Studio dell'attività biologica della proteina ORF-A del virus dell'immunodeficienza felina

Feline Immunodeficiency Virus (FIV), a non-primate lentivirus, causes an immunodeficiency syndrome in domestic cats that is strikingly similar to AIDS in humans. Although diverse in term of evolution, FIV is similar to Human Immunodeficiency Virus Type-1 (HIV-1) in many respects including genome organization, target cells infected in vivo, course of infection and disease state. Thus, for these reasons, FIV represents an attractive model for AIDS research. Similarly to HIV-1 and other lentivirus, the FIV genome encodes three large open reading frame gag, pol, env that encode for the structural and enzymatic proteins. HIV-1 encodes six additional small open reading frames that express regulatory and accessory proteins: vif, rev, tat, vpr, vpu and nef. However, the FIV genome contains only three additional open reading frames, two of which include rev, that encodes a viral post transcriptional regulatory protein and vif that encodes an accessory protein necessary for viral infectivity. The primary function of the third gene designated orf-A remains unclear but it is believed to represent a multifunctional accessory protein similar to HIV-1 Vpr, Tat and Nef accessory proteins. This study is part of a research project aimed to clarify FIV biology, focusing on the functions performed by ORF-A. Firstly, we evaluated Orf-A expression and its involvement in the cell cycle progression using different systems of over-expression. Secondly, in order to explore Orf-A involvement in cell cycle progression in the context of more physiological conditions, we obtained Petaluma proviral constructs containing a tagged version of Orf-A. Moreover, we employed the same constructs in order to evaluate i) the intracellular localization of Orf-A and Cyclin B1 proteins with immunofluorescence assays; ii) the replicative capacity of Orf-A tagged plus and minus viruses; iii) the incorporation of the Orf-A protein in viral particles. Taken together, the results suggest that Orf-A expression leads to a cell cycle arrest in G2/M phase, indicating a role of Orf-A similar to the one described for HIV-1 Vpr. Furthermore, for the first time we report the Orf-A incorporation into viral particles. This study contributes to dissect FIV Orf-A functions and to clarify the mechanisms underlying Feline Immunodeficiency Virus and, generally, lentiviruses biolog

The effect of FK506 on transforming growth factor beta signaling and apoptosis in chronic lymphocytic leukemia B cells.

Background Loss of response to transforming growth factor-beta (TGF-beta) is thought to contribute to the progression of chronic lymphocytic leukemia. Recent findings of over-activation of the TGF-beta signal in FKBP12-knockout mouse prompted us to investigate whether FK506, the canonical ligand of FKBP, can activate the TGF-beta signal in chronic lymphocytic leukemia. Design and Methods We studied 62 chronic lymphocytic leukemia samples from patients with Rai/Binet stage 0 to 4 disease. The TGF-beta signal was investigated by western blotting and flow cytometry. The levels of Bcl2-family members and death-associated-protein kinase were also investigated by western blotting, whereas apoptosis was studied in flow cytometry. Down-modulation of FKBP12 was obtained by gene silencing with short interfering RNA. Results Twenty-two out of 62 chronic lymphocytic leukemia samples were sensitive to TGF-beta-induced apoptosis. All but two of the responsive samples underwent apoptosis also when cultured with FK506, but not with cyclosporine. Thirteen samples that were not sensitive to TGF-beta were sensitive to FK506. Overall, response to FK506 occurred in 33 samples. FK506 induced Smad2 phosphorylation and nuclear translocation. Accordingly, death-associated-protein kinase, a transcriptional target of Smad, was induced. At the same time, Bcl-2 and Bcl-xL levels decreased whereas the levels of Bim and Bmf increased. A loss of mitochondrial membrane potential preceded caspase activation and cell death. FK506 removed FKBP12 from its binding to the TGF-beta-receptor. FKBP12 release activated the receptor-kinase activity as suggested by the enhanced levels of phospho-Smad found in cells depleted of FKBP12. Conclusions Our study shows that most chronic lymphocytic leukemia cells escape the homeostatic control of TGF-beta and that FK506 restores the TGF-beta signal in a proportion of non-responsive samples. We demonstrated that FK506 activates TGF-beta receptor I kinase activity in chronic lymphocytic leukemia, which transduces apoptosis by a mitochondrial-dependent pathway

Studio dell'attività biologica della proteina ORF-A del virus dell'immunodeficienza felina

