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Regulation of apoptosis by the p8/prothymosin alpha complex.

ABSTRACT: p8 is a small-stress protein involved in several cellular functions including apoptosis. To identify its putative partners, we screened a HeLa cDNA library by using the two-hybrid technique and found that p8 binds the antiapoptotic protein prothymosin alpha (ProTalpha). Fluorescence spectroscopy, circular dichroism, and NMR spectroscopy showed that p8 and ProTalpha formed a complex. Binding resulted in important changes in the secondary and tertiary structures of the proteins. Because p8 and ProTalpha form a complex, they could act in concert to regulate the apoptotic cascade. We induced apoptosis in HeLa cells by staurosporine treatment and monitored the effects of knocking down p8 and/or ProTalpha or overexpressing p8 and/or ProTalpha on caspase 3/7 and 9 activities and on cell death. Transfecting ProTalpha or p8 small interfering RNAs increased the activities of both caspases and the number of apoptotic nuclei. However, transfecting both small interfering RNAs resulted in no further increase. Overexpressing p8 or ProTalpha did not alter caspase activities, whereas overexpressing both resulted in a significant reduction of caspase activities. These results strongly suggest that the antiapoptotic response of HeLa cells upon staurosporine treatment requires expression of both p8 and ProTalpha.

SUBMITTER: Malicet C

PROVIDER: S-EPMC1413786 | biostudies-literature |

REPOSITORIES: biostudies-literature

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MicroRNA-1 induces apoptosis by targeting prothymosin alpha in nasopharyngeal carcinoma cells.

Project description:BACKGROUND:MiR-1 (microRNA-1) has been used as a positive control in some microRNA experiments. We found that miR-1 transfection of nasopharyngeal carcinoma cells reveals a typical apoptotic process as shown by time-lapse microscopy so we investigated the mechanisms of miR-1 inducing apoptosis. METHODS:To confirm that miR-1 induces apoptosis, we used Annexin V and TUNEL staining and caspase assay. To determine that miR-1 directly targets genes that involve in apoptosis, we analyzed microRNA and pathway databases, and cDNA expression microarrays from miR-1 transfected cells. To demonstrate candidate miR-1 targeted genes, we used qRT-PCR analysis and luciferase reporter vector assays. To assess the miR-1 target gene PTMA (prothymosin alpha, ProTalpha) involves in apoptosis, we used PTMA siRNA to knock down PTMA. RESULTS:Annexin V and TUNEL staining and caspase assay confirm that miR-1 induces nasopharyngeal carcinoma cell apoptosis. MiR-1 transfection of HeLa, Cal-27, KYSE30 and NPC-TW06 cell lines which express low levels of endogenous miR-1 also induces apoptosis. However, miR-1 transfection of cell lines such as SW620, HepG2, HEK-293T, SAS and PC-13 which express high levels of endogenous miR-1 does not result in apoptosis. MiR-1 directly targets PTMA gene. PTMA siRNA and miR-1 accelerate the apoptotic process in cells treated with apoptosis inducers. CONCLUSIONS:The exogenous expression of miR-1 induces apoptosis in a number of cell lines. This is a model of microRNA-induced cell apoptosis. The PTMA is one of miR-1 target genes which involve in miR-1 inducing apoptosis. The apoptotic inducers including actinomycin D, camptothecin and etoposide are also the chemotherapeutic drugs in clinical cancer therapy and PTMA siRNA can accelerate apoptotic progression in cells treated with those apoptosis inducers. Therefore PTMA siRNA may have potential applications as an adjuvant in cancer chemotherapy.

| S-EPMC3238347 | biostudies-literature

Structural analysis of the complex of Keap1 with a prothymosin alpha peptide.

