John M. Tomich

A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers

Subunit IV of yeast cytochrome oxidase is made in the cytoplasm with a transient pre‐sequence of 25 amino acids which is removed upon import of the protein into mitochondria. To study the function of this cleavable pre‐sequence in mitochondrial protein import, three peptides representing 15, 25 or 33 amino‐terminal residues of the subunit IV precursor were chemically synthesized. All three peptides were freely soluble in aqueous buffers, yet inserted spontaneously from an aqueous subphase into phospholipid monolayers up to an extrapolated limiting monolayer pressure of 40‐50 mN/m. The two longer peptides also caused disruption of unilamellar liposomes. This effect was increased by a diffusion potential, negative inside the liposomes, and decreased by a diffusion potential of opposite polarity. The peptides, particularly the two longer ones, also uncoupled respiratory control of isolated yeast mitochondria. The 25‐residue peptide had little secondary structure in aqueous buffer but became partly alpha‐helical in the presence of detergent micelles. Based on the amino acid sequence of the peptides, a helical structure would have a highly asymmetric distribution of charged and apolar residues and would be surface active. Amphiphilic helicity appears to be a general feature of mitochondrial pre‐sequences. We suggest that this feature plays a crucial role in transporting proteins into mitochondria.

Amphiphilicity is essential for mitochondrial presequence function.

We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non‐amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this ‘non‐amphiphilic’ presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non‐amphiphilic. One of the active presequence peptides was non‐helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.

A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis

Extracellular DNA (eDNA), a by‐product of cell lysis, was recently established as a critical structural component of the Enterococcus faecalis biofilm matrix. Here, we describe fratricide as the governing principle behind gelatinase (GelE)‐mediated cell death and eDNA release. GFP reporter assays confirmed that GBAP (gelatinase biosynthesis‐activating pheromone) quorum non‐responders (GelE–SprE–) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE+SprE+). The killing action is dependent on GelE, and the GelE producer population is protected from self‐destruction by the co‐production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, we address a mechanism by which GelE and SprE may modify the cell wall affinity of proteolytically processed AtlA resulting in either a pro‐ or anti‐lytic outcome.

Identification of an ion channel-forming motif in the primary structure of tetanus and botulinum neurotoxins

Synthetic peptides with amino acid sequences corresponding to predicted transmembrane segments of tetanus toxin were used as probes to identify a channel‐forming motif. A peptide denoted TeTx II, with sequence GVVLLLEYIPEITLPVIAALSIA, forms cation‐selective channels when reconstituted in planar lipid bilayers. The single channel conductance in 0.5 M NaCl or KCl is 28 ± 3 and 24 ± 2 pS, respectively. In contrast, a peptide with sequence NFIGALETTGVVLLLEYIPEIT, denoted as TeTx I. or a peptide with the same amino acid composition as TeTx II but with a randomized sequence, do not form channels. Conformational energy calculations show that a bundle of four amphipathic α‐helices is a plausible structural motif underlying observable pore properties. The identified functional module may account for the channel‐forming activity of both tetanus toxin and the homologous botulinum toxin A.

MEMBRANE TOPOGRAPHY AND NEAR-NEIGHBOR RELATIONSHIPS OF THE MITOCHONDRIAL ATP SYNTHASE SUBUNITS E, F, AND G

The well characterized subunits of the bovine ATP synthase complex are the α, β, γ, δ, and ϵ subunits of the catalytic sector, F1; the ATPase inhibitor protein; and subunits a, b, c, and d, OSCP (oligomycin sensitivity-conferring protein), F6, and A6L, which are present in the membrane sector, F0, and the 45-Å-long stalk that connects F1 to F0. It has been shown recently that bovine ATP synthase preparations also contain three small polypeptides, designated e, f, and g, with respective molecular masses of 8.2, 10.2, and 11.3 kDa. To ascertain their involvement as bona fide subunits of the ATP synthase and to investigate their membrane topography and proximity to the above ATP synthase subunits, polyclonal antipeptide antibodies were raised in the rabbit to the COOH-terminal amino acid residues 57-70 of e, 75-86 of f, and 91-102 of g. It was shown that (i) e, f, and g could be immunoprecipitated with anti-OSCP IgG from a fraction of bovine submitochondrial particles enriched in oligomycin-sensitive ATPase; (ii) the NH2 termini of f and g are exposed on the matrix side of the mitochondrial inner membrane and can be curtailed by proteolysis; (iii) the COOH termini of all three polypeptides are exposed on the cytosolic side of the inner membrane; and (iv) f cross-links to A6L and to g, and e cross-links to g and appears to form an e-e dimer. Thus, the bovine ATP synthase complex appears to have 16 unlike subunits, twice as many as its counterpart in Escherichia coli.