Feline Immunodeficiency Virus (FIV), a non-primate lentivirus, causes an immunodeficiency syndrome in domestic cats that is strikingly similar to AIDS in humans. Although diverse in term of evolution, FIV is similar to Human Immunodeficiency Virus Type-1 (HIV-1) in many respects including genome organization, target cells infected in vivo, course of infection and disease state. Thus, for these reasons, FIV represents an attractive model for AIDS research. Similarly to HIV-1 and other lentivirus, the FIV genome encodes three large open reading frame gag, pol, env that encode for the structural and enzymatic proteins. HIV-1 encodes six additional small open reading frames that express regulatory and accessory proteins: vif, rev, tat, vpr, vpu and nef. However, the FIV genome contains only three additional open reading frames, two of which include rev, that encodes a viral post transcriptional regulatory protein and vif that encodes an accessory protein necessary for viral infectivity. The primary function of the third gene designated orf-A remains unclear but it is believed to represent a multifunctional accessory protein similar to HIV-1 Vpr, Tat and Nef accessory proteins. This study is part of a research project aimed to clarify FIV biology, focusing on the functions performed by ORF-A. Firstly, we evaluated Orf-A expression and its involvement in the cell cycle progression using different systems of over-expression. Secondly, in order to explore Orf-A involvement in cell cycle progression in the context of more physiological conditions, we obtained Petaluma proviral constructs containing a tagged version of Orf-A. Moreover, we employed the same constructs in order to evaluate i) the intracellular localization of Orf-A and Cyclin B1 proteins with immunofluorescence assays; ii) the replicative capacity of Orf-A tagged plus and minus viruses; iii) the incorporation of the Orf-A protein in viral particles. Taken together, the results suggest that Orf-A expression leads to a cell cycle arrest in G2/M phase, indicating a role of Orf-A similar to the one described for HIV-1 Vpr. Furthermore, for the first time we report the Orf-A incorporation into viral particles. This study contributes to dissect FIV Orf-A functions and to clarify the mechanisms underlying Feline Immunodeficiency Virus and, generally, lentiviruses biologyIl Virus dell'Immunodeficienza Felina (FIV) è un Lentivirus dei non primati che causa nel gatto domestico una sindrome cronica progressiva molto simile alla Sindrome da Immunodeficienza Acquisita (AIDS) generata dal Virus dell'Immunodeficienza Umana di tipo 1 (HIV-1). Le analogie tra FIV ed HIV-1 coinvolgono numerosi aspetti, tra cui l'organizzazione genomica, il ciclo replicativo, i bersagli cellulari ed i meccanismi patogenetici. Per questi motivi FIV ed il suo ospite naturale rappresentano un interessante modello per la ricerca sull'AIDS. Similmente ad HIV-1, il genoma di FIV contiene i geni gag, pol, env che codificano le proteine strutturali ed enzimatiche del virione e i geni accessori e regolatori che giocano un ruolo chiave nella regolazione e nell'infettività virale. A differenza del genoma di HIV-1 caratterizzato dalla presenza di quattro geni accessori, il genoma di FIV ne presenta due: vif ed orf-A. Di questi, il primo codifica il fattore di infettività virale, molto simile a quello di HIV-1, mentre il secondo gene codifica la proteina Orf-A. Il ruolo biologico di Orf-A non è stato ancora chiarito, ma si ritiene rappresenti una proteina accessoria multifunzionale con caratteristiche simili alle proteine accessorie di HIV-1 quali Vpr, Tat e Nef. Il presente lavoro di dottorato si inserisce in un progetto di ricerca più ampio, volto a chiarire la biologia di base di FIV, focalizzandosi sulle funzioni svolte dalla proteina Orf-A. Inizialmente è stata valutata l'espressione della proteina ed il suo coinvolgimento nel ciclo cellulare mediante diversi sistemi di over-espressione. Successivamente sono stati condotti gli stessi esperimenti in un contesto più simile a quello fisiologico. Pertanto, sono stati ottenuti plasmidi basati sul genoma provirale, Orf-A plus e minus, dove l'estremità Carbossi-terminale della proteina Orf-A è stata fusa al tag HA. I vettori sono stati testati sia in cellule umane che feline per valutarne l'espressione ed il coinvolgimento nella progressione del ciclo cellulare. Gli stessi plasmidi sono stati impiegati per valutare i) la localizzazione della proteina Orf-A e della Ciclina B1 target del blocco del ciclo cellulare in saggi di immunofluorescenza; ii) la capacità replicativa del virus; iii) l'incorporazione della proteina Orf-A nelle particelle virali. Nel complesso, i risultati suggeriscono che, laddove la proteina Orf-A è espressa, si verifica un arresto del ciclo cellulare in fase G2/M indicando un ruolo di Orf-A simile a quello descritto per la proteina Vpr di HIV-1. Inoltre, per la prima volta in letteratura, è stata dimostrata l'incorporazione della proteina Orf-A nelle particelle virali. Questo studio contribuisce a dissezionare le funzioni della proteina Orf-A di FIV ed a chiarire i meccanismi alla base della biologia del Virus dell'Immunodeficienza Felina e, più in generale, di quella dei lentiviru

