon with respect to the other. Comparison of the translated nucleic acid sequences of the five remaining genes with those encoded in the mRNAs revealed 30-98% homology in the first 50 amino acids. These five genes appear to be processed genes and/or pseudogenes. The localization of prothymosin alpha mRNAs on free polysomes, together with the partial nucleotide sequences of the genes, strongly suggest an intracellular function for prothymosin alpha. Therefore, the possibility must be raised that prothymosin alpha and its peptide derivatives act as xenobiotics when introduced into assays of immune function.