Remo Perozzo

Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis.

Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis — a finding with potential importance for clinical anticancer therapies.

Thermodynamics of protein-ligand interactions: history, presence, and future aspects.

The understanding of molecular recognition processes of small ligands and biological macromolecules requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures involved. A quantitative description of the forces that govern molecular associations requires determination of changes of all thermodynamic parameters, including free energy of binding (ΔG), enthalpy (ΔH), and entropy (ΔS) of binding and the heat capacity change (ΔCp). A close insight into the binding process is of significant and practical interest, since it provides the fundamental know-how for development of structure-based molecular design strategies. The only direct method to measure the heat change during complex formation at constant temperature is provided by isothermal titration calorimetry (ITC). With this method one binding partner is titrated into a solution containing the interaction partner, thereby generating or absorbing heat. This heat is the direct observable that can be quantified by the calorimeter. The use of ITC has been limited due to the lack of sensitivity, but recent developments in instrument design permit to measure heat effects generated by nanomol (typically 10–100) amounts of reactants. ITC has emerged as the primary tool for characterizing interactions in terms of thermodynamic parameters. Because heat changes occur in almost all chemical and biochemical processes, ITC can be used for numerous applications, e.g., binding studies of antibody–antigen, protein–peptide, protein–protein, enzyme–inhibitor or enzyme–substrate, carbohydrate–protein, DNA–protein (and many more) interactions as well as enzyme kinetics. Under appropriate conditions data analysis from a single experiment yields ΔH, KB, the stoichiometry (n), ΔG and ΔS of binding. Moreover, ITC experiments performed at different temperatures yield the heat capacity change (ΔCp). The informational content of thermodynamic data is large, and it has been shown that it plays an important role in the elucidation of binding mechanisms and, through the link to structural data, also in rational drug design. In this review we will present a comprehensive overview to ITC by giving some historical background to calorimetry, outline some critical experimental and data analysis aspects, discuss the latest developments, and give three recent examples of studies published with respect to macromolecule–ligand interactions that have utilized ITC technology.

Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation

In the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. We describe a new proapoptotic pathway in which cathepsin D directly activates caspase-8. Cathepsin D is released from azurophilic granules in neutrophils in a caspase-independent but reactive oxygen species–dependent manner. Under inflammatory conditions, the translocation of cathepsin D in the cytosol is blocked. Pharmacological or genetic inhibition of cathepsin D resulted in delayed caspase activation and reduced neutrophil apoptosis. Cathepsin D deficiency or lack of its translocation in the cytosol prolongs innate immune responses in experimental bacterial infection and in septic shock. Thus, we identified a new function of azurophilic granules that is in addition to their role in bacterial defense mechanisms: to regulate the life span of neutrophils and, therefore, the duration of innate immune responses through the release of cathepsin D.

Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD+, and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Cαs, 0.30 Å). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.

Single-molecule pulling simulations can discern active from inactive enzyme inhibitors.

Understanding ligand-protein recognition and interaction processes is of primary importance for structure-based drug design. Traditionally, several approaches combining docking and molecular dynamics (MD) simulations have been exploited to investigate the physicochemical properties of complexes of pharmaceutical interest. Even if the geometric properties of a modeled protein-ligand complex can be well predicted by computational methods, it is challenging to rank a series of analogues in a consistent fashion with biological data. In the unique beta-hydroxyacyl-ACP dehydratase of Plasmodium falciparum (PfFabZ), the application of standard molecular docking and MD simulations was partially sufficient to shed light on the activity of previously discovered inhibitors. Complementing docking results with atomistic simulations in the steered molecular dynamics (SMD) framework, we devised an in silico approach to study molecular interactions and to compare the binding characteristics of ligand analogues. We hypothesized an interaction model that both explained the biological activity of known ligands, and provided insight into designing novel enzyme inhibitors. Mimicking single-molecule pulling experiments, we used SMD-derived force profiles to discern active from inactive compounds for the first time. A new compound was designed and its biological activity toward the PfFabZ enzyme predicted. Finally, the computational predictions were experimentally confirmed, highlighting the robustness of the drug design approach presented herein.

The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor

The two‐component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane‐bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE–CitAC) composed of the maltose‐binding protein and the CitA kinase domain (amino acids 327–547) showed constitutive autokinase activity and transferred the γ‐phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H‐350 and D‐56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB–D56N formed a stable complex with MalE–CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45–176) was overproduced as a soluble, cytoplasmic protein with a C‐terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri‐ and dicarboxylates tested, with high affinity (KD ≈ 5 μM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (ΔH = −76.3 kJ mol−1), whereas the entropy change was opposed (−TΔS = + 46.3 kJ mol−1). The pH dependency of the binding reaction indicated that the dianionic form H‐citrate2− is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg–citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.

Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

Background and Methodology Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. Principal Findings A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. Conclusions and Significance Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Substrate Diversity of Herpes Simplex Virus Thymidine Kinase IMPACT OF THE KINEMATICS OF THE ENZYME

Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches.

NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action

BACKGROUND Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood.

Nucleoside Binding Site of Herpes Simplex Type 1 Thymidine Kinase Analyzed by X-Ray Crystallography

The crystal structures of the full‐length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 Å resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme‐prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 Å resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water‐mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus‐directed enzyme‐prodrug gene therapy by enzyme engineering. Proteins 2000;41:545–553.