Project description:The Nrf2 transcription factor, which plays important roles in oxidative and xenobiotic stress, is negatively regulated by the cytoplasmic repressor Keap1. The beta-propeller/Kelch domain of Keap1, which is formed by the double-glycine repeat and C-terminal region domains (Keap1-DC), interacts directly with the Neh2 domain of Nrf2. The nuclear oncoprotein prothymosin alpha (ProTalpha) also interacts directly with Keap1 and may play a role in the dissociation of the Keap1-Nrf2 complex. The structure of Keap1-DC complexed with a ProTalpha peptide (amino acids 39-54) has been determined at 1.9 A resolution. The Keap1-bound ProTalpha peptide possesses a hairpin conformation and binds to the Keap1 protein at the bottom region of the beta-propeller domain. Complex formation occurs as a consequence of their complementary electrostatic interactions. A comparison of the present structure with recently reported Keap1-DC complex structures revealed that the DLG and ETGE motifs of the Neh2 domain of Nrf2 and the ProTalpha peptide bind to Keap1 in a similar manner but with different binding potencies.

| S-EPMC2374262 | biostudies-literature

Probiotic-derived p8 protein induce apoptosis via regulation of RNF152 in colorectal cancer cells.

Project description:Worldwide, colorectal cancer (CRC) is one of the most common cancers and is a leading cause of cancer-related deaths. Accumulating evidence suggests that probiotics suppress the development of various cancers including CRC. Recently, we reported a Lactobacillus rhamnosus (LR)-derived 8 kDa protein (p8) that displayed anti-cancer properties in CRC cells. However, the precise anti-cancer mechanism of p8 and its target genes has not been fully examined. In the present study, we reveal that p8 leads to apoptotic cells and cleaved PARP1 expression in a mouse xenograft model of CRC. Additionally, we identified Ring finger protein 152 (RNF152) as a putative target of p8 using RNA-sequencing. Furthermore, the expression levels of RNF152 were increased following in vivo and in vitro treatment with p8. We also found that p8 leads to the accumulation of cleaved PARP1 in CRC cells. These results suggest that p8 induces apoptosis via regulation of RNF152, thus inhibiting the development of CRC.

| S-EPMC7994155 | biostudies-literature

Prothymosin α accelerates dengue virus-induced thrombocytopenia.

Project description:Thrombocytopenia is the hallmark finding in dengue virus (DENV) infection. Prothymosin α (ProT) has both intracellular and extracellular functions involved in cell cycle progression, cell differentiation, gene regulation, oxidative stress response, and immunomodulation. In this study, we found that ProT levels were elevated in dengue patient sera as well as DENV-infected megakaryoblasts and their culture supernatants. ProT transgenic mice had reduced platelet counts with prolonged bleeding times. Upon treatment with DENV plus anti-CD41 antibody, they exhibited severe skin hemorrhage. Furthermore, overexpression of ProT suppressed megakaryocyte differentiation. Infection with DENV inhibited miR-126 expression, upregulated DNA (cytosine-5)-methyltransferase 1 (DNMT1), downregulated GATA-1, and increased ProT expression. Upregulation of ProT led to Nrf2 activation and reduced reactive oxygen species production, thereby suppressing megakaryopoiesis. We report the pathophysiological role of ProT in DENV infection and propose an involvement of the miR-126-DNMT1-GATA-1-ProT-Nrf2 signaling axis in DENV-induced thrombocytopenia.

| S-EPMC10783621 | biostudies-literature

Prothymosin-alpha mediates nuclear import of the INrf2/Cul3 Rbx1 complex to degrade nuclear Nrf2.

Project description: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.

| S-EPMC2679486 | biostudies-literature

Prothymosin alpha is a component of a linker histone chaperone.

Project description:Linker histone H1 binds with high affinity to naked and nucleosomal DNA in vitro but is rapidly exchanged between chromatin sites in vivo suggesting the involvement of one or more linker histone chaperones. Using permeabilized cells, we demonstrate that the small acidic protein prothymosin alpha (ProTalpha) can facilitate H1 displacement from and deposition onto the native chromatin template. Depletion of ProTalpha levels in vivo by siRNA-mediated mRNA degradation resulted in a decreased rate of exchange of linker histones as assayed by photobleaching techniques. These results indicate that ProTalpha is a component of a linker histone chaperone.

| S-EPMC2891112 | biostudies-literature

Prothymosin alpha interacts with the CREB-binding protein and potentiates transcription.

