Prothymosin Alpha Research Articles

Neuroprotective DAMPs member prothymosin alpha has additional beneficial actions against cerebral ischemia-induced vascular damages

Prothymosin alpha (ProTα) suppresses stress-induced necrosis of cultured cortical neurons. As neuroprotection alone could not explain the long-lasting protective actions against cerebral ischemia by ProTα, we further examined whether ProTα, in addition to neuroprotective effects, has other anti-ischemic activities. When recombinant mouse ProTα (rmProTα) at 0.3 mg/kg was intravenously (i.v.) given 2 h after the start of tMCAO, all mice survived for more than 14 days. In evaluation of CD31- and tomato lectin-labeling as well as IgG and Evans blue leakage, rmProTα treatment (0.1 mg/kg) largely blocked ischemia-induced vascular damages. Therefore, rmProTα has novel beneficial effects against ischemia-induced brain damage through vascular mechanisms.

Neuroprotective impact of prothymosin alpha-derived hexapeptide against retinal ischemia–reperfusion

Prothymosin alpha (ProTα) has robustness roles against brain and retinal ischemia or serum-starvation stress. In the ProTα sequence, the active core 30-amino acid peptide/P30 (a.a.49–78) is necessary for the original neuroprotective actions against ischemia. Moreover, the 9-amino acid peptide sequence/P9 (a.a.52–60) in P30 still shows neuroprotective activity against brain and retinal ischemia, though P9 is less potent than P30. As the previous structure–activity relationship study for ProTα may not be enough, the possibility still exists that any sequence smaller than P9 retains potent neuroprotective activity. When different P9- and P30-related peptides were intravitreally injected 24h after retinal ischemia in mice, the 6-amino acid peptide/P6 (NEVDEE, a.a.51–56) showed potent protective effects against ischemia-induced retinal functional deficits, which are equipotent to the level of P30 peptide in electroretinography (ERG) and histological damage in Hematoxylin and Eosin (HE) staining. Further studies using ERG and HE staining suggested that intravitreal or intravenous (i.v.) injection with modified P6 peptide/P6Q (NEVDQE) potently inhibited retinal ischemia-induced functional and histological damage. In an immunohistochemical analysis, the ischemia-induced loss of retinal ganglion, bipolar, amacrine and photoreceptor cells were inhibited by a systemic administration with P6Q peptide 24h after the ischemic stress. In addition, systemic post-treatment with P6Q peptide significantly inhibited retinal ischemia-induced microglia and astrocyte activation in terms of increased ionized calcium-binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) intensity, respectively, as well as their morphological changes, increased number and migration. Thus, this study demonstrates the therapeutic significance of modified P6 peptide P6Q (NEVDQE) derived from 6-amino acid peptide (P6) in ProTα against ischemic damage.

A flow cytometric approach for studying alterations in the cytoplasmic concentration of calcium ions in immune cells following stimulation with thymic peptides

[Ca2+]i alterations are vital in signaling pathways of cell activation. We tried to detect such changes, in intracellular signaling pathways downstream TLR4 in immune cells, following stimulation with prothymosin alpha (proTα) and its decapeptide proTα(100–109). Human leukocytes were activated with LPS, proTα or proTα(100–109), directly or after 24h stimulation, while neutrophils were directly challenged. Cells were loaded with Fluo-4 and cytoplasmic Ca2+ alterations were recorded by flow cytometry. Direct challenge with 20μg/mL LPS induced a measurable [Ca2+]i increase in macrophages and neutrophils. Monocytes and macrophages incubated for 24h with LPS, proTα or proTα(100–109) and challenged with LPS, displayed a robust response. Lymphocytes and iDCs exhibited no alterations. Conclusively, we assessed a flow cytometry-based method for monitoring Ca2+ ion influx changes in immune cells. Their stimulation with proTα or proTα(100–109) generates an activating background, similar to LPS, allowing for the detection of [Ca2+]i alterations induced upon subsequent challenge.

