Elena Sartori

vOX2 glycoprotein of human herpesvirus 8 modulates human primary macrophages activity.

Human herpesvirus 8 (HHV‐8) is a lymphotropic herpesvirus linked to several disorders such as Kaposis sarcoma, primary effusion lymphoma and multicentric Castlemans disease. Several HHV‐8 proteins regulate host innate and adaptive immune response; in particular, orfK14 is expressed as an immediate early gene during the viral lytic cycle and encodes a surface glycoprotein (vOX2), significantly homologous to the cellular OX2, which delivers inhibitory signals to macrophages. Although it has been suggested that vOX2 may down‐regulate basophil and neutrophil functions, its role in macrophages, a cell type lytically infected by HHV‐8 in vivo, is still controversial. Therefore, we investigated the effect of vOX2 expression in human primary monocyte‐derived macrophages (MDMs). In this report, we demonstrate that vOX2‐expressing MDMs in basal conditions are induced to produce inflammatory cytokines and display higher phagocytic activity with respect to mock cells. By contrast, an opposite effect is exhibited by vOX2 in MDMs undergoing IFN‐γ‐activation, with a down‐modulation of the cytokine production and phagocytic activity. Moreover, we observed that, when MDMs are co‐cultured with vOX2‐expressing cells, the inflammatory cytokine release is increased, independently from the MDM activation state. Interestingly, we could correlate our results with the mRNA transcript level of the vOX2 cellular CD200R receptor. Finally, we demonstrate a down‐regulation of the MHC class I and class II molecules on the cell surface of vOX2‐transduced MDMs. Our results provide new insights into the immunomodulatory effects of HHV‐8 vOX2 protein. J. Cell. Physiol. 219: 698–706, 2009.

Herpes simplex virus type 2 infection increases human immunodeficiency virus type 1 entry into human primary macrophages.

Epidemiological and clinical data indicate that genital ulcer disease (GUD) pathogens are associated with an increased risk of human immunodeficiency virus type 1 (HIV-1) acquisition and/or transmission. Among them, genital herpes simplex virus type 2 (HSV-2) seems to play a relevant role. Indeed, the ability of HSV-2 to induce massive infiltration at the genital level of cells which are potential targets for HIV-1 infection may represent one of the mechanisms involved in this process. Here we show that infection of human primary macrophages (MDMs) by HSV-2 results in an increase of CCR5 expression levels on cell surface and allows higher efficiency of MDMs to support entry of R5 HIV-1 strains. This finding could strengthen, at the molecular level, the evidence linking HSV-2 infection to an increased susceptibility to HIV-1 acquisition.

CIDEP Effects of Intramolecular Quenching of Singlet and Triplet Excited States by Nitroxide Radicals in Oligopeptides: A Potentially Useful New Method for Investigating Peptide Secondary Structures in Solution

Two hexapeptides, each bearing one photoactive alpha-amino acid (Bin or Bpa) and one nitroxide-containing TOAC residue, have been synthesized and fully characterized. FT-IR absorption measurements indicate that a 3(10)-helical conformation is adopted by these peptides in solution. As two amino acid units separate the photoactive residue from TOAC in the peptide sequences, the two moieties face each other at a distance of about 6 A after one complete turn of the ternary helix. Irradiation by a light pulse from an excimer laser populates the excited states localized on the chromophores. An intramolecular interaction between the singlet (Bin) or triplet (Bin and Bpa) excited states and the doublet state of the TOAC nitroxide makes a spin-selective decay pathway possible, that produces transient spin polarization. In addition, in order to determine whether the intramolecular exchange interaction occurs through-bond or through-space, we have prepared linear and cyclic TOAC-Bin dipeptide units. A CIDEP study revealed that a through-space intramolecular interaction is operative. The observation of spin polarization makes the two helical hexapeptides suitable models to test the possibility of application of this novel technique to conformational studies of peptides in solution.