Project description:Prothymosin alpha (ProTalpha) is a histone H1-binding protein localized in sites of active transcription in the nucleus. We report here that ProTalpha physically interacts with the CREB-binding protein (CBP), which is a versatile transcription co-activator. Confocal laser scanning microscopy reveals that ProTalpha partially colocalizes with CBP in discrete subnuclear domains. Using transient transfections, we show that ProTalpha synergizes with CBP and stimulates AP1- and NF-kappaB-dependent transcription. Furthermore, overexpression of ProTalpha enhances the transactivation potential of CBP. These findings reveal a new function for ProTalpha in transcription activation, probably through CBP-mediated recruitment to different promoters.

| S-EPMC1084059 | biostudies-literature

Antiapoptotic function of RNA-binding protein HuR effected through prothymosin alpha.

Project description:We report the antiapoptotic effect of RNA-binding protein HuR, a critical regulator of the post-transcriptional fate of target transcripts. Among the most prominent mRNAs complexing with HuR is that encoding prothymosin alpha (ProTalpha), an inhibitor of the apoptosome. In HeLa cells, treatment with the apoptotic stimulus ultraviolet light (UVC) triggered the mobilization of ProTalpha mRNA to the cytoplasm and onto heavier polysomes, where its association with HuR increased dramatically. Analysis of a chimeric ProTalpha mRNA directly implicated HuR in regulating ProTalpha production: ProTalpha translation and cytoplasmic concentration increased in HuR-overexpressing cells and declined in cells in which HuR levels were lowered by RNA interference. Importantly, the antiapoptotic influence engendered by HuR was vitally dependent on ProTalpha expression, since use of oligomers that blocked ProTalpha translation abrogated the protective effect of HuR. Together, our data support a regulatory scheme whereby HuR binds the ProTalpha mRNA, elevates its cytoplasmic abundance and translation, and thereby elicits an antiapoptotic program.

| S-EPMC1142594 | biostudies-literature

Effect of prothymosin α on neuroplasticity following cerebral ischemia‑reperfusion injury.

Project description:Prothymosin α (ProT), a highly acidic nuclear protein with multiple cellular functions, has shown potential neuroprotective properties attributed to its anti‑necrotic and anti‑apoptotic activities. The present study aimed to investigate the beneficial effect of ProT on neuroplasticity after ischemia‑reperfusion injury and elucidate its underlying mechanism of action. Primary cortical neurons were either treated with ProT or overexpressing ProT by gene transfection and exposed to oxygen‑glucose deprivation for 2 h in vitro. Immunofluorescence staining for ProT and MAP‑2 was performed to quantify ProT protein expression and assess neuronal arborization. Mice treated with vehicle or ProT (100 µg/kg) and ProT overexpression in transgenic mice received middle cerebral artery occlusion for 50 min to evaluate the effect of ProT on neuroplasticity‑associated protein following ischemia‑reperfusion injury. The results demonstrated that in cultured neurons ProT significantly increased neurite lengths and the number of branches, accompanied by an upregulation mRNA level of brain‑derived neurotrophic factor. Furthermore, ProT administration improved the protein expressions of synaptosomal‑associated protein, 25 kDa and postsynaptic density protein 95 after ischemic‑reperfusion injury in vivo. These findings suggested that ProT can potentially induce neuroplasticity effects following ischemia‑reperfusion injury.

| S-EPMC10902629 | biostudies-literature

Generation of mature Nα-terminal acetylated thymosin α 1 by cleavage of recombinant prothymosin α.

Project description:N(α)-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. N(α)-terminal acetylated thymosin α1 (Tα1), a 28-amino-acid peptide, is an immune modifier that has been used in the clinic to treat hepatitis B and C virus (HBV/HCV) infections. We previously documented N(α)-terminal acetylation of recombinant prothymosin α (ProTα) in E. coli. Here we present a method for production of N(α)-acetylated Tα1 from recombinant ProTα. The recombinant ProTα was cleaved by human legumain expressed in Pichia pastoris to release Tα1 in vitro. The N(α)-acetylated Tα1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant N(α)-acetylated Tα1 was 3108.79 in, which is identical to the mass of N(α)-acetylated Tα1 produced by total chemical synthesis. This mass corresponded to the nonacetylated Tα1 mass with a 42 Da increment. The retention time of recombinant N(α)-acetylated Tα1 and chemosynthetic N(α)-acetylated Tα1 were both 15.4 min in RP-high performance liquid chromatography (HPLC). These data support the use of an E. coli expression system for the production of recombinant human N(α)-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides in E. coli.

| S-EPMC3830889 | biostudies-literature

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