Prothymosin Alpha and Immune Responses: Are We Close to Potential Clinical Applications?

The thymus gland produces soluble molecules, which mediate significant immune functions. The first biologically active thymic extract was thymosin fraction V, the fractionation of which led to the isolation of a series of immunoactive polypeptides, including prothymosin alpha (proTα).ProTα displays a dual role, intracellularly as a survival and proliferation mediator and extracellularly as a biological response modifier. Accordingly, inside the cell, proTα is implicated in crucial intracellular circuits and may serve as a surrogate tumor biomarker, but when found outside the cell, it could be used as a therapeutic agent for treating immune system deficiencies. In fact, proTα possesses pleiotropic adjuvant activity and a series of immunomodulatory effects (eg, anticancer, antiviral, neuroprotective, cardioprotective). Moreover, several reports suggest that the variable activity of proTα might be exerted through different parts of the molecule. We first reported that the main immunoactive region of proTα is the carboxy-terminal decapeptide proTα(100–109). In conjunction with data from others, we also revealed that proTα and proTα(100–109) signal through Toll-like receptor 4. Although their precise molecular mechanism of action is yet not fully elucidated, proTα and proTα(100–109) are viewed as candidate adjuvants for cancer immunotherapy.Here, we present a historical overview on the discovery and isolation of thymosins with emphasis on proTα and data on some immune-related new activities of the polypeptide and smaller immunostimulatory peptides thereof. Finally, we propose a compiled scenario on proTα’s mode of action, which could eventually contribute to its clinical application.

Identification of the β-catenin/JNK/prothymosin-alpha axis as a novel target of sorafenib in hepatocellular carcinoma cells.

Sorafenib is a kinase inhibitor used as anticancer drug against various human tumors, including advanced hepatocellular carcinoma (HCC). β-Catenin and prothymosin alpha (PTMA) are overexpressed in HCC and other tumors. Previous studies have shown that PTMA expression modulates the response of HCC cells to sorafenib. However, the underlying mechanism of PTMA activity in this context remains unclear. We show here that sorafenib inhibits both β-catenin and PTMA in a dose-dependent manner. Silencing β-catenin reduces PTMA level and sensitizes HCC cells to sorafenib. In contrast, ectopic expression of β-catenin induces PTMA expression and cell resistance to the drug. Sorafenib inhibits PTMA expression at the transcriptional level by inhibiting the β-catenin pathway. Nucleotide deletion analysis of the PTMA gene promoter reveals that a DNA segment lying 1,500-1,600 bp upstream of the PTMA transcription start site represents an AP-1-binding site that is critical for β-catenin modulation of gene transcription in response to sorafenib. In addition, chemical inhibitors that target JNK abrogate β-catenin/AP-1 binding to the endogenous PTMA gene and reduces PTMA transcription and protein expression. Silencing of β-catenin or c-Fos induces similar effects on gene regulation and these are reversed by ectopic expression of β-catenin. Mutations in the PTMA promoter at the predicted β-catenin/AP-1 binding site partly abrogate sorafenib's effects on PTMA transcription. These results indicate that PTMA is induced by the oncoprotein β-catenin and protects HCC cells against sorafenib-induced cell death. The β-catenin/JNK/PTMA axis may thus represent a novel target for chemotherapy against HCC.

Prothymosin alpha expression and localization during the spermatogenesis of Danio rerio.