Nef and cell signaling transduction: a possible involvement in the pathogenesis of human immunodeficiency virus–associated dementia

Although the introduction of highly active antiretroviral therapy (HAART) has resulted in a significant decrease of acquired immunodeficiency syndrome (AIDS) morbidity and mortality, the prevalence of human immunodeficiency virus (HIV)-associated dementia (HAD) has actually risen, due to the increasing life expectancy of the infected subjects. To date, several aspects of the HAD pathogenesis remain to be dissected. In particular, the viral-cellular protein interplay is still under investigation. Given their specific features, two viral proteins, Tat and Nef, have been mainly hypothesized to play a role in HIV neuropathology. Here we show that HIV-1 Nef has an effect on the transcriptional levels of a cellular protein, anaplastic lymphoma kinase (ALK), that is preferentially expressed in the central and peripheral nervous system at late embryonic stages. By its overexpression along with Nef, the authors demonstrate ALK ability to influence, at least in the U87MG astrocytic glioma cells, the mytogen-activated protein kinase (MAP-K) dependent pathway. Moreover, although in the absence of a physical direct interaction, Nef and ALK activate matrix metalloproteinases (MMPs), which are likely to contribute to blood-brain barrier (BBB) damage in HAD. Finally, in the in vitro model of glioblastoma cells adopted, Nef and ALK show similar effects by increasing different cytochines/chemokines that may be relevant for HAD pathogenesis. If confirmed in vivo, these data may indicate that, thanks to its ability to interfere with specific cellular pathways involved in BBB damage and in central nervous system (CNS) integrity, Nef, along with specific cellular counterparts, could be one of the viral players implicated in HAD development.

A Simple Mean Field Model for Social Interactions: Dynamics, Fluctuations, Criticality

We study the dynamics of a spin-flip model with a mean field interaction. The system is non reversible, spacially inhomogeneous, and it is designed to model social interactions. We obtain the limiting behavior of the empirical averages in the limit of infinitely many interacting individuals, and show that phase transition occurs. Then, after having obtained the dynamics of normal fluctuations around this limit, we analyze long time fluctuations for critical values of the parameters. We show that random inhomogeneities produce critical fluctuations at a shorter time scale compared to the homogeneous system.

A Helical Peptide Receptor for [60]Fullerene

Two terminally blocked nonapeptides, each made up of six Aib residues, a Gly spacer and two L-Tyr residues in positions 2 and 8 (these are substituted in the side chain with either ferrocenoyl or methyl moieties), have been synthesized by solution methods and fully characterized. FT-IR absorption and two-dimensional NMR analyses indicate that a 3(10)-helical conformation is adopted by these rigid peptides in structure-supporting solvents. An X-ray diffraction investigation shows that the bis-L-Tyr(Me) nonapeptide in the crystal state is folded in a regular right-handed 3(10)-helical structure. As five amino acid units separate the two substituted L-Tyr residues in the peptide sequence, the two side chain moieties will-in solution-face each other after two complete turns of the ternary helix. By carrying out a detailed photophysical analysis, we have demonstrated that the electron-rich, hydrophobic and wide cavity generated by the nonapeptide template with two ferrocenoyloxybenzyl walls is able to host [60]fullerene. Further evidence for this superstructure has been provided in the gas phase by a mass spectrometric investigation.

Interaction between TOAC free radical and photoexcited triplet chromophores linked to peptide templates.

The intramolecular quenching of photoexcited triplet states by free radicals linked to peptide templates was studied by time-resolved electron paramagnetic resonance (EPR) with pulsed laser excitation. The systems investigated are 3(10)-helix forming peptides, having in the amino acid sequence the free radical 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and a triplet precursor, such as Bin, Bpa, or Trp, incorporated at different relative positions. Upon interaction with the excited triplet the TOAC radical spin sublevel populations assume values that differ from the Boltzmann equilibrium values. This spin polarization effect produces EPR lines in emission whose time evolution reflects the triplet quenching rate. In particular, in a series of peptides labeled with Bpa and TOAC at successive positions in the 3(10)-helix, radical-triplet interaction was observed in all cases. However, for the peptide where Bpa and TOAC are at positions 2 and 4 the rate of triplet quenching is lower than for the other peptides in the series. In addition, the radical-excited triplet complex in the quartet spin state was observed in a peptide containing fullerene (C(60)) as a triplet precursor and TOAC.

