Paola Fusi

Domain architecture of the polyglutamine protein ataxin-3: a globular domain followed by a flexible tail☆

Anomalous expansion of a polyglutamine (polyQ) tract in the protein ataxin‐3 causes spinocerebellar ataxia type 3, an autosomal dominant neurodegenerative disease. Very little is known about the structure and the function of ataxin‐3, although this information would undoubtedly help to understand why the expanded protein forms insoluble nuclear aggregates and causes neuronal cell death. With the aim of establishing the domain architecture of ataxin‐3 and the role of the polyQ tract within the protein context, we have studied the human and murine orthologues using a combination of techniques, which range from limited proteolysis to circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The two protein sequences share a highly conserved N‐terminus and differ only in the length of the glutamine repeats and in the C‐terminus. Our data conclusively indicate that ataxin‐3 is composed by a structured N‐terminal domain, followed by a flexible tail. Moreover, [15N]glutamine selectively labelled samples allowed us to have a direct insight by NMR into the structure of the polyQ region.

Bacteria-Induced Gap Junctions in Tumors Favor Antigen Cross-Presentation and Antitumor Immunity

Bacterial infection induces extra gap junctions in tumors, through which tumor antigens travel to antigen-presenting cells, triggering an effective antitumor immune response. Special Delivery: Immune Cells Receive a Package of Tumor-Specific Peptides Immune cells on patrol often recognize cancer cells as abnormal and eliminate them. But as tumors progress and proliferate, they can become invisible to these immune guardians of order. An injection of bacteria into the tumor can render them again visible to the immune system, thus promoting tumor-directed immune responses. If we understood how this reappearing act worked, we might be able to exploit it for cancer treatment. Saccheri et al. have discovered that the injected bacteria perform a key function: They reactivate connexin 43, a protein often suppressed in cancer cells that forms tiny communication channels—gap junctions—between cells. Tumor peptides escape through these channels and enter immune cells, which display the peptides on their surfaces, successfully triggering a specific immune response against the cancer. The authors showed that the bacteria Salmonella or its components elicited increased amounts of connexin 43 in melanoma cell lines from mice or humans. This connexin was used by the cells to form new gap junctions, allowing small molecules of the dye Lucifer yellow to pass between tumor cells or from tumor cells into antigen-presenting dendritic cells, which could also transfer dye among themselves. When the authors artificially inserted the protein ovalbumin into the melanoma cells in culture, it was broken down into peptides by the tumor proteasome, and these peptides passed through the gap junctions into cocultured dendritic cells, where they were displayed on the surface. Ovalbumin-specific T cells could be activated in response, and this activation depended on connexin 43. The authors then tested in mice whether this gap junction route for getting tumor peptides to dendritic cells operates in living animals. Although bacteria injected into tumors caused the eradication of that particular tumor independent of connexin and gap junctions, the regression of distant metastases, mediated by cytotoxic T cells, required the infection-induced gap junction mechanism. The delivery of tumor peptides to dendritic cells through gap junctions can be harnessed to generate tumor-specific dendritic cells ex vivo, a way to create tumor-specific immune cells for infusion into patients. Incubation of bacteria-infected melanoma cells with dendritic cells in vitro allowed loading of the immune cells with tumor peptides, conferring an effective antitumor response when the dendritic cells were injected into tumor-bearing mice. And this approach could have even more practical potential: These in vitro–loaded immune cells protected mice against developing tumors from seeded cancer cells, a “vaccination”-style preventive strategy. Antigen-presenting dendritic cells (DCs) trigger the activation of cytotoxic CD8 T cells that target and eliminate cells with the antigen on their surface. Although DCs usually pick up and process antigens themselves, they can also receive peptide antigens from other cells via gap junctions. We demonstrate here that infection with Salmonella can induce, in both human and murine melanoma cells, the up-regulation of connexin 43 (Cx43), a ubiquitous protein that forms gap junctions and that is normally lost during melanoma progression. Bacteria-treated melanoma cells can establish functional gap junctions with adjacent DCs. After bacterial infection, these gap junctions transferred preprocessed antigenic peptides from the tumor cells to the DCs, which then presented those peptides on their surface. These peptides activated cytotoxic T cells against the tumor antigen, which could control the growth of distant uninfected tumors. Melanoma cells in which Cx43 had been silenced, when infected in vivo with bacteria, failed to elicit a cytotoxic antitumor response, indicating that this Cx43 mechanism is the principal one used in vivo for the generation of antitumor responses. The Cx43-dependent cross-presentation pathway is more effective than standard protocols of DC loading (peptide, tumor lysates, or apoptotic bodies) for generating DC-based tumor vaccines that both inhibit existing tumors and prevent tumor establishment. In conclusion, we exploited an antimicrobial response present in tumor cells to activate cytotoxic CD8 T cells specific for tumor-generated peptides that could directly recognize and kill tumor cells.

Complexity in influenza virus targeted drug design: interaction with human sialidases

With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.

Study of subcellular localization and proteolysis of ataxin-3.