Prothymosin α (PTMA) is a highly acidic, intrinsically disordered protein, which is widely expressed and conserved throughout evolution; its uncommon features are reflected by its involvement in a variety of processes, including chromatin remodelling, transcriptional regulation, cell proliferation and death, immunity. PTMA has also been implicated in spermatogenesis: during vertebrate germ cell progression in the testis the protein is expressed in meiotic and post-meiotic stages, and it is associated with the acrosome system of the differentiating spermatids in mammals. Then, it finally localizes on the inner acrosomal membrane of the mature spermatozoa, suggesting its possible role in both the maturation and function of the gametes. In the present work we studied PTMA expression during the spermatogenesis of the adult zebrafish, a species in which two paralogs have been described. Our data show that ptma transcripts are expressed in the testis, and localize in meiotic and post-meiotic germ cells, namely spermatocytes and spermatids. Consistently, the protein is expressed in spermatocytes, spermatids, and spermatozoa: its initial perinuclear distribution is extended to the chromatin region during cell division and, in haploid phases, to the cytoplasm of the developing and final gametes. The nuclear localization in the acrosome-lacking spermatozoa suggests a role for PTMA in chromatin remodelling during gamete differentiation. These data further provide a compelling starting point for the study of PTMA functions during vertebrate fertilization.

Tongue sole (Cynoglossus semilaevis) prothymosin alpha: Cytokine-like activities associated with the intact protein and the C-terminal region that lead to antiviral immunity via Myd88-dependent and -independent pathways respectively

Prothymosin alpha (ProTα) is a small protein that in mammals is known to participate in diverse biological processes including immunomodulation. In teleost, the immunological function of ProTα is unknown. In the current study, we investigated the expression and function of the ProTα (named CsProTα) from the teleost fish tongue sole (Cynoglossus semilaevis). We found that CsProTα expression was abundant in immune relevant tissues and upregulated by megalocytivirus infection. Immunoblot detected secretion of CsProTα by peripheral blood leukocytes. Recombinant CsProTα (rCsProTα) as well as the C-terminal 11-residue (Ct11) were able to bind head kidney monocytes (HKM) and induce immune gene expression; however, the induction patterns caused by rCsProTα and Ct11 differed considerably. When introduced in vivo, rCsProTα and Ct11 significantly reduced megalocytivirus infection in fish tissues, whereas rCsProTα antibody significantly promoted viral replication. Blocking of Myd88 activity abolished the virus-inhibitory effect of rCsProTα but not Ct11. Taken together, these results demonstrate for the first time that both the intact protein and the C-terminal segment of a teleost ProTα can act like cytokines and induce antiviral immunity via, however, distinct signaling pathways that differ in the requirement of Myd88.

Sorafenib Induces Apoptosis in Hepatocellular Carcinoma Cells by Inhibiting c-Myc and Prothymosin-Alpha

The anti-apoptotic protein prothymosin alpha (PTMA) is overexpressed in various cancers, including hepatocellular carcinoma (HCC). Earlier studies have shown that PTMA blocks apoptosis in cancer cells by inhibiting caspase-9 activation and apoptosome formation. Our recent study shows that silencing of PTMA potentiates the mitochondria-dependent apoptosis pathway in sorafenib-treated HCC cells, leading to Bax translocation, pBad dephosphorylation, and cytochrome c release. Our findings also indicate that the pERK/c-Myc/Max/PTMA axis represents a newly identified target of sorafenib in chemotherapy against HCC.

Expression of prothymosin α predicts early recurrence and poor prognosis of hepatocellular carcinoma

