Biomedical subjects

M G Masucci

Publications and source records attributed to M G Masucci.

At least 19 recordsLinked to original sources

PubMed2006

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins.

The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate.

Actins↗

PubMed2004

Differential regulation of MHC class-I-restricted and unrestricted cytotoxicity by the Us3 protein kinase of herpes simplex virus-1.

Modulation of cytotoxic responses by viral immunoevasins plays an important role in the establishment of latent and persistent viral infections. Together with MHC class-I-restricted CD8T-lymphocytes, non-MHC-restricted natural killer (NK) and lymphokine-activated killer (LAK) cells participate in this anti-viral control. The Us3 protein kinase of herpes simplex virus-1 (HSV-1) inhibits CD8T-cell cytotoxicity, which correlates with the inhibition of granzyme-B (GrB)-induced activation of pro-apoptotic Bid. We have investigated the effect of Us3 on NK and LAK cytotoxicity, because these effectors are believed to share common mechanisms for inducing cell death. We show that, in contrast to their lower sensitivity to CD8T-cell lysis, HSV-1-infected cells are lysed by NK cells or LAK cells as efficiently as the uninfected controls. Both CD8T and NK/LAK effectors were dependent on the activity of GrB and were efficiently blocked by means of treatment with a GrB inhibitor. However, unlike CD8T cells, LAK cells and NK cells failed to induce Bid cleavage, suggesting that various GrB downstream targets be involved in the induction of cell lysis. This finding explains their various sensitivities to viral modulation, which is likely to be important for the respective role of MHC-restricted and non-restricted effectors in the control of HSV-1 infection.

CD8-Positive T-Lymphocytes↗

PubMed2003

The herpes simplex virus-1 Us3 protein kinase blocks CD8T cell lysis by preventing the cleavage of Bid by granzyme B.

The Us3 kinase is part of the antiapoptotic arsenal that salvages herpes simplex virus (HSV)-1-infected cells from damage caused by different stimuli. We demonstrate that Us3 protects HSV-1-infected cells from lysis by MHC class I-restricted CD8T cells without affecting antigen presentation. Expression of Us3 was associated with inhibition of caspase activation and reduced cleavage of the proapoptotic protein Bid. Recombinant granzyme B (GrB) failed to cleave Bid in cytosolic extracts from Us3 positive cells, while recombinant Bid served as substrate for Us3 phosphorylation, suggesting that modification of Bid by Us3 blocks its processing by GrB. Our data illustrate a new strategy of viral escape, where modification of a cellular proapoptotic substrate may prevent lysis of the infected cells without affecting other T-cell functions.

Antigen Presentation↗

PubMed2003

Pharmacological disintegration of lipid rafts decreases specific tetramer binding and disrupts the CD3 complex and CD8 heterodimer in human cytotoxic T lymphocytes.

Accumulating evidence strongly supports the role of lipid rafts in the regulation of T-lymphocyte activation, but the organization and molecular composition of these cholesterol- and sphingolipid-rich membrane microdomains in different subsets of T cells remain poorly investigated. Here, we show that pharmacological disruption of lipid rafts in human CD8+ cytotoxic T-lymphocyte (CTL) clones disturbs the integrity of CD3 complex and CD8 heterodimer, without affecting the reactivity with T-cell receptor (TCR)-specific antibodies. This demonstrates that interaction with completely assembled CD3 complex is not required for the stable expression of TCR at the cell surface. The effect of raft disruption on CD3 and CD8 expression correlates with failure to bind specific tetrameric complexes by a proportion of surface TCR molecules. However, the interaction of specific tetramer with the rest of surface TCR pools appears to be unaffected, demonstrating that TCR-signalling complexes may differ in their requirement for cholesterol to stably maintain their composition and to rearrange for efficient tetramer binding. Together with previously published data, our results support the existence of molecular and/or structural heterogeneity of lipid rafts that may play an important role in controlling distinct functional properties of T-cell subsets.

Antifungal Agents↗

PubMed2002

Avoiding proteasomal processing: the case of EBNA1.

