Unusual architecture of the p7 channel from hepatitis C virus.

The hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations. We wanted to examine the structural solution that the viroporin adopts in order to achieve selective cation conduction, because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The activity of p7 can be inhibited by amantadine and rimantadine, which are potent blockers of the influenza M2 channel and licensed drugs against influenza infections. The adamantane derivatives have been used in HCV clinical trials, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here we determine the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbours, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also points to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter, whereas an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording shows that these residues are critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide a molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching.

Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or 'uncoupling' of the proton electrochemical potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1-4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.

A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth.

Tumorigenesis results from events that impinge on a variety of collaborating metabolic pathways. To assess their role in this process, we utilized a cell-based assay to perform a high-throughput, chemical library screen. In so doing, we identified F16, a small molecule that selectively inhibits proliferation of mammary epithelial, neu-overexpressing cells, as well as a variety of mouse mammary tumor and human breast cancer cell lines. F16 belongs to a group of structurally similar molecules with a delocalized positive charge. The compound is accumulated in mitochondria of responsive cells, driven by the membrane potential, and it compromises their functional integrity. Mitochondrial hyperpolarization is a shared feature of many tumor cell lines, explaining the broad action spectrum of this novel delocalized lipophilic cation.

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: competition kinetics as a tool for enzyme specificity investigation.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide-substrate's N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.

A bifunctional targeted peptide that blocks HER-2 tyrosine kinase and disables mitochondrial function in HER-2-positive carcinoma cells.

The HER-2 oncoprotein is commonly overexpressed in a variety of human malignancies and has become an attractive antitumor target. A number of strategies to inhibit the HER-2 receptor tyrosine kinase are currently the focus of intensive preclinical and clinical research. In the present study, we have engineered a bifunctional peptide, BHAP, which consists of two modular domains: a HER-2-targeting/neutralizing domain and a mitochondriotoxic, proapoptotic domain. The chimeric peptide is biologically active and capable of selectively triggering apoptosis of HER-2-overexpressing cancer cells in culture, even those previously described as Herceptin resistant. Furthermore, BHAP slows down growth of HER-2-overexpressing human mammary xenografts established in SCID mice. This approach can be extended to the development of tailored targeted chimeric peptides against a number of overexpressed cellular receptors implicated in the development and progression of cancer.

Fatty acid flippase activity of UCP2 is essential for its proton transport in mitochondria.

Modulation of cellular energy expenditure is fundamental to normal and pathological cell growth and differentiation. Mitochondria stores energy as a proton gradient across their inner membrane. Uncoupling proteins (UCPs) can dissipate the gradient to produce heat or regulate metabolite fluxes. UCP-mediated proton currents require fatty acids (FAs) and are blocked by nucleotides, but the molecular basis of these processes is unknown. We find, by nuclear magnetic resonance and functional mutagenesis, that UCP2 can bind FAs laterally through its peripheral site, and this intramembrane molecular recognition is essential for UCP2-catalyzed FA flipping across the membrane, which in turn is essential for proton translocation. The antagonist GDP binds inside the UCP2 cavity and perturbs its conformation, which can displace FA from the peripheral site as a mean of inhibiting proton currents. Our data provide a biophysical perspective of the intricate interplay of UCPs, FA, and nucleotides in determining proton fluxes in mitochondria.

Survival of the fittest: metabolic adaptations in cancer.

Malignant transformation is often a multistep process characterized by an initial period of avascular growth. Rapid cell proliferation creates areas within the emerging preneoplastic lesion with limited diffusion of oxygen and nutrients. In this context, activation of oncogenes, loss of tumor suppressors as well as additional adaptive mechanisms drive a profound metabolic rewiring to overcome the environmental constraints. The emerging cells are in principle better suited to proliferate and survive in the hostile tumor microenvironment. Furthermore, some of the acquired metabolic traits impact their metastatic behavior and response to therapy. It is becoming increasingly clear that malignant cells are highly dependent on certain nutrients, an Achilles' heel of cancer and an opportunity for therapeutic intervention.