Functional Interaction Between the ESCRT-I Component TSG101 and the HSV-1 Tegument Ubiquitin Specific Protease

Similar to phosphorylation, transient conjugation of ubiquitin to target proteins (ubiquitination) mediated by the concerted action of ubiquitin ligases and de‐ubiquitinating enzymes (DUBs) can affect substrate function. As obligate intracellular parasites, viruses rely on different cellular pathways for their own replication and the well conserved ubiquitin conjugating/de‐conjugating system is not an exception. Viruses not only usurp the host proteins involved in the ubiquitination/de‐ubiquitination process, but they also encode their own ubiquitin ligases and DUBs. Here we report that an N‐terminal variant of the herpes simplex virus (HSV) type‐1 large tegument protein VP1/2 (VP1/21–767), encompassing an active DUB domain (herpesvirus tegument ubiquitin specific protease, htUSP), and TSG101, a component of the endosomal sorting complex required for transport (ESCRT)‐I, functionally interact. In particular, VP1/21–767 modulates TSG101 ubiquitination and influences its intracellular distribution. Given the role played by the ESCRT machinery in crucial steps of both cellular pathways and viral life cycle, the identification of TSG101 as a cellular target for the HSV‐1 specific de‐ubiquitinating enzyme contributes to the clarification of the still under debate function of viral encoded DUBs highly conserved throughout the Herpesviridae family. J. Cell. Physiol. 230: 1794–1806, 2015.

Cryptochrome Is a Regulator of Synaptic Plasticity in the Visual System of Drosophila melanogaster

Drosophila CRYPTOCHROME (CRY) is a blue light sensitive protein with a key role in circadian photoreception. A main feature of CRY is that light promotes an interaction with the circadian protein TIMELESS (TIM) resulting in their ubiquitination and degradation, a mechanism that contributes to the synchronization of the circadian clock to the environment. Moreover, CRY participates in non-circadian functions such as magnetoreception, modulation of neuronal firing, phototransduction and regulation of synaptic plasticity. In the present study we used co-immunoprecipitation, yeast 2 hybrid (Y2H) and in situ proximity ligation assay (PLA) to show that CRY can physically associate with the presynaptic protein BRUCHPILOT (BRP) and that CRY-BRP complexes are located mainly in the visual system. Additionally, we present evidence that light-activated CRY may decrease BRP levels in photoreceptor termini in the distal lamina, probably targeting BRP for degradation.

Isoform-specific interactions of the von Hippel-Lindau tumor suppressor protein.

Deregulation of the von Hippel-Lindau tumor suppressor protein (pVHL) is considered one of the main causes for malignant renal clear-cell carcinoma (ccRCC) insurgence. In human, pVHL exists in two isoforms, pVHL19 and pVHL30 respectively, displaying comparable tumor suppressor abilities. Mutations of the p53 tumor suppressor gene have been also correlated with ccRCC insurgence and ineffectiveness of treatment. A recent proteomic analysis linked full length pVHL30 with p53 pathway regulation through complex formation with the p14ARF oncosuppressor. The alternatively spliced pVHL19, missing the first 53 residues, lacks this interaction and suggests an asymmetric function of the two pVHL isoforms. Here, we present an integrative bioinformatics and experimental characterization of the pVHL oncosuppressor isoforms. Predictions of the pVHL30 N-terminus three-dimensional structure suggest that it may exist as an ensemble of structured and disordered forms. The results were used to guide Yeast two hybrid experiments to highlight isoform-specific binding properties. We observed that the physical pVHL/p14ARF interaction is specifically mediated by the 53 residue long pVHL30 N-terminal region, suggesting that this N-terminus acts as a further pVHL interaction interface. Of note, we also observed that the shorter pVHL19 isoform shows an unexpected high tendency to form homodimers, suggesting an additional isoform-specific binding specialization.