Human sorCS1 binds sortilin and hampers its cellular functions.

Sortilin and sorCS1 [sortilin-related Vps10p (vacuolar protein sorting/targeting protein 10) domain-containing receptor 1], both members of the Vps10p-D (Vps10p-domain) receptor family, are synthesized as precursor proteins and are converted into their mature form by enzymatic cleavage of a short N-terminal propeptide. SorCS1 does not bind its propeptide, but sortilin is able to bind not just its own propeptide, but also that of sorCS1. In the present study we show that the propeptide region of sorCS1 contains two separate sites for binding to sortilin and that only one of these sites is removed from human (as opposed to mouse) sorCS1 during processing. This leaves mature human sorCS1 with a sortilin-binding N-terminus, which allows formation of a complex between the two receptors in solution and on cell membranes. Furthermore, we find that the interaction with sorCS1 has a pronounced effect on sortilin's ability to mediate the cellular uptake of alternative ligands, and to hamper its facilitation of CNTF (ciliary neutrophic factor) signalling and the induction of phosphorylated STAT3 (signal transducer and activator of transcription 3). Thus the present study reveals a novel regulatory mechanism and suggest an entirely new role for sorCS1 as a modulator of sortilin function.

A suicidal and extensively disordered luciferase with a bright luminescence.

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

Hyperactivity of the Ero1α oxidase elicits endoplasmic reticulum stress but no broad antioxidant response.

Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.

Role of dairy proteins in the reduction of capsaicin-induced oral burning pain.

Capsaicin-induced burning sensation in the oral mucosa can be relieved by skimmed and whole milk. The mechanism behind this effect, however, is unknown. This study aimed to asses the role of milk proteins in reducing capsaicin-induced oral burning sensation. 24 healthy participants were included in this single-blinded cross-over study consisting of four sessions. In each sessions, mucosal burning sensation was evoked by having the participants dip their tongues in a cup containing 0.1% capsaicin gel for a total of 8 min. The perceived levels of unpleasantness and burning intensity were scored on two different numerical rating scales. After capsaicin exposure, the participants rinsed their mouth for 10 s with a different solution in each session (3.5% casein, 3.5% whey, 3.5% lactose (non-protein control) and skimmed milk (active/positive control)). Mechanical (64, 128 and 256 mN pinprick) and thermal (5, 40, 45 and 50 ̊C) sensitivity of the tongue was measured using semi-quantative sensory testing at baseline, immediately after capsaicin exposure and when the scores for unpleasantness and burning intensity reached the minimum value of 0. Thermographic images of the tongue were taken at the same time-points. Overall, no statistically significant difference in unpleasantness and burning intensity was found between the four sessions (P ≥ 0.070). Explorative pair-wise comparisons, however, showed slight short-term reduction in unpleasantness and burning intensity when comparing the casein solution with the lactose solution (P ≤ 0.020). Scores for burning intensity and unpleasantness varied over time (P ≤ 0.001). Statistically significant changes in heat and mechanical sensitivity was observed between time-points (P < 0.001) but not sessions (P ≥ 0.410). An increased sensitivity towards heat and a decreased sensitivity towards mechanical stimuli was observed after capsaicin exposure compared with baseline (P < 0.001). Similarly, changes in thermographic temperature of the tongue was observed between time-points (P < 0.001), but not sessions (P ≥ 0.827). An increased maximum, minimum and average temperature of the tongue was observed immediately after capsaicin exposure compared with baseline (P < 0.001). In conclusion, short-term rinsing with room temperature milk proteins did not robustly alter capsaicin-induced oral burning sensation, unpleasantness, somatosensory changes or tongue temperature compared with control. Further studies exploring the effects of increased rinsing time and concentrations are needed in the future.

Prevention of Initial Bacterial Attachment by Osteopontin and Other Bioactive Milk Proteins.

A considerable body of work has studied the involvement of osteopontin (OPN) in human physiology and pathology, but comparably little is known about the interaction of OPN with prokaryotic cells. Recently, bovine milk OPN has been proposed as a therapeutic agent to prevent the build-up of dental biofilms, which are responsible for the development of caries lesions. Bioactive milk proteins are among the most exciting resources for caries control, as they hamper bacterial attachment to teeth without affecting microbial homeostasis in the mouth. The present work investigated the ability of OPN to prevent the adhesion of three dental biofilm-forming bacteria to saliva-coated surfaces under shear-controlled flow conditions in comparison with the major milk proteins α-lactalbumin, ß-lactoglobulin, αs1-casein, ß-casein and κ-casein, as well as crude milk protein. OPN was the most effective single protein to reduce the adhesion of Actinomyces naeslundii, Lactobacillus paracasei subsp. paracasei and Streptococcus mitis. ß-casein and crude milk protein also had a pronounced effect on all three species, which suggests binding to different microbial surface structures rather than the blocking of a specific bacterial adhesin. Bioactive milk proteins show potential to delay harmful biofilm formation on teeth and hence the onset of biofilm-related oral disease.

DisCoTune: versatile auxiliary plasmids for the production of disulphide-containing proteins and peptides in the E. coli T7 system.

Secreted proteins and peptides hold large potential both as therapeutics and as enzyme catalysts in biotechnology. The high stability of many secreted proteins helps maintain functional integrity in changing chemical environments and is a contributing factor to their commercial potential. Disulphide bonds constitute an important post-translational modification that stabilizes many of these proteins and thus preserves the active state under chemically stressful conditions. Despite their importance, the discovery and applications within this group of proteins and peptides are limited by the availability of synthetic biology tools and heterologous production systems that allow for efficient formation of disulphide bonds. Here, we refine the design of two DisCoTune (Disulphide bond formation in E. coli with tunable expression) plasmids that enable the formation of disulphides in the highly popular Escherichia coli T7 protein production system. We show that this new system promotes significantly higher yield and activity of an industrial protease and a conotoxin, which belongs to a group of disulphide-rich venom peptides from cone snails with strong potential as research tools and pharmacological agents.