JAIDS Journal of Acquired Immune Deficiency Syndromes:
Letter to the Editor
Data on the Interaction Between Prothymosin α and TLR4 May Help to the Design of New Antiviral Compounds
Cordero, Oscar J PhD
Department of Biochemistry and Molecular Biology, School of Biology, University of Santiago de Compostela, Santiago de Compostela, Spain
To the Editors:
Mosoian et al1,2 identified in macrophages that prothymosin-alpha (ProTα) has a potent postentry HIV-1-inhibitory activity, which is dependent on type I interferon (IFN) induction. They proposed that ProTα may provide, therefore, therapeutic leads for IFN-sensitive viral infections. Interestingly, Ueda et al3,4 has recently suggested that ProTα may have neuroprotective roles in brain stroke or traumatic damage because it is a unique cell death regulatory molecule that converts the intractable cell death necrosis into controllable apoptosis that can be inhibited therapeutically by growth factors.
Human prothymosin-alpha is a small 109 amino acid highly acidic protein that it was initially described as the precursor molecule for several peptides originally regarded as thymic factors/hormones, including its N-terminal region (Δ1-29) thymosin-apha1 (Tα1).5 Many of them have been used as therapeutics in late-stage clinical trials in cancer and immunodeficiency disorders, including ProTα.6 Whether ProTα, a nuclear protein involved in transcription and chromatin decondensation and in the apoptotic process,7,8 was secreted itself (it can be found in human serum) and/or served as precursor of Tα1 was disputed for several years.1,7-10
It is well established that a lot of intracellular molecules, including DNA and RNA, can be secreted via several alternative (nonclassical) secretion pathways.11 Endogenous molecules that are released in response to injury, infection, or other inflammatory stimuli and initiate inflammatory responses are so-called damage-associated molecular patterns and/or alarmins.11 It has been shown that under apoptotic stimuli, ProTα is released from the nucleus to the cytosol, where inhibits apoptosome formation.12 Very recently, Ueda et al described how necrosis-inducing stress induces an extracellular release of ProTα from neurons and astrocytes in a nonvesicular manner, whereas apoptosis-inducing stress does not, through interaction with cargo molecule for extracellular release S100A13, a member of the Ca2+-binding S100 family.13 It is probable that virus-infected CD8+ T cells secrete ProTα in a similar way.1
ProTα exerts its effect on macrophages via TLR4-dependent signal transduction2,14 and Mosoian et al2 demonstrated that ProTα activity is lost in the absence of TLR4. Because our laboratory and others had observed many immunomodulatory activities for ProTα,5,9,10,15,16 we looked for cell surface ProTα receptors on cells from the immune system and were the first group to report their existence.17-19 The presence of a cell surface ProTα receptor has also been confirmed in cortical neurons.4 Although we failed to identify TLR4 at the time, reevaluating these previously reported results under the light of new data,4,20-22 a model that may help to design new ProTα-related therapeutic compounds that can fight against IFN-sensitive viral infections, including HIV-1, is suggested.
We looked for ProTα cell-surface receptors by using a direct approach, radiolabeling the ligand and analyzing steady state binding data.17-19 Important data in which our results agree with those found by Mosoian et al2 is the fact that Tα1 neither displays anti-HIV-1 activity nor competes with ProTα for its receptors.19 Therefore, the ProTα binding to TLR4 is through its non-N-terminal part.10 ProTα mutants that lacked the N-terminal region (Δ1-29), including Tα1, or the C-terminal region (Δ102-112), including the nuclear localization signal TKKQKK, also retained the original antinecrosis activity of ProTα.4 Tα1 has shown a variety of effects on cells and pathways of the immune system by modulating dendritic cell (DC) function via the TLR9/toll IL-1 receptor (TIR) domain-containing adaptor inducing IFN-/Â-dependent viral recognition signal transduction mechanism.23
Our results and those of Mosoian et al2 also agree in that ProTα effects are not unique to macrophages because it induced IFNβ in DCs as well and it bound sites in the natural killer (NK) cell-derived YT cell line and T lymphocytes,18,19 these subsets expressing TLR4 at least in some cases21,24,25 and where ProTα can modulate several pathways.5,9,10,15,16 This means that the physiological context of a clinical trial with ProTα as therapy will be more complex, although not necessary for worse, than the ProTα effects described on macrophages treated in vitro.2
