Marco Schiavone

Ca2+ binding to F‐ATP synthase β subunit triggers the mitochondrial permeability transition

F‐ATP synthases convert the electrochemical energy of the H+ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F‐ATP synthases can also undergo a Ca2+‐dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca2+ binding site and the mechanism(s) through which Ca2+ can transform the energy‐conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the “Ca2+‐trigger site” of the PTP to the catalytic site of the F‐ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca2+‐ATP hydrolysis, confer resistance to Ca2+‐induced, PTP‐dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.

NIM811, a cyclophilin inhibitor without immunosuppressive activity, is beneficial in collagen VI congenital muscular dystrophy models

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.

Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk

Brutons tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5′RACE and 3′RACE we identified three IBTKα, IBTKβ and IBTKγ mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk.

In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder.

B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-beta cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.

Physical and Functional Interaction of HIV-1 Tat with E2F-4, a Transcriptional Regulator of Mammalian Cell Cycle

Tat protein of the human immunodeficiency virus type-1 (HIV-1) plays a critical role in the regulation of viral transcription and replication. In addition, Tat regulates the expression of a variety of cellular genes and could account for AIDS-associated diseases including Kaposis Sarcoma and non-Hodgkins lymphoma by interfering with cellular processes such as proliferation, differentiation, and apoptosis. The molecular mechanisms underlying the pleiotropic activities of Tat may include the generation of functional heterodimers of Tat with cellular proteins. By screening a human B-lymphoblastoid cDNA library in the yeast two-hybrid system, we identified E2F-4, a member of E2F family of transcription factors, as a Tat-binding protein. The interaction between Tat and E2F-4 was confirmed by GST pull-down experiments performed with cellular extracts as well as with in vitro translated E2F-4. The physical association of Tat and E2F-4 was confirmed by in vivobinding experiments where Tat·E2F-4 heterodimers were recovered from Jurkat cells by immunoprecipitation and immunoblotting. By using plasmids expressing mutant forms of Tat and E2F-4, the domains involved in Tat·E2F-4 interaction were identified as the regions encompassing amino acids 1–49 of Tat and amino acids 1–184 of E2F-4. Tat·E2F-4 complexes were shown to bind to E2F cis-regions with increased efficiency compared with E2F-4 alone and to mediate the activity of E2F-dependent promoters including HIV-1 long terminal repeat and cyclin A. The data point to Tat as an adaptor protein that recruits cellular factors such as E2F-4 to exert its multiple biological activities.

A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development.

TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left-right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate.

Discovery, Synthesis, and Optimization of Diarylisoxazole-3-carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

The mitochondrial permeability transition pore (mtPTP) is a Ca2+‐requiring mega‐channel which, under pathological conditions, leads to the deregulated release of Ca2+ and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1, 5‐(3‐hydroxyphenyl)‐N‐(3,4,5‐trimethoxyphenyl)isoxazole‐3‐carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC50<0.39 μM) and showed no interference on the inner mitochondrial membrane potential (rhodamine 123 uptake EC50>100 μM). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N‐(3‐chloro‐2‐methylphenyl)‐5‐(4‐fluoro‐3‐hydroxyphenyl)isoxazole‐3‐carboxamide (60), was validated in a biologically relevant zebrafish model of collagen VI congenital muscular dystrophies.

Zebrafish reporter lines reveal in vivo signaling pathway activities involved in pancreatic cancer

Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. This condition is usually driven by oncogenic KRAS point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and unbalanced expression of genes involved in the specific signaling pathways. To examine in vivo the effects of KRASG12D during pancreatic cancer progression and time correlation with cancer signaling pathway activities, we have generated a zebrafish model of pancreatic adenocarcinoma in which eGFP-KRASG12D expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic KRASG12D line with transgenic zebrafish reporters, harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFβ, Notch, Bmp and Shh activities during tumor development. Zebrafish transgenic lines expressing eGFP-KRASG12D showed normal exocrine pancreas development until 3 weeks post fertilization (wpf). From 4 to 24 wpf we observed several degrees of acinar lesions, characterized by an increase in mesenchymal cells and mixed acinar/ductal features, followed by progressive bowel and liver infiltrations and, finally, highly aggressive carcinoma. Moreover, live imaging analysis of the exocrine pancreatic tissue revealed an increasing number of KRAS-positive cells and progressive activation of TGFβ and Notch pathways. Increase in TGFβ, following KRASG12D activation, was confirmed in a concomitant model of medulloblastoma (MDB). Notch and Shh signaling activities during tumor onset were different between MDB and pancreatic adenocarcinoma, indicating a tissue-specific regulation of cell signaling pathways. Moreover, our results show that a living model of pancreatic adenocarcinoma joined with cell signaling reporters is a suitable tool for describing in vivo the signaling cascades and molecular mechanisms involved in tumor development and a potential platform to screen for novel oncostatic drugs.

Cancer-Associated CD43 Glycoforms as Target of Immunotherapy

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer–mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy. Mol Cancer Ther; 13(3); 752–62. ©2013 AACR.

Design and characterization of a peptide mimotope of the HIV-1 gp120 bridging sheet.

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV+ broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.