Aqueous Solubilization of Transmembrane Peptide Sequences with Retention of Membrane Insertion and Function

We recently reported that the peptide C-K4-M2GlyR mimics the action of chloride channels when incorporated into the apical membrane of cultured renal epithelial monolayers. C-K4-M2GlyR is one of a series of peptides that were prepared by the addition of lysine residues to the N- or C-terminus of the M2 transmembrane sequence of the brain glycine receptor. This study addresses how such modifications affect physical properties such as aqueous solubility, aggregation, and secondary structure, as well as the ability of the modified peptides to form channels in epithelial monolayers. A graded improvement in solubility with a concomitant decrease in aggregation in aqueous media was observed for the M2GlyR transmembrane sequences. Increases in short-circuit current (I(SC)) of epithelial monolayers were observed after treatment with some but not all of the peptides. The bioactivity was higher for the more soluble, less aggregated M2GlyR peptides. As described in our previous communication, sensitivity of channel activity to diphenylamine-2-carboxylate, a chloride channel blocker, and bumetanide, an inhibitor of the Na/K/2Cl cotransporter, was used to assess changes in chloride selectivity for the different assembled channel-forming peptides. The unmodified M2GlyR sequence and the modified peptides with less positive charge are more sensitive to these agents than are the more highly charged forms. This study shows that relatively insoluble transmembrane sequences can be modified such that they are easier to purify and deliver in the absence of organic solvents with retention of membrane association, insertion, and assembly.

Tribolium castaneum Larval Gut Transcriptome and Proteome: A Resource for the Study of the Coleopteran Gut

Tribolium castaneum is an important agricultural pest and an advanced genetic model for coleopteran insects. We have taken advantage of the recently acquired T. castaneum genome to identify T. castaneum genes and proteins in one of the more critical environmental interfaces of the insect, the larval alimentary tract. Genetic transcripts isolated from the T. castaneum larval gut were labeled and hybridized to a custom array containing oligonucleotides from predicted genes in the T. castaneum genome. Through a ranking procedure based on relative labeling intensity, we found that approximately 17.6% of the genes represented in the array were predicted to be highly expressed in gut tissue. Several genes were selected to compare relative expression levels in larval gut, head, or carcass tissues using quantitative real-time PCR, and expression levels were, with few exceptions, consistent with the gut rankings. In parallel with the microarrays, proteins extracted from the T. castaneum larval gut were subjected to proteomic analysis. Two-dimensional electrophoretic analysis combined with MALDI-TOF resulted in the identification of 37 of 88 selected protein samples. As an alternative strategy, one-dimensional electrophoretic separation of T. castaneum larval gut proteins followed by two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of 98 proteins. A comparison of the proteomic studies indicated that 16 proteins were commonly identified in both, whereas 80 proteins from the proteomic analyses corresponded to genes with gut rankings indicative of high expression in the microarray analysis. These data serve as a resource of T. castaneum transcripts and proteins in the larval gut and provide the basis for comparative transcriptomic and proteomic studies related to the gut of coleopteran insects.