Incardinated with this issue is the fact that only one binding site was found on the NK-like cells 19 versus two sites, with high and low affinity, on T cells and monocytes.17,18 Furthermore, although there were binding sites with higher affinity in T cells, there were more lower affinity sites per cell in monocytes (macrophage precursors).17,18 Equilibrium Binding Data Analysis, Ligand, and Kinetic programs did not detect cooperation between both binding sites and both 1 receptor/2 chains and the 2 receptor model fitted with our data.17 These data support the role of the participation of TLR4 with its extracellularly associated MS-2 protein (they may form TLR4/MS-2 complexes that dimerize in the presence of ligands), with lipopolysaccharide (LPS) binding protein or with CD14,20,21,26 in ProTα's anti-HIV-1 activity.2 Although the participation of lipopolysaccharide (LPS) binding protein or CD14 cannot be totally excluded, the big difference between LPS and ProTα sizes, and certain data about the MW of the ProTα binding molecule (around 30 KDa),22 support MD-2. However, the formation of different types of complexes, and their number, in each cell type may explain the diverse affinities of ProTα.17-19
If neuron cell surface ProTα receptor is confirmed as TLR4, the existence of different complexes may also explain that ProTα survival activity is mediated by a Gαi/o-coupled receptor that activates phospholipase C and PKCβII, inducing GLUT1/4 transport to membranes, whereas ProTα apoptotic effect is mediated by PKCβI and PKCβII activation, leading to I-κB kinase complex and necrosis factor κB activation.4 These signal transduction pathways can be mediated by one of the major TLR4 signaling adaptors, toll IL-1 receptor (TIR) domain-containing adaptor inducing IFN-/Â, whereas type I IFN induction is dependent of the other main downstream TLR4 adaptor, toll IL-1 receptor (TIR) domain-containing adaptor inducing IFN-/Â.2,27 Perhaps, toll IL-1 receptor (TIR) domain-containing adaptor inducing IFN-/Â is not expressed in neurons, or the adaptor may respond to the type of extracellular complex formed pending on the type of ligand bound.
One possible explanation for the loss of activity of the mutant peptide ProTα (Δ50-89)2 could be because the size of the peptide is not enough to interact with TLR4 and MD-2 protein or to dimerize the complex.20,21 The role of extracellularly cosecreted cargo molecule S100A13 also remains to be elucidated.13 Interestingly, synthetic peptides of different lengths spanning ProTα C-terminal (Δ94-109) sequence showed different degrees of activity on enhancing NK cell cytolytic activity and on inducing immature DC maturation (but not monocyte differentiation), the best being the decapeptide (Δ100-109) that includes the nuclear localization signal.28 This sequence lacks the antinecrosis activity of ProTα on neurons.4 Altogether these facts may help in the design of those new therapeutic compounds, even several with different characteristics.29 For this purpose, it is also important to know, for the dynamics of ligand-receptor turnover, that ProTα is internalized after binding for a short period and then degraded.17 Again, our results support recent data on TLR trafficking.30
Finally, it should be taken into consideration that anti-ProTα autoantibodies, different to anti-Tα1 autoantibodies, have been found in certain diseases,19,31 and although ProTα is poorly immunogenic, particularly its non-Tα1 sequence, this fact should be also taken into account in future clinical trials.
Oscar J. Cordero, PhD
Department of Biochemistry and Molecular Biology, School of Biology, University of Santiago de Compostela, Santiago de Compostela, Spain
1. Mosoian A, Teixeira A, High AA, et al. Novel function of prothymosin alpha as a potent inhibitor of human immunodeficiency virus type 1 gene expression in primary macrophages. J Virol
2. Mosoian A, Teixeira A, Burns CS, et al. Prothymosin-alpha inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction. Proc Natl Acad Sci U S A
3. Ueda H, Matsunaga H, Uchida H, et al. Prothymosin alpha as robustness molecule against ischemic stress to brain and retina. Ann N Y Acad Sci
4. Ueda H, Fujita R, Yoshida A, et al. Identification of prothymosin-alpha1, the necrosis-apoptosis switch molecule in cortical neuronal cultures. J Cell Biol
5. Cordero OJ, Nogueira M. Thymic hormones and peptides. In: Delves PJ, Roitt IM, eds. Encyclopedia of Immunology
.2nd ed. London, United Kingdom: Academic Press; 1998:2300-2304.