Ubiquitin/proteasome-dependent proteolysis is involved in the regulation of a large variety of cellular processes including cell cycle progression, tissue development and atrophy, flux of substrates through metabolic pathways, selective elimination of abnormal proteins and processing of intracellular antigens for major histocompatibility complex (MHC) class I-restricted T-cell responses. Many viruses tamper with this proteolytic machinery by encoding proteins that interact with various components of the pathway. A particularly interesting example of a viral protein that interferes with proteasomal processing is the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1). EBNA1 contains an internal repeat exclusively composed of glycines and alanines that inhibits in cis the presentation of MHC class I-restricted T-cell epitopes and prevents ubiquitin/proteasome-dependent proteolysis in vitro and in vivo. The glycine-alanine repeat acts as a transferable element on a variety of proteasomal substrates and may therefore provide a new approach to the modification of cellular proteins for therapeutic purposes.

Alanine↗

PubMed2001

cis-Inhibition of proteasomal degradation by viral repeats: impact of length and amino acid composition.

The Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen 1 is a cis acting inhibitor of ubiquitin-proteasome proteolysis. We have investigated the capacity of various repeats to inhibit the turnover of the proteasomal substrate IkappaBalpha. Inhibition of TNFalpha-induced degradation was achieved by insertion of octamers containing three alanines or valines, interspersed by no more then three consecutive glycines. The inhibitory activity was abolished by increasing the length of the spacer, by eliminating the spacers, or by substitution of a single hydrophobic residue with a polar or charged residue. A serine containing octamer was inactive but inhibition was partially restored by insertion of three consecutive repeats. These findings suggest a model where inhibition requires the interaction of at least three alanine residues of the GAr in a beta-strand conformation with adjacent hydrophobic binding pockets of a putative receptor.

Alanine↗

PubMed2001

Different programs of activation-induced cell death are triggered in mature activated CTL by immunogenic and partially agonistic peptide ligands.

Activation-induced cell death (AICD) of mature T cells plays an important role in the control of immune homeostasis and peripheral tolerance. TNFRs and Fas have been implicated in the induction of AICD. However, these molecules were shown to be dispensable, at least in some experimental systems, for downsizing of Ag-induced T cell expansions and development of tolerance in vivo. The conditions of T cell activation leading to T cell deletion in a death receptor-independent manner are not well characterized. Here we show that human CTLs die through a death receptor-independent apoptotic program upon triggering with a partially agonistic peptide ligand. This apoptotic process exhibits some features of T cell death due to lymphokine deprivation and is blocked by exogenous IL-2. Our data demonstrate that engagement of TCR by MHC-peptide complexes can trigger diverse apoptotic programs of AICD and that the choice between these programs is determined by the agonistic potency of MHC-peptide ligand.

Animals↗

PubMed2001

c-myc overexpression activates alternative pathways for intracellular proteolysis in lymphoma cells.

Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein-Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

Aminopeptidases↗

PubMed2001

Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity.

The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells.

Catalysis↗

PubMed2000

Variations in proteasome subunit composition and enzymatic activity in B-lymphoma lines and normal B cells.

We investigated the expression of interferon gamma (IFN-gamma)-regulated subunits and the enzymatic activity of proteasomes purified from tumor-derived and normal B lymphocytes representing different stages of B-cell activation/differentiation. The catalytic beta subunits (Lmp2 and Lmp7) and the regulatory subunits (PA28alpha and PA28beta) were expressed at equally high levels in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), freshly isolated B-chronic lymphocytic leukemia (B-CLL) cells and normal CD23(-) B lymphocytes. Lmp2 and Lmp7 were selectively down-regulated in germinal center cell-derived Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HD) cell lines. There was a direct correlation between the expression of Lmp2/7 and the chymotrypsin and trypsin-like activities in proteasomes purified from LCLs, BLs and CLL cells, whereas 5 HD cell lines expressing B or T-cell markers exhibited a variable pattern of subunit expression and enzymatic activity. Poor hydrolysis of the fluorogenic substrates by proteasomes from BL cells correlated with a distinct pattern of cleavage of a reference 50mer peptide, production of different sets of degradation products and significantly reduced recovery of a known cytotoxic T-lymphocyte (CTL) target epitope. The enzymatic activity of proteasomes from normal CD23(-) "resting" B lymphocytes resembled that of BL cells in spite of high Lmp2/7 expression. This pattern was not reversed by treatment with the B-cell mitogen, lipopolysaccharide (LPS). The results suggest that different stages of B-cell activation/differentiation are associated with distinct profiles of IFN-gamma-regulated subunit composition and enzymatic activity of the proteasome. This may have important implications for the analysis and manipulation of tumor-specific immune responses.

