Development of affinity beads-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione

For a better understanding of metal-ligand interaction and its function in cells, we developed an easy, sensitive, and high-throughput method to quantify ligand-metal(loid) binding affinity under physiological conditions, by combining ligand-attached affinity beads and inductively coupled plasma-optical emission spectrometry (ICP-OES). Glutathione (GSH) and two phytochelatins (PC2 and PC3, small peptides with different thiol numbers) were employed as model ligands and attached to affinity beads. The principal of the assay resembles that of affinity purification of proteins in biochemistry: metals binding to the ligand on the beads and the rest in the buffer are separated by a spin-column and quantified by ICP-OES. The binding assay using the GSH-attached beads and various metal(loid)s suggested that the assay can detect the different affinity of the metal-ligand interactions, in accordance with the order of the Irving–Williams series and the reported stability constants. The binding assay using PC2 or PC3-attached beads suggested positive binding between PCs and nickel, copper, zinc, cadmium, and arsenite ions in a thiol-dependent manner. We then conducted the competition assay using cadmium, manganese, iron, copper, and zinc and the results suggested a better binding affinity of PC2 with cadmium than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and cadmium compared to GSH and cadmium. These results suggested the dominance of PC-Cd complex formation in vitro, supporting the physiological importance of PCs for the detoxification of cadmium in vivo. We also discuss the further potential application of the assay.

Proteomic analysis of haem-binding protein from Arabidopsis thaliana and Cyanidioschyzon merolae

Chloroplast biogenesis involves the coordinated expression of the plastid and nuclear genomes, requiring information to be sent from the nucleus to the developing chloroplasts and vice versa. Although it is well known how the nucleus controls chloroplast development, it is still poorly understood how the plastid communicates with the nucleus. Currently, haem is proposed as a plastid-to-nucleus (retrograde) signal that is involved in various physiological regulations, such as photosynthesis-associated nuclear genes expression and cell cycle in plants and algae. However, components that transduce haem-dependent signalling are still unidentified. In this study, by using haem-immobilized high-performance affinity beads, we performed proteomic analysis of haem-binding proteins from Arabidopsis thaliana and Cyanidioschyzon merolae . Most of the identified proteins were non-canonical haemoproteins localized in various organelles. Interestingly, half of the identified proteins were nucleus proteins, some of them have a similar function or localization in either or both organisms. Following biochemical analysis of selective proteins demonstrated haem binding. This study firstly demonstrates that nucleus proteins in plant and algae show haem-binding properties. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

Ubiquitination of renal ENaC subunits in vivo

Ubiquitination of the epithelial Na+ channel (ENaC) in epithelial cells may influence trafficking and hormonal regulation of the channels. We assessed ENaC ubiquitination (ub-ENaC) in mouse and rat kidneys using affinity beads to capture ubiquitinated proteins from tissue homogenates and Western blot analysis with anti-ENaC antibodies. Ub-αENaC was observed primarily as a series of proteins of apparent molecular mass of 40–70 kDa, consistent with the addition of variable numbers of ubiquitin molecules primarily to the NH2-terminal cleaved fragment (~30 kDa) of the subunit. No significant Ub-βENaC was detected, indicating that ubiquitination of this subunit is minimal. For γENaC, the protein eluted from the affinity beads had the same apparent molecular mass as the cleaved COOH-terminal fragment of the subunit (~65 kDa). This suggests that the ubiquitinated NH2 terminus remains attached to the COOH-terminal moiety during isolation through disulfide bonds. Consistent with this, under nonreducing conditions, eluates contained material with increased molecular mass (90–150 kDa). In mice with a Liddle syndrome mutation (β566X) deleting a putative binding site for the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2, the amount of ub-γENaC was reduced as expected. To assess aldosterone dependence of ubiquitination, we fed rats either control or low-Na+ diets for 7 days before kidney harvest. Na+ depletion increased the amounts of ub-αENaC and ub-γENaC by three- to fivefold, probably reflecting increased amounts of fully cleaved ENaC. We conclude that ubiquitination occurs after complete proteolytic processing of the subunits, contributing to retrieval and/or disposal of channels expressed at the cell surface. Diminished ubiquitination does not appear to be a major factor in aldosterone-dependent ENaC upregulation.

One-step artificial antigen presenting cell-based vaccines induce potent effector CD8 T cell responses

The production and wide use of artificial antigen presenting cells (aAPCs) in the clinic as cancer immunotherapeutics are hindered by the need of identifying immunogenic cancer antigens and production of recombinant patient-specific major histocompatibility complexes (MHC) loaded with these peptides. To overcome these limitations, in this study, we tested the idea of whether peptide-MHCs can directly be captured from cell lysates, including cancer cells using affinity beads, and used to initiate T cell responses. In theory, these affinity beads covered with the unknown peptide-MHC repertoire captured from the cancer cells could interact with a wide range of antigen-specific T cells and promote anti-cancer responses. Indeed, we found that we can successfully pull-down peptide-MHCs from cell lysates and the aAPCs generated using this technique were able to induce antigen-specific cytotoxic effector T cell responses that led to in vitro and in vivo tumor cell killing. In summary, we present here a novel technique to generate patient-specific aAPCs, that might have the potential to revolutionize the field of cancer vaccines, and provide patients with a vaccine in matters of days at minimal costs.

Catalyst-proximity protein chemical labelling on affinity beads targeting endogenous lectins

Catalyst-proximity labelling on affinity beads enables the identification of ligand-binding proteins such as lectins, which cannot be analyzed by conventional techniques. 1-Methyl-4-arylurazole (MAUra) efficiently labels proteins bound to the beads.