Prothymosin alpha (PTMA) is a nuclear oncoprotein-transcription factor essential for cell cycle progression and proliferation. PTMA was overexpressed in several human malignancies including hepatocellular carcinoma (HCC). However, the prognostic significance of PTMA protein expression in HCC remains unclear. In the present study, we evaluated PTMA protein expression by immunohistochemistry in order to elucidate the prognostic roles of PTMA in HCC patients. By immunohistochemistry, we investigated the expression of PTMA protein in tumor tissue from 226 HCC patients who underwent curative hepatectomy. Univariate and multivariate analyses were performed to evaluate its predictive value for tumor recurrence and survival of patients. The median follow-up period was 120 months. PTMA expression was observed in 162 (71.7%) of the 226 HCC patients and was significantly associated with higher Edmondson grade, microvascular invasion, intrahepatic metastasis, higher American Joint Committee on Cancer (AJCC) T-stage, and lower albumin level. PTMA expression was an independent predictor of early recurrence (P=0.001). PTMA expression showed an unfavorable influence on recurrence-free survival (RFS) (P<0.001). Subgroup analysis showed that among patients with tumor size ≤5.0 cm (140 patients), patients at AJCC T-stage 1 (95 patients) and patients with alpha-fetoprotein ≤20 ng/mL (83 patients), the differences in RFS between PTMA-positive and PTMA-negative groups were also statistically significant (P=0.017, P=0.002 and P=0.002, respectively). In addition, PTMA expression was an independent predictor of shorter RFS (P=0.011). PTMA expression showed an unfavorable influence on overall survival (P=0.014), but was not an independent predictor of shorter overall survival (P=0.161). PTMA protein expression might be a novel predictor of early recurrence and RFS in HCC patients, even those at early stage or with alpha-fetoprotein-negative after curative hepatectomy. PTMA could be used as an immunohistochemical biomarker to detect patients with a high risk of recurrence.

Specific in vitro binding of a new 99mTc-radiolabeled derivative of the C-terminal decapeptide of prothymosin alpha on human neutrophils

Prothymosin alpha (ProTα) is a conserved mammalian polypeptide with intracellular functions associated with cell proliferation and apoptosis and an extracellular role associated with immunopotentiation. The N-terminal fragment [1–28], which is identical with the immunostimulating peptide thymosin α1 (Tα1), was earlier considered as the immunoactive region of the polypeptide; however, recent data suggest that ProTα may exert a discrete immunomodulating action through its central or C-terminal region, via targeting Toll-like receptor- 4 (TLR4). In this work, a derivative of the C-terminal fragment ProTα[100-109] (ProTα-D1) that can be radiolabeled with 99mTc was developed. The biological activity of the non-radioactive 185/187rhenium-complex of this derivative ([185/187Re]ProTα-D1, structurally similar with [99mTc]ProTα-D1) was verified through suitable in vitro bioassays on human neutrophils. Subsequent cell-binding studies revealed specific, time-dependent and saturable binding of [99mTc]ProTα-D1 on neutrophils, which was inhibited by intact ProTα and ProTα[100-109], as well as by a “prototype” TLR4-ligand (lipopolysaccharide from Escherichia coli). Overall, our results support the existence of ProTα-binding sites on human neutrophils, recognizing [99mTc]ProTα-D1, which might involve TLR4. [99mTc]ProTα-D1 may be a useful tool for conducting further in vitro and in vivo studies, aiming to elucidate the extracellular mode of action of ProTα and, eventually, develop ProTα-based immunotherapeutics.

Evidence for ProTα-TLR4/MD-2 binding: molecular dynamics and gravimetric assay studies

Objective: During preconditioning, lipopolysaccharide (LPS) selectively activates TLR4/MD-2/Toll/IL-1 receptor-domain-containing adaptor inducing IFN-β (TRIF) pathway instead of pro-inflammatory myeloid differentiation protein-88 (MyD88)/MyD88-adaptor-like protein (MAL) pathway. Extracellular prothymosin alpha (ProTα) is also known to selectively activate the TLR4/MD2/TRIF–IRF3 pathway in certain diseased conditions. In the current study, biophysical evidence for ProTα/TLR4/MD-2 complex formation and its interaction dynamics have been studied.Research design and methods: Gravimetric assay was used to investigate ProTα/TLR4/MD-2 complex formation while molecular dynamics (MD) simulation was used to study its interaction dynamics.Results: Through electrostatic interaction, full-length ProTα (F-ProTα) C-terminal peptide (aa 91 – 111) superficially interacts with similar TLR4/MD-2 (KD = 273.36 nm vs 16.07 μg/ml [LPS]) conformation with LPS at an overlapping three-dimensional space while F-ProTα is hinged to the TLR4 scaffold by one-amino acid shift-Mosoian domain (aa-51 – 90). Comparatively, F-ProTα better stabilizes MD-2 metastable states transition and mediates higher TLR4/MD-2 interaction than LPS.Conclusions: ProTα via its C-terminal peptide (aa 91 – 111) exhibits in vitro biophysical contact with TLR4/MD-2 complex conformation recognized by LPS at overlapping LPS-binding positions.