B-Lymphocytes↗

PubMed2000

Inhibition of proteasomal degradation by the gly-Ala repeat of Epstein-Barr virus is influenced by the length of the repeat and the strength of the degradation signal.

The Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen-1 is a transferable element that inhibits in cis ubiquitin/proteasome-dependent proteolysis. We have investigated this inhibitory activity by using green fluorescent protein-based reporters that have been targeted for proteolysis by N end rule or ubiquitin-fusion degradation signals, resulting in various degrees of destabilization. Degradation of the green fluorescent protein substrates was inhibited on insertion of a 25-aa GAr, but strongly destabilized reporters were protected only partially. Protection could be enhanced by increasing the length of the repeat. However, reporters containing the Ub-R and ubiquitin-fusion degradation signals were degraded even in the presence of a 239-aa GAr. In accordance, insertion of a powerful degradation signal relieved the blockade of proteasomal degradation in Epstein-Barr virus nuclear antigen-1. Our findings suggest that the turnover of natural substrates may be finely tuned by GAr-like sequences that counteract targeting signals for proteasomal destruction.

Alanine↗

PubMed2000

Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells.

A semiquantitative polymerase chain reaction assay was used to monitor the blood levels of Epstein-Barr virus (EBV)-DNA in 9 patients receiving allogeneic bone marrow transplants (BMT). Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 10(7) EBV-specific cytotoxic T-lymphocytes (CTLs)/m(2) starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBV-specific component, progressed to fatal EBV-positive lymphoma. Administration of EBV-CTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas 1 patient with Wiskott-Aldrich syndrome reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease.

Adolescent↗

PubMed2000

Supermotif peptide binding and degeneracy of MHC: peptide recognition in an EBV peptide-specific CTL response with highly restricted TCR usage.

We have investigated the presentation and CTL recognition of an HLA A*1101-restricted CTL peptide epitope AVFDRKSDAK (AVF)(3), derived from the EBV nuclear antigen (EBNA) 4, in the context of alleles belonging to the A3-supertype, A*0101, 0301, 1101, 3101, 3301, and 6801. The peptide binds to a A*6801 molecule as efficiently as to A*1101. The A*6801:AVF complex is recognized by some A*1101-restricted AVF- specific CTL clones. However, A*6801-positive (A*6801+) EBV-transformed lymphoblastoid cell lines (LCLs) are not killed by the same effectors. Furthermore, two A*6801+ donors did not mount an AVF-specific CTL response in vitro and lacked detectable AVF-specific effectors. Thus, this epitope is either subdominant, or non-immunogenic in the context of A*6801. These characteristics correlate with low stability of this MHC:peptide complex in living cells. We also demonstrate that a highly conserved AVF-specific TCR that dominates the AVF-specific CTL response in the majority of A*1101+ individuals recognizes the A*6801 molecule as a crossreactive alloantigen. Therefore, deletion of AVF-specific T cells may contribute to the non-immunogenicity or subdominance of the peptide in A*6801+ individuals.

Alleles↗

PubMed2000

Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.

The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.

Cysteine Endopeptidases↗

PubMed2000

Effect of interleukin-7 on the in vitro development and maturation of monocyte derived human dendritic cells.

We have compared the cell phenotype and functional properties of monocyte/macrophage derived dendritic cells (DCs) obtained by culture of human adherent peripheral blood mononuclear cells (PBMCs) in medium containing granulocyte macrophage colony stimulating factor (GM-CSF) either alone (GM-CSF-DCs), or in combination with interleukin (IL)-4 (IL4-DCs) or IL-7 (IL7-DCs). The cell surface phenotype of GM-CSF-DCs and IL-7-DCs was characterized by a high expression of major histocompatibility complex (MHC) class I and II, CD80, CD86 and CD40. In contrast to 'classical' IL-4-DCs, these two types of DCs expressed CD14 and a CD21-like molecule detected by two out of four CD21-specific monoclonal antibodies (MoAb) tested. The same pattern of reactivity with CD21 specific antibodies was observed in freshly isolated adherent PBMCs but not in B lymphocytes. This reactivity was upregulated by IL-7 in a dose dependent manner. Lipopolysaccharide (LPS) treatment induced the upregulation of CD40, CD80, CD86 and the T-cell stimulatory capacity in IL-4-DCs and, to a lesser extent, in the IL-7-DCs whereas GM-CSF-DCs responded very poorly to such treatment. Our data indicate that, together with GM-CSF, the IL-7 drives macrophage precursors to a differentiation stage that is close to but distinct from the phenotype of IL-4-DCs. Comparison of DC development in the presence of IL-7 or IL-4 may help in dissecting signalling pathways that regulate the expression of functionally relevant DC markers.

