Roberta Carbone

EPS8 and E3B1 transduce signals from Ras to Rac

The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1 (refs 4, 5). Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.

PML NBs associate with the hMre11 complex and p53 at sites of irradiation induced DNA damage.

PML nuclear bodies (PML NBs) respond to many cellular stresses including viral infection, heat shock, arsenic and oncogenes and have been implicated in the regulation of p53-dependent replicative senescence and apoptosis. Recently, the hMre11/Rad50/NBS1 repair complex, involved in Double Strand Breaks (DSBs) repair, was found to colocalize within PML NBs, suggesting a role for these nuclear sub-domains in the DNA repair signalling pathway. We report here that in normal human fibroblasts, after ionizing radiation (IR), the PML NBs are modified and recognize sites of DNA breaks (ssDNA breaks and DSBs). Eight to 12 h after radiation PML NBs associate with hMre11 Ionizing Radiation-Induced Foci (IRIF), and subsequently with p53 within discrete foci. The PML, hMre11 and p53 colocalizing structures mark sites of DSBs as identified by immunolocalization with anti phosphorylated histone γ-H2AX. Furthermore, we demonstrate that ionizing radiation induces the stable association of p53 with hMre11 and PML. These results suggest that the PML NBs are involved in the recognition and/or processing of DNA breaks and possibly in the recruitment of proteins (p53 and hMre11) required for both checkpoint and DNA-repair responses.

The effect of surface nanometre-scale morphology on protein adsorption.

BACKGROUND Protein adsorption is the first of a complex series of events that regulates many phenomena at the nano-bio interface, e.g. cell adhesion and differentiation, in vivo inflammatory responses and protein crystallization. A quantitative understanding of how nanoscale morphology influences protein adsorption is strategic for providing insight into all of these processes, however this understanding has been lacking until now. METHODOLOGY/PRINCIPAL FINDINGS Here we introduce novel methods for quantitative high-throughput characterization of protein-surface interaction and we apply them in an integrated experimental strategy, to study the adsorption of a panel of proteins on nanostructured surfaces. We show that the increase of nanoscale roughness (from 15 nm to 30 nm) induces a decrease of protein binding affinity (<or=90%) and a relevant increase in adsorbed proteins (<or=500%) beyond the corresponding increase of specific area. We demonstrate that these effects are caused by protein nucleation on the surface, which is promoted by surface nanoscale pores. CONCLUSIONS/SIGNIFICANCE These results show that the adsorption of proteins depends significantly on surface nanostructure and that the relevant morphological parameter regulating the protein adsorption process is the nanometric pore shape. These new findings improve our understanding of the role of nanostructures as a biomaterial design parameter and they have important implications for the general understanding of cell behavior on nanostructured surfaces.

Impairment of p53 acetylation, stability and function by an oncogenic transcription factor.

Mutations of p53 are remarkably rare in acute promyelocytic leukemias (APLs). Here, we demonstrate that the APL‐associated fusion proteins PML–RAR and PLZF‐RAR directly inhibit p53, allowing leukemic blasts to evade p53‐dependent cancer surveillance pathways. PML–RAR causes deacetylation and degradation of p53, resulting in repression of p53 transcriptional activity, and protection from p53‐dependent responses to genotoxic stress. These phenomena are dependent on the expression of wild‐type PML, acting as a bridge between p53 and PML–RAR. Recruitment of histone deacetylase (HDAC) to p53 and inhibition of p53 activity were abrogated by conditions that either inactivate HDACs or trigger HDAC release from the fusion protein, implicating recruitment of HDAC by PML–RAR as the mechanism underlying p53 inhibition.

Recruitment of the Histone Methyltransferase SUV39H1 and Its Role in the Oncogenic Properties of the Leukemia-Associated PML-Retinoic Acid Receptor Fusion Protein

ABSTRACT Leukemia-associated fusion proteins establish aberrant transcriptional programs, which result in the block of hematopoietic differentiation, a prominent feature of the leukemic phenotype. The dissection of the mechanisms of deregulated transcription by leukemia fusion proteins is therefore critical for the design of tailored antileukemic strategies, aimed at reestablishing the differentiation program of leukemic cells. The acute promyelocytic leukemia (APL)-associated fusion protein PML-retinoic acid receptor (RAR) behaves as an aberrant transcriptional repressor, due to its ability to induce chromatin modifications (histone deacetylation and DNA methylation) and silencing of PML-RAR target genes. Here, we indicate that the ultimate result of PML-RAR action is to impose a heterochromatin-like structure on its target genes, thereby establishing a permanent transcriptional silencing. This effect is mediated by the previously described association of PML-RAR with chromatin-modifying enzymes (histone deacetylases and DNA methyltransferases) and by recruitment of the histone methyltransferase SUV39H1, responsible for trimethylation of lysine 9 of histone H3.

Regulation of the tyrosine kinase substrate Eps8 expression by growth factors, v-Src and terminal differentiation.