In this work we investigate subcellular localization and proteolytic cleavage of different forms of ataxin-3 (AT-3), the protein responsible for spinocerebellar ataxia type 3. Normal (AT-3Q6 and AT-3Q26) and pathological (AT-3Q72) ataxins-3, as well as two truncated forms lacking poly-Q, were studied. Full-length proteins were also expressed as C14A mutants, in order to assess whether AT-3 autoproteolytic activity was involved in its fragmentation. We found that both normal and pathological proteins localized in the cytoplasm and in the nucleus, as expected, but also in the mitochondria. Microsequencing showed that all ataxins-3 underwent the same proteolytic cleavage, removing the first 27 amino acids. Interestingly, while normal ataxins were further cleaved at a number of caspase sites, pathological AT-3 was proteolyzed to a much lesser extent. This may play a role in the pathogenesis, hampering degradation of aggregation-prone expanded AT-3. In addition, autolytic cleavage was apparently not involved in AT-3 proteolysis.

Glycolipids and Benzylammonium Lipids as Novel Antisepsis Agents: Synthesis and Biological Characterization

New glycolipids and a benzylammonium lipid were rationally designed by varying the chemical structure of a D-glucose-derived hit compound active as lipid A antagonist. We report the synthesis of these compounds, their in vitro activity as lipid A antagonists on HEK cells, and the capacity to inhibit LPS-induced septic shock in vivo. The lack of toxicity and the good in vivo activity suggest the use of some compounds of the panel as hits for antisepsis drug development.

Human sialidase NEU4 long and short are extrinsic proteins bound to outer mitochondrial membrane and the endoplasmic reticulum, respectively

Sialidases are widely distributed glycohydrolytic enzymes removing sialic acid residues from glycoconjugates. In mammals, several sialidases with different subcellular localizations and biochemical features have been described. NEU4, the most recently identified member of the human sialidase family, is found in two forms, NEU4 long and NEU4 short, differing in the presence of a 12-amino-acid sequence at the N-terminus. Contradictory data are present in the literature about the subcellular distribution of these enzymes, their membrane anchoring mechanism being still unclear. In this work, we investigate the human NEU4 long and NEU4 short membrane anchoring mechanism and their subcellular localization. Protein extraction with Triton X-114 and sodium carbonate and cross-linking experiments demonstrate that both forms of NEU4 are extrinsic membrane proteins, anchored via protein-protein interactions. Moreover, through confocal microscopy and subcellular fractionation, we show that the long form localizes in mitochondria, while the short form is also associated with the endoplasmic reticulum. Finally, mitochondria subfractionation experiments suggest that NEU4 long is bound to the outer mitochondrial membrane.

Synthesis and biological evaluation of 1,4-diaryl-2-azetidinones as specific anticancer agents: activation of adenosine monophosphate-activated protein kinase and induction of apoptosis

A series of novel 1,4-diaryl-2-azetidinones were synthesized and evaluated for antiproliferative activity, cell cycle effects, and apoptosis induction. Strong cytotoxicity was observed with the best compounds (±)-trans-20, (±)-trans-21, and enantiomers (+)-trans-20 and (+)-trans-21, which exhibited IC(50) values of 3-13 nM against duodenal adenocarcinoma cells. They induced inhibition of tubulin polymerization and subsequent G2/M arrest. This effect was accompanied by activation of AMP-activated protein kinase (AMPK), activation of caspase-3, and induction of apoptosis. Additionally, the most potent compounds displayed antiproliferative activity against different colon cancer cell lines, opening the route to a new class of potential therapeutic agents against colon cancer.

Extreme heat- and pressure-resistant 7-kDa protein P2 from the archaeon Sulfolobus solfataricus is dramatically destabilized by a single-point amino acid substitution.

This study reports the characterization of the recombinant 7‐kDa protein P2 from Sulfolobus solfataricus and the mutants F31A and F31Y with respect to temperature and pressure stability. As observed in the NMR, FTIR, and CD spectra, wild‐type protein and mutants showed substantially similar structures under ambient conditions. However, midpoint transition temperatures of the denaturation process were 361, 334, and 347 K for wild type, F31A, and F31Y mutants, respectively: thus, alanine substitution of phenylalanine destabilized the protein by as much as 27 K. Midpoint transition pressures for wild type and F31Y mutant could not be accurately determined because they lay either beyond (wild type) or close to (F31Y) 14 kbar, a pressure at which water undergoes a phase transition. However, a midpoint transition pressure of 4 kbar could be determined for the F31A mutant, implying a shift in transition of at least 10 kbar. The pressure‐induced denaturation was fully reversible; in contrast, thermal denaturation of wild type and mutants was only partially reversible. To our knowledge, both the pressure resistance of protein P2 and the dramatic pressure and temperature destabilization of the F31A mutant are unprecedented. These properties may be largely accounted for by the role of an aromatic cluster where Phe31 is found at the core, because interactions among aromatics are believed to be almost pressure insensitive; furthermore, the alanine substitution of phenylalanine should create a cavity with increased compressibility and flexibility, which also involves an impaired pressure and temperature resistance. Proteins 29:381–390, 1997.

Casuarine-6-O-α-D-glucoside and its analogues are tight binding inhibitors of insect and bacterial trehalases

Two novel casuarine-6-alpha-D-glucoside analogues, as well as the parent compound, were synthesized and tested as inhibitors towards Chironomus riparius, mammalian pig kidney and Escherichia coli trehalases. Their potent and selective activity is promising for the development of new insecticides.

The powerful high pressure tool for protein conformational studies

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2 degrees, 3 degrees and 4 degrees structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.