Use of Membrane Technology for Concentration of Bioactive Peptides From Geoduck (Panopea Zelandica)

Enzymatic digestion of proteins generates biologically active peptides which can be concentrated by filtration with the appropriate membranes. This work uses a hydrolysate derived from the New Zealand geoduck Panopea zelandica and compares the effectiveness of membranes with varying molecular weight cut-offs (MWCOs) and surface charges for carrying out selective separations of the peptides. A membrane with a MWCO of 2.5 kDa was found to be most effective for obtaining the greatest differences between the permeate and retentate from the membranes when separating by size alone. For separating peptides based on charge the choice of membrane and pH was important. The results obtained suggested that each hydrolysate has different optimum conditions for achieving separation. A challenge when analysing the effects of membranes on peptide mixtures is quantifying the changes that have occurred. The work presented here introduced a multivariate approach to interpretation of complete sets of HPLC data that had not previously been used for analysis of complex peptide mixtures. This approach was used to measure the effectiveness of the membrane separations, and allowed comparisons to be drawn between these separations and the peptide bioactivity as determined using the FRAP assay

Detailed Distribution of Lipids in Greenshell™ Mussel (Perna canaliculus)

Greenshell™ mussels (GSM–Perna canaliculus) are a source of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA). Farmed GSM are considered to be a sustainable source of LC-PUFA as they require no dietary inputs, gaining all of their oil by filter-feeding microorganisms from sea water. GSM oil is a high-value product, with a value as much as 1000 times that of fish oils. GSM oil has important health benefits, for example, anti-inflammatory activity. It also contains several minor lipid components that are not present in most fish oil products, and that have their own beneficial effects on human health. We have shown the lipid content of the female GSM (1.9 g/100 g ww) was significantly greater than that of the male (1.4 g/100 g ww). Compared with male GSM, female GSM contained more n-3 LC-PUFA, and stored a greater proportion of total lipid in the gonad and mantle. The higher lipid content in the female than the male GSM is most likely related to gamete production. This information will be useful to optimize extraction of oils from GSM, a local and sustainable source of n-3 LC-PUFA

Smac Mimetics Activate the E3 Ligase Activity of cIAP1 Protein by Promoting RING Domain Dimerization*

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways

Birinapant, a Smac-Mimetic with Improved Tolerability for the Treatment of Solid Tumors and Hematological Malignancies

Birinapant (<b>1</b>) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that <b>1</b> stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic <b>1</b>-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound <b>A</b> (<b>2</b>) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and <b>2</b> mimicked features of triple IAP knockout cells in vitro. The improved tolerability of <b>1</b> was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P₂′ position of <b>1</b> was critical to this differential activity, and this improved tolerability has allowed <b>1</b> to proceed into clinical studies