Characterization of two thymosins as immune-related genes in common carp (Cyprinus carpio L.)

Prothymosin alpha (ProTα) and thymosin beta (Tβ) belong to thymosin family, which consists of a series of highly conserved peptides involved in stimulating immune responses. ProTα b and Tβ are still poorly studied in teleost. Here, the full-length cDNAs of ProTα b and Tβ-like (Tβ-l) were cloned and identified in common carp (Cyprinus carpio L.). The expressions of carp ProTα b and Tβ-l exhibited rise-fall pattern and then trended to be stable during early development. After spring viraemia of carp virus (SVCV) infection, the carp ProTα b and Tβ-l transcripts were significantly up-regulated in some immune-related organs. When transiently over-expressed carp ProTα b and Tβ-l in zebrafish, these two proteins up-regulated the expressions of T lymphocytes-related genes (Rag 1, TCR-γ, CD4 and CD8α). These results suggest that carp ProTα b and Tβ may ultimately enhance the immune response during viral infection and modulate the development of T lymphocytes in teleost.

Oncogenic c-Myc and prothymosin-alpha protect hepatocellular carcinoma cells against sorafenib-induced apoptosis

Prothymosin alpha (PTMA) is overexpressed in various human tumors, including hepatocellular carcinoma (HCC). The significance of PTMA overexpression and its underlying mechanism remain unclear. We show here that silencing PTMA sensitizes HCC cells to the kinase inhibitor sorafenib. In contrast, ectopic expression of PTMA induces cell resistance to the drug. While inhibitors targeting JNK, ERK or PI3K reduce PTMA expression, only ERK activation is suppressed by sorafenib. In addition, inhibition of ERK produces a dramatic decrease in both endogenous PTMA level and promoter activation. Ectopic expression of active MKK1/2 considerably induces PTMA expression. We also identify a sorafenib-responsive segment lying 1000–1500-bp upstream of the PTMA transcription start site and observe that it is controlled by c-Myc and ERK. Mutation in the PTMA promoter at the predicted c-Myc binding site and silencing of c-Myc both abrogate sorafenib's effect on PTMA transcription. We also find that silencing PTMA potentiates Bax translocation to mitochondria in response to sorafenib and this is associated with increased cytochrome c release from mitochondria and enhanced caspase-9 activation. These results indicate that PTMA is positively regulated by the oncoprotein c-Myc and protects HCC cells against sorafenib-induced cell death, thus identifying PTMA as a new target for chemotherapy against HCC.

Nadekspresja protymozyny alfa w gruczolakach przysadki jest związana z nawrotowością a nie z proliferacją komórek i inwazyjnością guza

