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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.)

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.

Thymosin beta-10 (TB10) is an actin monomer-sequestering peptide that consists of 43 amino acid residues and that tends to form alpha-helical structures. Previously, we showed that the overexpression of TB10 dramatically increases the frequency of apoptosis in human ovarian cancer cells. To identify the critical residues responsible for TB10-mediated apoptosis, we used a series of computational methods. First, a three-dimensional structure of human TB10 was constructed using the homology modeling method with the calf thymosin beta-9 NMR structure as a template. Although the sequences of both of these structures are almost identical, 200-ps molecular dynamics simulation results showed that their secondary structures differ. Analyses of molecular dynamics snapshot structures suggested that the TB10 structure is conformationally more complicated than the TB9 structure. The conserved 17LKKTET(22 motif region of TB10 was tested by Ala and Ser scanning mutagenesis using computational and biochemical methods, and 12 mutants were transfected into cancer cell lines and tested for their effects on growth arrest. Of the 12 mutants examined, only the Thr20 to Ser20 mutation showed reduced growth arrest. These results strongly suggest that Thr20 is specifically required for actin sequestration by TB10 in ovarian cancer cells. These results may provide useful information for the development of a new ovarian cancer therapy.

The free actin concentration at steady state, Ac, is a variable that determines how actin regulatory proteins influence the extent of actin polymerization. We describe a novel method employing fluorescence anisotropy to directly measure Ac in any sample after the addition of a trace amount of labeled thymosin beta4 or thymosin beta4 peptide. Using this assay, we confirm earlier theoretical work on the helical polymerization of actin and confirm the effects of actin filament-stabilizing drugs and capping proteins on Ac, thereby validating the assay. We also confirm a controversial prior observation that profilin lowers the critical concentration of Mg2+-actin. A general mechanism is proposed to explain this effect, and the first quantitative dose-response curve for the effect of profilin on Ac facilitates its evaluation. This mechanism also predicts the effect of profilin on critical concentration in the presence of the limited amount of capping protein, which is the condition often found in cells, and the effect of profilin on critical concentration in cell extracts is demonstrated for the first time. Additionally, nonlinear effects of thymosin beta4 on the steady state amount of F-actin are explained by the observed changes in Ac. This assay has potential in vivo applications that complement those demonstrated in vitro.

Thymosin beta4 is a major actin-sequestering protein, yet the structural basis for its biological function is still unknown. This study provides insight regarding the way this 43-amino acid peptide, mostly unstructured in solution, binds to monomeric actin and prevents its assembly in filaments. We show here that the whole backbone of thymosin beta4 is highly affected upon binding to G-actin. The assignment of all amide protons and nitrogens of thymosin in the bound state, obtained using a combination of NMR experiments and selective labelings, shows that thymosin folds completely upon binding and displays a central extended region flanked by two N- and C-terminal helices. The cleavage of actin by subtilisin in the DNase I binding loop does not modify the structure of thymosin beta4 in the complex, showing that the backbone of the peptide is not in close proximity to segment 42-47 of actin. The combination of our NMR results and previously published mutation and cross-link data allows a better characterization of the binding mode of thymosins on G-actin.

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.

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.

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.

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.

In the present work we have investigated the macromolecules that contribute to the brain 1H NMR spectrum. The cerebral cortex showed distinct resonances at the uncrowded methyl- and methylene chemical shift scale of the spin-echo 1H NMR spectrum. The peaks at 1.22 and 1.40 ppm (relative to the methyl protons of N-acetyl aspartate at 2.02 ppm) arise from cerebral macromolecules without evidence for co-resonances from low molecular weight metabolites as shown by the spin-spin relaxation decays of these resonances. In addition to these NMR signals, peaks at 0.9 and 1.7 ppm from macromolecules were detected. These resonances are from proteins, and we have identified the polypeptides that contributed to the 1H NMR peaks. Two proteins that were present at concentrations of 250 and 350 micrograms/g of dryed tissue showed 1H NMR spectra that resembled the macromolecular pattern in the cerebral 1H NMR spectrum. They were identified as thymosin beta 4 and histone H1, respectively. Thymosin beta 4 was present in soluble high speed cytoplasmic fraction and in P2 pellet, whereas histone H1 was detected in nuclear enriched fraction. A chemical shift-correlated two-dimensional 1H NMR spectrum of thymosin beta 4 in vitro revealed a coupling pattern that matched the macromolecule in the cerebral cortex which we have previously noted (Kauppinen R. A., Kokko, H., and Williams, S. R. (1992) J. Neurochem. 58, 967-974). On the basis of both one- and two-dimensional NMR evidence, subcellular distribution and high concentration, we assign the 1H NMR signals at 0.9, 1.22, 1.40, and 1.7 ppm in the cerebral cortex to thymosin beta 4.

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.