Role of anthocyanin‐enriched purple‐fleshed sweet potato p40 in colorectal cancer prevention

SCOPE Anthocyanins, the natural pigments in plant foods, have been associated with cancer prevention. However, the content of anthocyanins in staple foods is typically low and the mechanisms by which they exert anticancer activity is not yet fully defined. METHODS AND RESULTS We selected an anthocyanin-enriched purple-fleshed sweet potato clone, P40, and investigated its potential anticancer effect in both in vitro cell culture and in vivo animal model. In addition to a high level of total phenolics and antioxidant capacity, P40 possesses a high content of anthocyanins at 7.5 mg/g dry matter. Treatment of human colonic SW480 cancer cells with P40 anthocyanin extracts at 0-40 μM of peonidin-3-glucoside equivalent resulted in a dose-dependent decrease in cell number due to cytostatic arrest of cell cycle at G1 phase but not cytotoxicity. Furthermore, dietary P40 at 10-30% significantly suppressed azoxymethane-induced formation of aberrant crypt foci in the colons of CF-1 mice in conjunction with, at least in part, a lesser proliferative PCNA and a greater apoptotic caspase-3 expression in the colon mucosal epithelial cells. CONCLUSION These observations, coupled with both in vitro and in vivo studies reported here, suggest anthocyanin-enriched sweet potato P40 may protect against colorectal cancer by inducing cell-cycle arrest, antiproliferative, and apoptotic mechanisms.

Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor

Nicotinic cholinergic receptors are membrane proteins composed of five subunits organized around a central aqueous pore. A pentameric channel protein, T5M2δ, that emulates the presumed pore‐forming structure of this receptor was generated by assembling five helix‐forming peptide modules at the lysine ϵ‐amino groups of the 11‐residue template [K∗AK∗KK∗PGK∗EK∗G], where ∗ indicates attachment sites. Helical modules represent the sequence of the M2 segment of the Torpedo californica acetylcholine receptor (AChR) δ subunit; M2 segments are considered involved in pore‐lining. Purified T5M2δ migrates in SDS‐PAGE with an apparent M r~14,000, concordant with a protein of 126 residues. T5M2δ forms cation‐selective channels when reconstituted in planar lipid bilayers. The single channel conductance in symmetric 0.5 M K.C1 is 40 pS. This value approximates the 45 pS single channel conductance characteristic of authentic purified Torpedo AChR, recorded under otherwise identical conditions. These results, together with conformational energy calculations, support the notion that a bundle of five amphipathic a‐helices is a plausible structural motif underlying the inner bundle that forms the pore of the pentameric AChR channel.

A synthetic channel-forming peptide induces Cl(-) secretion: modulation by Ca(2+)-dependent K(+) channels.

A synthetic Cl(-) channel-forming peptide, C-K4-M2GlyR, applied to the apical membrane of human epithelial cell monolayers induces transepithelial Cl(-) and fluid secretion. The sequence of the core peptide, M2GlyR, corresponds to the second membrane-spanning region of the glycine receptor, a domain thought to line the pore of the ligand-gated Cl(-) channel. Using a pharmacological approach, we show that the flux of Cl(-) through the artificial Cl(-) channel can be regulated by modulating basolateral K(+) efflux through Ca(2+)-dependent K(+) channels. Application of C-K4-M2GlyR to the apical surface of monolayers composed of human colonic cells of the T84 cell line generated a sustained increase in short-circuit current (I(SC)) and caused net fluid secretion. The current was inhibited by the application of clotrimazole, a non-specific inhibitor of K(+) channels, and charybdotoxin, a potent inhibitor of Ca(2+)-dependent K(+) channels. Direct activation of these channels with 1-ethyl-2-benzimidazolinone (1-EBIO) greatly amplified the Cl(-) secretory current induced by C-K4-M2GlyR. The effect of the combination of C-K4-M2GlyR and 1-EBIO on I(SC) was significantly greater than the sum of the individual effects of the two compounds and was independent of cAMP. Treatment with 1-EBIO also increased the magnitude of fluid secretion induced by the peptide. The cooperative action of C-K4-M2GlyR and 1-EBIO on I(SC) was attenuated by Cl(-) transport inhibitors, by removing Cl(-) from the bathing solution and by basolateral treatment with K(+) channel blockers. These results indicate that apical membrane insertion of Cl(-) channel-forming peptides such as C-K4-M2GlyR and direct activation of basolateral K(+) channels with benzimidazolones may coordinate the apical Cl(-) conductance and the basolateral K(+) conductance, thereby providing a pharmacological approach to modulating Cl(-) and fluid secretion by human epithelia deficient in cystic fibrosis transmembrane conductance regulator Cl(-) channels.