Prothymosin alpha (ProTα) is a peptide initially considered as a thymic hormone, but further studies have shown its wide distribution in different tissues and organs. It has a prevalent nuclear localisation and is thought to be involved in the control of proliferation and apoptosis. In earlier studies, the overexpression of ProTα was found in several human tumours, including pituitary adenomas. The present study deals with the relations of ProTα to the pituitary adenoma hormonal phenotype, proliferation, recurrence and invasiveness. Sixty two pituitary adenomas were included in the study. The invasiveness of the tumours was estimated before surgery by means of magnetic resonance imaging. The paraffin sections of the tumours were immunostained with an antibody against the C-terminal fragment (101-109) of ProTα and with anti-Ki-67 antibody. The hormonal phenotype of the investigated pituitary adenomas had been established previously by means of immunostaining with antibodies to pituitary hormones (GH, PRL, FSH, LH, TSH, ACTH and α-subunit). Strong immunostaining with anti-ProTα antibody occurred in the subpopulation of cell nuclei and the walls of intratumoural blood vessels. ProTα index is higher in clinically non-functioning pituitary adenomas (CNFPA) compared to any type of functioning adenomas. There was no difference in the percentage of ProTα- positive cell nuclei in non-invasive vs. invasive adenomas, but it was significantly more frequent in recurrent than in primary tumours. Moreover, the decrease of ProTα index was found in somatotroph tumours treated with somatostatin analogues vs. untreated ones. The percentage of ProTα nuclei did not correlate with Ki-67 index. The overexpression of nuclear ProTα in pituitary adenomas is related to tumour recurrence, but not to proliferation or invasiveness.

Differential modulation of host genes in the kidney of brown trout Salmo trutta during sporogenesis of Tetracapsuloides bryosalmonae (Myxozoa)

Tetracapsuloides bryosalmonae (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids in Europe and North America. In Europe, spores of T. bryosalmonae develop in the kidney of infected brown trout Salmo trutta and are released via urine to infect the freshwater bryozoan Fredericella sultana. The transcriptomes of kidneys of infected and non-infected brown trout were compared by suppressive subtractive hybridization. Differential screening and a subsequent NCBI BLAST analysis of expressed sequence tags revealed 21 transcripts with functions that included cell stress and cell growth, ribonucleoprotein, signal transduction, ion transporter, immune response, hemoglobin and calcium metabolisms. Quantitative real time PCR was used to verify the presence of these selected transcripts in brown trout kidney at sporogonic stages of T. bryosalmonae development. Expression of cold-inducible RNA-binding protein, cyclin-dependent kinase inhibitor 2A, prothymosin alpha, transforming protein RhoA, immunoglobulin light chain and major histocompatibility complex class I were up-regulated significantly in infected brown trout. Expression of both the hemoglobin subunit beta and stanniocalcin precursor were down-regulated significantly in infected brown trout. This study suggests that cell stress and cell growth processes, signal transduction activities, erythropoiesis and calcium homeostasis of the host are modulated during sporogonic stages of parasite development, which may support the sporogenesis of T. bryosalmonae in the kidney of brown trout.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0101-z) contains supplementary material, which is available to authorized users.

Immune responses induced by the TLR-4 agonist-based adjuvant prothymosin alpha.

11131 Background: The immunomodulator prothymosin α (proTα) stimulates immune subpopulations via generating an optimized cytokine milieu. The decapeptide proTα(100-109) restores the deficient immun...

Prothymosin alpha and Ki-67 expression in pituitary adenomas

Prothymosin alpha and Ki-67 expression in pituitary adenomas

S100A7 overexpression is a predictive marker for high risk of malignant transformation in oral dysplasia

Early detection of oral lesions (OLs) at high risk of cancer development is of utmost importance for intervention. There is an urgent unmet clinical need for biomarkers that allow identification of high-risk OLs. Recently, we identified and verified a panel of five candidate protein biomarkers namely S100A7, prothymosin alpha, 14-3-3ζ, 14-3-3σ and heterogeneous nuclear ribonucleoprotein K using proteomics to distinguish OLs with dysplasia and oral cancers from normal oral tissues. The objective of our study was to evaluate the potential of these candidate protein biomarkers for identification of oral dysplastic lesions at high risk of cancer development. Using immunohistochemistry, we analyzed expressions of these five candidate protein biomarkers in 110 patients with biopsy-proven oral dysplasia and known clinical outcome and determined their correlations with p16 expression and HPV 16/18 status. Kaplan-Meier survival analysis showed reduced oral cancer-free survival (OCFS) of 68.6 months (p = 0.007) in patients showing cytoplasmic S100A7 overexpression when compared to patients with weak or no S100A7 immunostaining in cytoplasm (mean OCFS = 122.8 months). Multivariate Cox regression analysis revealed cytoplasmic S100A7 overexpression as the most significant candidate marker associated with cancer development in dysplastic lesions (p = 0.041, hazard ratio = 2.36). In conclusion, our study suggested the potential of S100A7 overexpression in identifying OLs with dysplasia at high risk of cancer development.

