Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors. Our results suggest that these novel peptides may provide an avenue to deliver diagnostic and therapeutic reagents directly to TRPV6-rich tumors and, as such, have potential applications for a range of carcinomas including ovarian, breast, thyroid, prostate and colon, as well as certain leukemia's and lymphomas.

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly 76 ) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly 76 to Cys 76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.The ubiquitin-dependent proteolysis pathway controls the removal of damaged and misfolded proteins in the cell. One of the key steps in this pathway is the assembly of a polyubiquitin chain that ultimately targets a substrate for degradation (1, 2). This process involves tagging of a substrate protein with an arrangement of four to eight ubiquitin molecules linked in series through the C-terminal Gly 76 of one ubiquitin molecule and the side-chain ⑀-NH 2 from Lys 48 of another (3). Once tagged, the polyubiquitinated protein is recognized by the 26 S proteasome, where it is degraded. The building of a polyubiquitin chain and subsequent labeling of a substrate protein is a complex process potentially involving the passage of ubiquitin through three different enzymes (1, 4, 5). Initially, ubiquitin is activated in an ATP-dependent step forming a high energy E1 1 -ubiquitin thioester complex. Ubiquitin is then transferred to an E2 or ubiquitin-conjugating enzyme forming a thioester intermediate. E2 proteins have been demonstrated recently to bind to the ubiquitin-like domain of the E1 providing insight into the mechanism in which the thioester-bound ubiquitin is passed to the E2 (6 -8). Two different E3 ligase proteins can catalyze the final passage of ubiquitin to the substrate. For RING E3 ligases, ubiquitin or a polyubiquitin chain is transferred directly from the E2 to the substrate, whereas HECT E3 ligases form a...

In this study, we constructed and evaluated a target-specific, salt-resistant antimicrobial peptide (AMP) that selectively targeted Streptococcus mutans, a leading cariogenic pathogen. The rationale for creating such a peptide was based on the addition of a targeting domain of S. mutans ComC signaling peptide pheromone (CSP) to a killing domain consisting of a portion of the marine-derived, broad-spectrum AMP pleurocidin to generate a target-specific AMP. Here, we report the results of our assessment of such fusion peptides against S. mutans and two closely related species. The results showed that nearly 95% of S. mutans cells lost viability following exposure to fusion peptide IMB-2 (5.65 M) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii cells were killed following the same exposure. Similar results were also observed in dual-species mixed cultures of S. mutans with S. sanguinis or S. gordonii. The peptide-guided killing was further confirmed in S. mutans biofilms and was shown to be dose dependent. An S. mutans mutant defective in the CSP receptor retained 60% survival following exposure to IMB-2, suggesting that the targeted peptide predominantly bound to the CSP receptor to mediate killing in the wild-type strain. Our work confirmed that IMB-2 retained its activity in the presence of physiological or higher salt concentrations. In particular, the fusion peptide showed a synergistic killing effect on S. mutans with a preventive dose of NaF. In addition, IMB-2 was relatively stable in the presence of saliva containing 1 mM EDTA and did not cause any hemolysis. We also found that replacement of serine-14 by histidine improved its activity at lower pH. Because of its effectiveness, salt resistance, and minimal toxicity to host cells, this novel target-specific peptide shows promise for future development as an anticaries agent.

E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an ␣/␤ fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.Labeling of proteins with the molecule ubiquitin is an important cellular function that is required for protein degradation, cell cycle control, stress response, DNA repair, signal transduction, transcriptional regulation, and vesicular traffic (1-4). In this process, the number and topology of the ubiquitin molecule chains underscores the fate of the substrate and determines the biochemical pathway followed. For example, labeling the substrate with a single ubiquitin (monoubiqutination) is important for cellular regulation (5, 6). On the other hand ubiquitin-dependent proteolysis, the process responsible for the turnover of damaged or misfolded proteins in the cell, involves labeling the substrate protein with a polyubiquitin chain that is subsequently recognized by the 26 S proteasome facilitating substrate degradation. The most common polyubiquitin chains are formed via isopeptide bond linkages between the C-terminal (Gly 76 ) of one ubiquitin molecule and the side chain ⑀-NH 2 from Lys 48 of another. However, other configurations are possible including the Lys 63 linkage, which are important in the postreplicative DNA repair pathway (7).The ubiquitination degradation pathway is described as a cascade of events in which ubiquitin is passed through three enzymes until it reaches a protein selected for degradation. The first step involves an ATP-dependent activation of ubiquitin by an ubiquitin-activating enzyme (E1) 1 forming a high energy E1-ubiquitin thiol ester complex. The activated ubiquitin is then passed from the E1 to an ubiquitin-conjugating enzyme (E2) forming a second thiol ester intermediate between the E2 and ubiquitin. Labeling the target protein with ubiquitin is catalyzed by an E3 ligase protein, either by direct transfer of the ubiquitin to the substrate from the E2 (RING E3) or by thiol ester formation between ubiquitin to an E3 (HECT E...

Metabolic changes in spikelets of wheat varieties FL62R1, Stettler, Muchmore and Sumai3 following Fusarium graminearum infection were explored using NMR analysis. Extensive 1D and 2D 1H NMR measurements provided information for detailed metabolite assignment and quantification leading to possible metabolic markers discriminating resistance level in wheat subtypes. In addition, metabolic changes that are observed in all studied varieties as well as wheat variety specific changes have been determined and discussed. A new method for metabolite quantification from NMR data that automatically aligns spectra of standards and samples prior to quantification using multivariate linear regression optimization of spectra of assigned metabolites to samples’ 1D spectra is described and utilized. Fusarium infection-induced metabolic changes in different wheat varieties are discussed in the context of metabolic network and resistance.