Prothymosin alpha, a mammalian c-myc-regulated acidic nuclear protein, provokes the decondensation of human chromosomes in vitro

Prothymosin (ProT alpha) is an acidic nuclear protein, widely distributed in mammalian cells, whose expression is regulated by c-myc and linked to cell proliferation. ProT alpha interacts with histone H1 via its acidic domain, and its overexpression provokes the unfolding of chromatin fibers. Here we show that incubation of human native metaphase chromosomes with ProT alpha induces their extensive unravelling suggesting a function of this protein in chromosome decondensation.     

Prothymosin alpha is processed to thymosin alpha 1 and thymosin alpha 11 by a lysosomal asparaginyl endopeptidase

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.     

Identification of nuclear-import and cell-cycle regulatory proteins that bind to prothymosin alpha.

Prothymosin alpha (ProT alpha) is a nuclear protein that is widely distributed in mammalian tissues, and is thought to play a role in cell proliferation. In an attempt to shed light on this role, affinity chromatography on ProT alpha-Sepharose columns was used to identify proteins in subcellular extracts of transformed human lymphocytes (NC37 cells) that interact with ProT alpha in vitro, and thus may interact with ProT alpha in vivo. Immunoblotting techniques were used to screen the ProT alpha-binding fractions for histones and other proteins involved in nuclear transport and cell-cycle control. The most abundant ProT alpha-binding proteins were histones H2A, H2B, H3, and H4. Of the nuclear-transport proteins, karyopherin beta1, Rch-1, Ran, and RCC1 were detected at high concentrations; NTF2, nucleoporin p62, and Hsp70 were detected at low concentrations; while tranportin, CAS, and Ran BPI were not detected. Of the cell-cycle control proteins, PCNA, Cdk2, and cyclin A were detected at high concentrations; cdc2, Cdk4, and cyclin B were detected at very low concentrations; while cyclin D1, cyclin D3, Cip1, and Kip1 were not detected. These results suggest (i) that ProT alpha is transported into the nucleus by the karyopherin beta1-Rch-1 complex, and (ii) that ProT alpha may interact in the nucleus with proteins involved in DNA metabolism and cell-cycle control.     

Expression of the rat prothymosin alpha gene during T-lymphocyte proliferation and liver regeneration.

Prothymosin alpha (ProT alpha) is a widely distributed acidic protein whose function has been related to cell proliferation. We have analyzed the expression of the rat ProT alpha gene in several proliferative systems: concanavalin A (ConA)/interleukin-2-stimulated thymocytes, ConA-stimulated splenic T-lymphocytes, and hepatocytes proliferating during liver regeneration. In these systems, ProT alpha mRNA was detected in all stages of the cell cycle, with maximal increments (2-4-fold) at the beginning of the S phase. By contrast, the mRNAs for proliferating cell nuclear antigen/cyclin and histone H3, two cell-cycle-regulated proteins, were hardly detected in resting cells but increased notably at the G1/S boundary and in the S phase, respectively. Treatment of T-cells with the calcium ionophore A23187 increased ProT alpha mRNA levels 2.5-fold, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, had no effect on ProT alpha gene expression. Incubation of ConA-stimulated T-cells with hydroxyurea, a DNA synthesis inhibitor, did not decrease the levels of ProT alpha mRNA, indicating that its expression is independent of DNA synthesis. These findings suggest that ProT alpha is required throughout all the stages of the cell cycle, resembling a constitutively expressed gene rather than one strictly involved in cell proliferation.     

The M2-type isoenzyme of pyruvate kinase phosphorylates prothymosin α in proliferating lymphocytes

Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity.     

Prothymosin alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation.