Antigens, CD↗

PubMed1999

CD40 cross-linking enhances the immunogenicity of Burkitt's-lymphoma cell lines.

Epstein-Barr-virus (EBV)-positive Burkitt's-lymphoma (BL) cell lines are not recognized by EBV-specific T cells, due to their non-immunogenic phenotype and restricted expression of latent EBV genes. We tested whether triggering of CD40 can alter the phenotype of the tumor cells with regard to: (i) expression of surface markers, (ii) expression of viral antigens, (iii) presentation of endogenous antigens to MHC-class-1 restricted cytotoxic T lymphocytes (CTLs), (iv) stimulatory capacity in allogeneic mixed-lymphocyte cultures (MLCs), (v) sensitivity to natural-killer (NK)-cell-mediated lysis. Co-culture of EBV-positive BL cells with CD40-ligand-transfected L cells induced up-regulation of CD54 and CD80 but did not affect the expression of viral genes. In spite of significant up-regulation of TAP1 and TAP2, and increased expression of MHC class 1, the BL cells remained unable to present endogenously expressed viral antigens to EBV-specific CTL. However, the up-regulation of adhesion and co-stimulatory molecules was associated with increased stimulatory capacity in MLC and enhanced sensitivity to NK cells. These findings indicate that, while inducing only a modest phenotype shift, cross-linking of CD40 under physiologic conditions may selectively enhance the sensitivity of BL cells to anti-tumor immune responses.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

A partially agonistic peptide acts as a selective inducer of apoptosis in CD8+ CTLs.

We have analyzed the effect of partially agonistic peptides on the activation and survival of CTL clones specific for a highly immunogenic HLA A11-restricted peptide epitope derived from the EBV nuclear Ag-4. Several analogues with substitutions of TCR contact residues were able to trigger cytotoxic activity without induction of IL-2 mRNA and protein or T cell proliferation. Triggering with these partial agonists in the absence of exogenous IL-2 resulted in down-regulation of the cytotoxic potential of the specific CTLs. One analogue selectively triggered apoptosis as efficiently as the original epitope, subdividing the partial agonists into apoptosis-inducing and noninducing ligands. Analysis of early T cell activation events, induction of Ca2+ influx, and acid release did not reveal significant differences between the two types of analogue peptides. These results demonstrate that some partial agonists can dissociate the induction of CTL death from CTL activation. Peptides with such properties may serve as useful tools to study signal transduction pathways in CD8+ lymphocytes and as therapeutic agents modulating natural immune responses.

Apoptosis↗

PubMed1999

A single specific amino acid residue in peptide antigens is sufficient to activate memory CTL: potential role of cross-reactive peptides in memory T cell maintenance.

In the present study, we examined the structural requirements of peptide Ags for productive interactions with the TCR of CTL. For this purpose, we used as a model a previously identified immunodominant epitope that represents the target of EBV-specific HLA-A11-restricted CTL responses. By the use of peptides having minimal sequence homology with the wild-type epitope, we demonstrated that it is possible to selectively expand and reactivate memory CTL precursors without triggering the lytic mechanisms of wild-type specific effectors. In fact, stimulation of PBL from EBV-seropositive donors by polyalanine analogues, sharing only the putative TCR contact residue with the natural epitope, exclusively induced clonal expansion and reactivation of EBV-specific memory CTL precursors. Interestingly, these polyalanine peptides failed to trigger the cytotoxic function of CTLs specific for the wild-type viral epitope. This clearly indicates that reactivation of memory CTL precursors and triggering of the cytotoxic function have different requirements. The same phenomenon was observed using as stimulators naturally occurring peptides carrying the appropriate TCR contact residue. These data strongly suggest that cross-reactive peptides may play an important role in the expansion and reactivation of CTL clones from the memory T cell pool, and may be involved in long-term maintenance of T cell memory.

Amino Acid Substitution↗