SH3-containing proteins are involved in signal transduction by a number of growth factor receptors and in the organization of the cytoskeleton. The recently identified Eps8 protein, which contains an SH3 domain, is coupled functionally and physically to the EGFR and is tyrosine phosphorylated by this receptor and other receptors as well. Here, we examined the regulation of eps8 expression in response to mitogenic or differentiative signals. We show that Eps8 is expressed at low levels in resting fibroblasts, but its expression is strongly induced during activation by serum, phorbol esters and the v-src oncogene. Conversely, expression of Eps8, but not of other EGFR substrates such as Shc or Eps15, is virtually extinguished in non-proliferating, terminally differentiated murine myogenic cells. The putative role of Eps8 protein as a v-Src substrate was analysed in murine fibroblasts and in quail myogenic cells expressing a temperature-sensitive variant of the tyrosine kinase. Tyrosine phosphorylation of Eps8 was detected only at the permissive temperature. A non-myristylated, transformation-defective mutant of v-Src did not phosphorylate Eps8, whereas it phosphorylated Shc. Together, these findings indicate that Eps8 may be a critical substrate of v-Src. They further establish Eps8 as an example of a signal transducer whose expression senses the balance between growth and differentiation and might, therefore, be involved in the determination of the phenotype.

CDK9 has the intrinsic property to shuttle between nucleus and cytoplasm, and enhanced expression of CyclinT1 promotes its nuclear localization

CDK9 in association with cyclin T constitutes the P‐TEFb complex that stimulates transcription elongation of RNAPII transcripts by phosphorylation of the CTD of RNAPII. Here we report subcellular distribution of P‐TEFb in terms of localization of CDK9 and cyclin T1. We found that cyclin T1 is exclusively nuclear and it is present in nuclear‐speckled structures. CDK9, albeit mainly nuclear, was also visualized in the cytoplasm. We determined that CDK9 is actively exported from the nucleus, and that leptomycin B (LMB), a specific inhibitor of nuclear export, inhibits this process. Interestingly, enforced expression of cyclin T1 enhances nuclear localization of CDK9. These findings reveal a novel control mechanism for the function of the P‐TEFb complex.

Miniaturized FISH for screening of onco-hematological malignancies

Fluorescence in situ hybridization (FISH) represents a major step in the analysis of chromosomal aberrations in cancer. It allows the precise detection of specific rearrangements, both for diagnostic and prognostic purposes. Here we present a miniaturized FISH method performed on fresh and fixed hematological samples. This procedure has been developed together with a microfluidic device that integrates cluster-assembled nanostructured TiO2 (ns-TiO2) as a nanomaterial promoting hematopoietic cell immobilization in conditions of shear stress. As a result of miniaturization, FISH can be performed with at least a 10-fold reduction in probe usage and minimal cell requirements, creating the possibility of using FISH in genetic screening applications. We developed the protocol on tumor cells and bone marrow (BM) from a normal donor using commercially sex-specific and onco-hematology probes. The procedure was then validated using either BM or peripheral blood (PB) from six patients with hematological diseases, each associated with different genetic lesions. Miniaturized FISH demonstrated comparable performance to standard FISH, indicating that it is suitable for genetic screenings, in research, and in clinical settings for the diagnosis of samples from onco-hematological malignancies.

Characterization of cluster-assembled nanostructured titanium oxide coatings as substrates for protein arrays.

Protein microarray technologies are rapidly expanding to fulfill current needs of proteome discovery for disease management. Nanostructured materials have been shown to present interesting features when used in biological settings: nanostructured titanium oxide film (ns-TiOx), synthesized by supersonic cluster beam deposition (SCBD), has recently emerged as a biocompatible substrate in different biological assays. The ns-TiOx surface is characterized by a morphology at the nanoscale that can be tuned to modulate specific biomolecule-material interactions. Here we present a systematic characterization of ns-TiOx coatings as protein binding surfaces, comparing their performances with those of most common commercial substrates in protein and antibody microarray assays. Through a robust statistical evaluation of repeatability in terms of coefficient of variation (CV) analysis, we demonstrate that ns-TiOx can be used as reliable substrate for biochips in analytical protein microarray application.

Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches

BackgroundPirin (PIR) is a highly conserved nuclear protein originally isolated as an interactor of NFI/CTF1 transcription/replication factor. It is a member of the functionally diverse cupin superfamily and its activity has been linked to different biological and molecular processes, such as regulation of transcription, apoptosis, stress response and enzymatic processes. Although its precise role in these functions has not yet been defined, PIR expression is known to be deregulated in several human malignancies.ResultsWe performed immunohistochemical analysis of PIR expression in primary samples from normal human tissues and tumors and identified a dislocation of PIR to the cytoplasm in a subset of melanomas, and a positive correlation between cytoplasmic PIR levels and melanoma progression. PIR localization was subsequently analyzed in vitro in melanoma cell lines through a high content immunofluorescence based approach (ImmunoCell-Array).ConclusionsThe high consistency between in vivo and in vitro results obtained by immunohistochemistry and ImmunoCell-Array provides a validation of the potential of ImmunoCell-Array technology for the rapid screening of putative biological markers, and suggests that cytoplasmic localization of PIR may represent a characteristic of melanoma progression.