Prothymosin alpha (ProT alpha) is an acidic polypeptide with potentiating effects on HLA-DR-restricted in vitro cellular immune response systems such as T cell proliferative responses to soluble proteins and cellular auto- or alloantigens. Experiments were performed to investigate the effect of ProT alpha on MHC class II Ag expression in human monocytes, murine splenocytes, and tumor cell lines at both protein and molecular levels. RIA and immunofluorescence analysis revealed that ProT alpha enhances HLA-DR surface Ag expression whereas Northern blot analysis demonstrated that ProT alpha causes significant accumulation of MHC class II mRNA. The enhancing effect of ProT alpha was demonstrated convincingly using precultured human peripheral monocytes, which are known to express decreased amounts of surface HLA-DR Ag, and HLA-DR-positive human cell lines. Moreover, ProT alpha was shown to induce HLA-DR Ag expression in a priori HLA-DR-negative tumor cells. Furthermore, ProT alpha was shown to be active in vivo. Splenocytes from mice pretreated with ProT alpha expressed more surface Ilpha Ag and contained more I alpha-specific mRNA. These findings suggest that ProT alpha may be important in the regulation of the immune response by enhancing MHC class II Ag expression in APC.     

Prothymosin α is a component of a linker histone chaperone

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 α (ProTα) can facilitate H1 displacement from and deposition onto the native chromatin template. Depletion of ProTα 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 ProTα is a component of a linker histone chaperone.     

Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.     

Tissue-specific and differential expression of prothymosin alpha gene during rat development.

We have analyzed the RNA expression of prothymosin alpha (ProT alpha) gene during rat development in several tissues and compared it to that of two proteins related to cell proliferation: proliferating cell nuclear antigen (PCNA)/cyclin and histone H3 (H3). The expression of ProT alpha gene was found to be regulated in a developmental and tissue-specific manner. The mRNA levels of ProT alpha followed a similar time-course in liver, brain, kidney, and testis, being highly increased in the early periods of postnatal development. However, in thymus ProT alpha mRNA showed only moderate changes throughout development. Our findings suggest that ProT alpha participates in developmental processes like cell proliferation and/or differentiation.     

Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation

MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.     

Prothymosin alpha lacking the nuclear localization signal as an effective gene therapeutic strategy in collagen-induced arthritis

Prothymosin alpha (ProT) is regulated by c-Myc, an oncoprotein overexpressed in synovium of rheumatoid arthritis, and is associated with cell proliferation. However, ProT also exerts immunomodulatory activities. The growth-promoting activity of ProT can be abolished by deleting its nuclear localization signal (NLS). In this study, we showed that AdProTDeltaNLS, an adenoviral vector encoding ProT lacking the NLS, did not enhance the proliferation of synovial fibroblasts. AdProTDeltaNLS treatment abolished the up-regulation of the MIP-1alpha promoter activity induced by TNF-alpha in synovial fibroblasts. AdProTDeltaNLS suppressed macrophage chemotaxis and reduced macrophage infiltration into the ankle joints in rats with collagen-induced arthritis (CIA). Neutralization test confirmed the involvement of MIP-1alpha in macrophage chemotaxis. Administration of AdProTDeltaNLS reduced the severity of CIA in the clinical, radiographic, and histological aspects. The levels of TNF-alpha (mean +/- SEM, 1261.9 +/- 107.9 vs 2880.1 +/- 561.4 pg/mg total protein; p < 0.05), IL-1beta (56.8 +/- 8.0 vs 109.2 +/- 4.9 pg/mg total protein; p < 0.01), and MIP-1alpha (41.7 +/- 3.6 vs 55.2 +/- 1.1 pg/mg total protein; p < 0.05) in the ankle joints were lower in the AdProTDeltaNLS-treated rats with CIA than those in their control counterparts. In the AdProTDeltaNLS-treated ankle joints, matrix metalloproteinase-9 expression was decreased by 40% and infiltrating macrophages reduced by 50%. Our results demonstrate that intra-articular delivery of AdProTDeltaNLS significantly ameliorated the clinical course of CIA in rats. This study is the first to suggest that ProT lacking the NLS may have therapeutic potential for the management of rheumatoid arthritis.     