Pleiotropic roles in cancer biology for multifaceted proteins FKBPs

FK506 binding proteins (FKBP) are multifunctional proteins highly conserved across the species and abundantly expressed in the cell. In addition to a well-established role in immunosuppression, FKBPs modulate several signal transduction pathways in the cell, due to their isomerase activity and the capability to interact with other proteins, inducing changes in conformation and function of protein partners. Increasing literature data support the concept that FKBPs control cancer related pathways

Molecular Aspects of FKBP51 that Enable Melanoma Dissemination

FKBP51 (FKBP5 Official Symbol) is large molecular weight member of the FK506 binding protein family, a subfamily of the immunophilin proteins. FKBP51 exerts multiple biological functions in the cell, including modulation of steroid hormone response, immune regulation, cell proliferation, regulation of pAkt levels and control of NF-κB activation. Several lines of evidence support a role for this protein in cancer biology, especially in resistance to chemo- and radio-therapy. Recent research studies highlighted functions of FKBP51 in promoting the epithelial to mesenchymal transition (EMT) transdifferentiation program in melanoma. This process, which is classically regulated by Transforming Growth Factor (TGF)-β, enables cancer cells to disseminate from primary tumors and spread to distant locations, acquiring resistance to therapy and self-renewal capability. This last, in turn, is crucial to their subsequent expansion at sites of dissemination. The aim of the present article is to review recent literature data that involve FKBP51 in the mechanisms that switch the TGF-β from a tumor suppressor to a pro-metastatic invader

Molecular Aspects of FKBP51 that Enable Melanoma Dissemination.

FKBP51 (FKBP5 Official Symbol) is large molecular weight member of the FK506 binding protein family, a subfamily of the immunophilin proteins. FKBP51 exerts multiple biological functions in the cell, including modulation of steroid hormone response, immune regulation, cell proliferation, regulation of pAkt levels and control of NF-κB activation. Several lines of evidence support a role for this protein in cancer biology, especially in resistance to chemo- and radio-therapy. Recent research studies highlighted functions of FKBP51 in promoting the epithelial to mesenchymal transition (EMT) transdifferentiation program in melanoma. This process, which is classically regulated by Transforming Growth Factor (TGF)-β, enables cancer cells to disseminate from primary tumors and spread to distant locations, acquiring resistance to therapy and self-renewal capability. This last, in turn, is crucial to their subsequent expansion at sites of dissemination. The aim of the present article is to review recent literature data that involve FKBP51 in the mechanisms that switch the TGF-β from a tumor suppressor to a pro-metastatic invader

Cellular and molecular background underlying the diversity in therapeutic responses between primary tumours and metastases.

Metastasis, also called secondary neoplastic disease, is a tumour newly formed in a site different from that of origin, as a consequence of cancer progression and dissemination largely through blood and lymphatic vessels. The ability to form metastases is the main property that distinguishes malignant from benign tumours. Treatments for metastatic cancer are similar in practice to those for primary tumours, but such treatments are mostly palliative; indeed, almost all deaths caused by solid tumours occur in the metastatic phase. Increasing evidence supports the concept that therapies for primary tumours are inadequate to treat metastasis and can even promote formation of metastases, while exerting local growth control. Furthermore, recurrent tumours, which are denoted by increased aggressiveness and therapy resistance in comparison with the primary tumour, have an increased metastatic potential. Genetic modifications occurring during tumour progression lead to substantial differences between the primary and metastatic tumours. This emphasises the importance of designing novel therapies for metastasis. In the last decade, a number of studies have contributed to the understanding of the genetic rearrangements underlying the conversion of cancer cells into the metastasis founder cells. The present article aims at reviewing recent advances in metastasis research and attempts to discuss the reasons for which the therapeutic strategies against primary tumours may not satisfactorily address their metastatic counterparts