Prothymosin α and a prothymosin α-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes

BackgroundActive cancer immunotherapies are beginning to yield clinical benefit, especially those using peptide-pulsed dendritic cells (DCs). Different adjuvants, including Toll-like receptor (TLR) agonists, commonly co-administered to cancer patients as part of a DC-based vaccine, are being widely tested in the clinical setting. However, endogenous DCs in tumor-bearing individuals are often dysfunctional, suggesting that ex vivo educated DCs might be superior inducers of anti-tumor immune responses. We have previously shown that prothymosin alpha (proTα) and its immunoreactive decapeptide proTα(100–109) induce the maturation of human DCs in vitro. The aim of this study was to investigate whether proTα- or proTα(100–109)-matured DCs are functionally competent and to provide preliminary evidence for the mode of action of these agents.ResultsMonocyte-derived DCs matured in vitro with proTα or proTα(100–109) express co-stimulatory molecules and secrete pro-inflammatory cytokines. ProTα- and proTα(100–109)-matured DCs pulsed with HER-2/neu peptides induce TH1-type immune responses, prime autologous naïve CD8-positive (+) T cells to lyse targets expressing the HER-2/neu epitopes and to express a polyfunctional profile, and stimulate CD4+ T cell proliferation in an HER-2/neu peptide-dependent manner. DC maturation induced by proTα and proTα(100–109) is likely mediated via TLR-4, as shown by assessing TLR-4 surface expression and the levels of the intracellular adaptor molecules TIRAP, MyD88 and TRIF.ConclusionsOur results suggest that proTα and proTα(100–109) induce both the maturation and the T cell stimulatory capacity of DCs. Although further studies are needed, evidence for a possible proTα and proTα(100–109) interaction with TLR-4 is provided. The initial hypothesis that proTα and the proTα-derived immunoactive decapeptide act as “alarmins”, provides a rationale for their eventual use as adjuvants in DC-based anti-cancer immunotherapy.

Knockdown of prothymosin α leads to apoptosis and developmental defects in zebrafish embryos

Prothymosin alpha (ProTα) is an abundant nuclear protein involved in cellular processes intricately linked to development, such as cell proliferation and apoptosis. Although it is known that ProTα inhibits the formation of apoptosome and blocks caspase-3 activity, its mechanism of function in the apoptotic machinery is still under investigation. We have studied the cellular role of ProTα by knocking down its expression in HeLa cells with small hairpin RNA (shRNA) in the absence of apoptotic stimuli. Flow cytometric analysis showed that the live cell population was significantly decreased with a concomitant increase of the apoptotic populations. To understand the physiological role of ProTα within the context of embryonic development, we knocked down the Ptmab zebrafish ortholog using 2 specific morpholino oligonucleotides. Ptmab morphants exhibited growth retardation, bended trunks, and curly tails. The frequency of occurrence of the phenotypic defects was increased in a morpholino dose-dependent manner. Co-injection of ptmaa mRNA with ptmab morpholino partially rescued the morphological defects. Immunostaining with the anti-phospho-histone H3 (pH3) antibody suggested that the abnormalities of Ptmab morphants could be due to defective cell proliferation that results in growth imbalances. TUNEL fluorescent labelling and Acridine Orange staining of the morphants showed high rates of cell death in the head and tail regions. Concomitantly, the active form of caspase-3 was detected in Ptmab morphants. Our data suggest a conserved anti-apoptotic role of ProTα between zebrafish and humans, and provide the first evidence that ProTα is important for early embryogenesis.