Preliminary methylation analysis of prothymosin α genomic sequences

Prothymosin α is a mammalian nuclear protein involved in cell proliferation and differentiation. Here, we carried out the first study of the methylation status of ProTα genomic sequences in cell lines during differentiation as well as in tumoral tissues. We found that there is hypermethylation in all cell lines analyzed with a pattern that is characteristic of each cell type revealing specific genomic reorganizations. The decrease of ProTα mRNA during differentiation was not accompanied by changes in the methylation status. Remarkably, we found that there is hypomethylation in gastrointestinal tumors when compared with the peritumoral tissue. The biological implications of these findings are discussed.     

Neuron‐specific non‐classical release of prothymosin alpha: a novel neuroprotective damage‐associated molecular patterns

Prothymosin alpha (ProTα), a nuclear protein devoid of signal sequence, has been shown to possess a number of cellular functions including cell survival. Most recently, we demonstrated that ProTα is localized in the nuclei of neurons, while it is found in both nuclei and cytoplasm in the astrocytes and microglia of adult brain. However, the cell type‐specific non‐classical release of ProTα under cerebral ischemia is yet unknown. In this study, we report that ProTα is non‐classically released along with S100A13 from neurons in the hippocampus, striatum and somatosensory cortex at 3 h after cerebral ischemia, but amlexanox (an anti‐allergic compound) reversibly blocks this neuronal ProTα release. We found that none of ProTα is released from astrocytes and microglia under ischemic stress. Indeed, ProTα intensity is increased gradually in astrocytes and microglia through 24 h after the cerebral ischemia. Interestingly, Z‐Val‐Ala‐Asp fluoromethyl ketone, a caspase 3 inhibitor, pre‐treatment induces ProTα release from astrocytes in the ischemic brain, but this release is reversibly blocked by amlexanox. However, Z‐Val‐Ala‐Asp fluoromethyl ketone as well as amlexanox has no effect on ProTα distribution in microglia upon cerebral ischemia. Taken together, these results suggest that only neurons have machineries to release ProTα upon cerebral ischemic stress in vivo.     

Prothymosin alpha expression in lymph nodes and tonsils: an optical and ultrastructural study.

Prothymosin alpha (ProT alpha) distribution in human and rat lymph nodes and human tonsils was studied by means of immunohistochemical methods, using specific antibodies raised against thymosin alpha 1. We observed ProT alpha immunoreactivity in lymphoid cells of the germinal centers both in humans and rats. In human tonsils, positive cells were also seen in the basal layer of the surface epithelium. These results support the hypothesis that ProT alpha expression is restricted to actively proliferating cells. Immunoelectron microscopy revealed that ProT alpha was located in the nucleus, mainly in the border between euchromatin and heterochromatin.     

Human pescadillo induces large-scale chromatin unfolding

The human pescadillo gene encodes a protein with a BRCT domain. Pescadillo plays an important role in DNA synthesis, cell proliferation and transformation. Since BRCT domains have been shown to induce chromatin large-scale unfolding, we tested the role of Pescadillo in regulation of large-scale chromatin unfolding. To this end, we isolated the coding region of Pescadillo from human mammary MCF10A cells. Compared with the reported sequence, the isolated Pescadillo contains in-frame deletion from amino acid 580 to 582. Targeting the Pescadillo to an amplified, lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity maps to the BRCT domain of Pescadillo. These data provide a new clue to understanding the vital role of Pescadillo.     

The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair

SUMO conjugation is known to occur in response to double‐stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3‐specific peptidase, SENP7, interacts with the chromatin repressive KRAB‐associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA‐damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.     

Human pescadillo induces large-scale chromatin unfolding

Pescadillo was initially identified in a genetic screen for mutations that affect embryonic develop-ment of the zebrafish Daniorerio[1]. Pescadillo-/- ze-brafish mutants showed abnormal embryonic devel-opment such as reduced brain, eye size and a lack of extension of the jaw on developmental day 3. Further study showed that the Pescadillo protein is mainly distributed in tissues containing a significant number of proliferating cells and dramatically elevated in ma-lignant human astrocytomas an…