6. Cordero OJ, Maurer HM, Nogueira M. Novel approaches to immunotherapy using thymic peptides. Immunol Today
7. Letsas KP, Frangou-Lazaridis M. Surfing on prothymosin alpha proliferation and anti-apoptotic properties. Neoplasma
8. Gómez-Márquez J. Function of prothymosin alpha in chromatin decondensation and expression of thymosin beta-4 linked to angiogenesis and synaptic plasticity. Ann N Y Acad Sci
9. Piñeiro A, Cordero OJ, Nogueira M. Fifteen years of prothymosin alpha: contradictory past and new horizons. Peptides
10. Hannappel E, Huff T. The thymosins. Prothymosin alpha, parathymosin, and beta-thymosins: structure and function. Vitam Horm
11. Butler GS, Overall CM. Proteomic identification of multitasking proteins in unexpected locations complicates drug targeting. Nat Rev Drug Discov
12. Jiang X, Kim HE, Shu H, et al. Distinctive roles of PHAP proteins and prothymosin-alpha in a death regulatory pathway. Science
13. Matsunaga H, Ueda H. Stress-induced non-vesicular release of prothymosin-α initiated by an interaction with S100A13, and its blockade by caspase-3 cleavage. Cell Death Differ
14. Skopeliti M, Kratzer U, Altenberend F, et al. Proteomic exploitation on prothymosin alpha-induced mononuclear cell activation. Proteomics
15. Garbin F, Eckert K, Immenschuh P, et al. Prothymosin alpha 1 effects, in vitro, on the antitumor activity and cytokine production of blood monocytes from colorectal tumor patients. Int J Immunopharmacol
16. Skopeliti M, Voutsas IF, Klimentzou P, et al. The immunologically active site of prothymosin alpha is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients. Cancer Immunol Immunother
17. Cordero OJ, Sarandeses C, Nogueira M. Prothymosin alpha receptors on peripheral blood mononuclear cells. FEBS Lett
18. Cordero OJ, Sarandeses C, Nogueira M. Prothymosin alpha receptors on lymphocytes. J Interferon Cytokine Res
19. Cordero OJ, Sarandeses C, Nogueira M. Binding of 125I-prothymosin alpha to lymphoblasts through the non-thymosin alpha 1 sequence. Life Sci
20. Park BS, Song DH, Kim HM, et al. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature
21. Jin MS, Lee JO. Structures of the toll-like receptor family and its ligand complexes. Immunity
22. Piñeiro A, Bugía MB, Arias MP, et al. Identification of receptors for prothymosin alpha on human lymphocytes. Biol Chem
23. Romani L, Bistoni F, Montagnoli C, et al. Thymosin alpha1: an endogenous regulator of inflammation, immunity, and tolerance. Ann N Y Acad Sci
24. Hammond T, Lee S, Watson MW, et al. Toll-like receptor (TLR) expression on CD4+ and CD8+ T-cells in patients chronically infected with hepatitis C virus. Cell Immunol
25. Saikh KU, Lee JS, Kissner TL, et al. Toll-like receptor and cytokine expression patterns of CD56+ T cells are similar to natural killer cells in response to infection with Venezuelan equine encephalitis virus replicons. J Infect Dis
26. Visintin A, Iliev DB, Monks BG, et al. MD-2. Immunobiology
27. Kawai T, Akira S. TLR signaling. Semin Immunol
28. Skopeliti M, Iconomidou VA, Derhovanessian E, et al. Prothymosin alpha immunoactive carboxyl-terminal peptide TKKQKTDEDD stimulates lymphocyte reactions, induces dendritic cell maturation and adopts a beta-sheet conformation in a sequence-specific manner. Mol Immunol
29. Erridge C. Endogenous ligands of TLR2 and TLR4: agonists or assistants? J Leukoc Biol
30. McGettrick AF, O'Neill LA. Localisation and trafficking of Toll-like receptors: an important mode of regulation. Curr Opin Immunol
31. Vlachoyiannopoulos PG, Frillingos S, Tzioufas AG, et al. Circulating antibodies to prothymosin alpha in systemic lupus erythematosus. Clin Immunol Immunopathol
© 2011 Lippincott Williams & Wilkins, Inc.
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