Nuclear localisation of the G-actin sequestering peptide thymosin beta4.

Thymosin beta4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin beta4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin beta4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin beta4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin beta4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin beta4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.

HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane.

The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.

Sox8 is a specific marker for muscle satellite cells and inhibits myogenesis.

Sox8 belongs to a family of transcription regulators characterized by a unique DNA-binding domain known as the high mobility group box. Many Sox proteins play fundamental roles in vertebrate development and differentiation processes. Expression of Sox8 is strong during embryonic muscle development and gradually declines postnatally. In this study, we report that in adult skeletal muscle Sox8 is confined to satellite cells. Down-regulation during myogenic differentiation was also detected in cell culture systems and occurred in parallel with down-regulation of the related Sox9. Overexpression of Sox8 or Sox9 on the other hand disrupted myoblasts in their ability to form myotubes. Concomitantly, expression of MyoD and myogenin decreased and basal as well as MyoD-induced activities of the myogenin promoter were strongly reduced in a Sox8-dependent manner. Our data suggest that Sox8 acts as a specific negative regulator of skeletal muscle differentiation, possibly by interfering with the function of myogenic basic helix-loop-helix proteins.

Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation.

Sox10 belongs to a family of transcription regulators characterized by a DNA-binding domain known as the HMG box. It plays fundamental roles in neural crest development, peripheral gliogenesis, and terminal differentiation of oligodendrocytes. In accord with its function as transcription factor, Sox10 contains two nuclear localization signals and is most frequently detected in the nucleus. In this study, we report that Sox10 is an active nucleocytoplasmic shuttle protein, competent of both entering and exiting the nucleus. We identified a functional Rev-type nuclear export signal within the DNA-binding domain of Sox10. Mutational inactivation of this nuclear export signal or treatment of cells with the CRM1-specific export inhibitor leptomycin B inhibited nuclear export and consequently nucleocytoplasmic shuttling of Sox10. Importantly, the inhibition of the nuclear export of Sox10 led to decreased transactivation of transfected reporters and endogenous target genes, arguing that continuous nucleocytoplasmic shuttling is essential for the function of Sox10. To our knowledge this is the first time that nuclear export has been reported and shown to be functionally relevant for any Sox protein.

Identification of two nuclear import signals in the alpha-gene product ICP22 of herpes simplex virus 1.

The herpes simplex virus 1 (HSV-1) infected cell protein 22 (ICP22) is a multifunctional viral regulator that localizes in the nucleus of infected cells. ICP22 is required for optimal virus replication in certain cell types and is subject to extensive posttranslational modification. To map the signals in ICP22 which mediate its efficient nuclear localization, we investigated the nuclear import of fusion proteins comprising various fragments of ICP22 fused to green fluorescent protein (GFP) or beta-galactosidase (beta-Gal). These data demonstrated that ICP22 contains two independent regions with nuclear localization signal (NLS) activity. NLS1 maps to ICP22 amino acid position 16-31 and closely resembles the classical bipartite NLS of the type originally identified in nucleoplasmin. In contrast, NLS2 maps to ICP22 amino acid position 118-131 and contains multiple critical basic residues. Furthermore, fusion of both NLSs to chimeric glutathione-S-transferase (GST)-GFP protein and subsequent cytoplasmic microinjection of the respective transport substrates allowed us to monitor nuclear import in real-time. These data demonstrated that both ICP22-derived NLSs mediated efficient nuclear import with identical kinetics, resulting in complete nuclear accumulation of the chimeric transport cargoes at approximately 30 min postinjection. Finally, our data provide new insights into the domain structure of the multifunctional alpha-gene product ICP22 of HSV-1.

Hemofiltrate CC chemokine 1[9-74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages.

Proteolytic processing of the abundant plasmatic human CC chemokine 1 (HCC-1) generates a truncated form, HCC-1[9-74], which is a potent agonist of CCR1, CCR3, and CCR5; promotes calcium influx and chemotaxis of T lymphoblasts, monocytes, and eosinophils; and inhibits infection by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. In the present study we demonstrate that HCC-1[9-74] interacts with the second external loop of CCR5 and inhibits replication of CCR5-tropic HIV-1 strains in both primary T cells and monocyte-derived macrophages. Low concentrations of the chemokine, however, frequently enhanced the replication of CCR5-tropic HIV-1 isolates but not the replication of X4-tropic HIV-1 isolates. Only HCC-1[9-74] and HCC-1[10-74], but not other HCC-1 length variants, displayed potent anti-HIV-1 activities. Fluorescence-activated cell sorter analysis revealed that HCC-1[9-74] caused up to 75% down-regulation of CCR5 cell surface expression, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) achieved a reduction of only about 40%. Studies performed with green fluorescent protein-tagged CCR5 confirmed that both HCC-1[9-74] and RANTES, but not full-length HCC-1, mediated specific internalization of the CCR5 HIV-1 entry cofactor. Our results demonstrate that the interaction with HCC-1[9-74] causes effective intracellular sequestration of CCR5, but they also indicate that the effect of HCC-1[9-74] on viral replication is subject to marked cell donor- and HIV-1 isolate-dependent variations.