Novel role for linear ubiquitination in regulating NFAT1 stability.

Linear ubiquitination is an important post-translational modification regulating the activation of numerous proinflammatory signalling mediators. Deregulated linear ubiquitination has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. In this issue, Miao et al. have identified a novel role for linear ubiquitination in the stabilisation of the NFAT1 transcription factor, leading to enhanced NFAT1-mediated gene expression, which might have functional implications in patients with Kawasaki disease.

Investigation of the Potential of Selected Food-Derived Antioxidants to Bind and Stabilise the Bioactive Blue Protein C-Phycocyanin from Cyanobacteria Spirulina.

Blue C-phycocyanin (C-PC), the major Spirulina protein with innumerable health-promoting benefits, is an attractive colourant and food supplement. A crucial obstacle to its more extensive use is its relatively low stability. This study aimed to screen various food-derived ligands for their ability to bind and stabilise C-PC, utilising spectroscopic techniques and molecular docking. Among twelve examined ligands, the protein fluorescence quenching revealed that only quercetin, coenzyme Q10 and resveratrol had a moderate affinity to C-PC (Ka of 2.2 to 3.7 × 105 M-1). Docking revealed these three ligands bind more strongly to the C-PC hexamer than the trimer, with the binding sites located at the interface of two (αß)3 trimers. UV/VIS absorption spectroscopy demonstrated the changes in the C-PC absorption spectra in a complex with quercetin and resveratrol compared to the spectra of free protein and ligands. Selected ligands did not affect the secondary structure content, but they induced changes in the tertiary protein structure in the CD study. A fluorescence-based thermal stability assay demonstrated quercetin and coenzyme Q10 increased the C-PC melting point by nearly 5 °C. Our study identified food-derived ligands that interact with C-PC and improve its thermal stability, indicating their potential as stabilising agents for C-PC in the food industry.

Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states.

Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily. However, ENTs lack functionally-essential features of that superfamily, suggesting that they may use a different transport mechanism. Understanding the molecular basis of their transport requires insight into diverse conformational states. Differences between intermediate states may be discrete and mediated by subtle gating interactions, such as salt bridges. We identified four variants of human equilibrative nucleoside transporter isoform 1 (hENT1) at the large intracellular loop (ICL6) and transmembrane helix 7 (TM7) that stabilise the apo-state (∆T m 0.7-1.5°C). Furthermore, we showed that variants K263A (ICL6) and I282V (TM7) specifically stabilise the inhibitor-bound state of hENT1 (∆∆T m 5.0 ± 1.7°C and 3.0 ± 1.8°C), supporting the role of ICL6 in hENT1 gating. Finally, we showed that, in comparison with wild type, variant T336A is destabilised by nitrobenzylthioinosine (∆∆T m -4.7 ± 1.1°C) and binds it seven times worse. This residue may help determine inhibitor and substrate sensitivity. Residue K263 is not present in the solved structures, highlighting the need for further structural data that include the loop regions.

Stabilisation of Viral Membrane Fusion Proteins in Prefusion Conformation by Structure-Based Design for Structure Determination and Vaccine Development.

The membrane surface of enveloped viruses contains dedicated proteins enabling the fusion of the viral with the host cell membrane. Working with these proteins is almost always challenging because they are membrane-embedded and naturally metastable. Fortunately, based on a range of different examples, researchers now have several possibilities to tame membrane fusion proteins, making them amenable for structure determination and immunogen generation. This review describes the structural and functional similarities of the different membrane fusion proteins and ways to exploit these features to stabilise them by targeted mutational approaches. The recent determination of two herpesvirus membrane fusion proteins in prefusion conformation holds the potential to apply similar methods to this group of viral fusogens. In addition to a better understanding of the herpesviral fusion mechanism, the structural insights gained will help to find ways to further stabilise these proteins using the methods described to obtain stable immunogens that will form the basis for the development of the next generation of vaccines and antiviral drugs.

Human TRMT112-Methyltransferase Network Consists of Seven Partners Interacting with a Common Co-Factor.

Methylation is an essential epigenetic modification mainly catalysed by S-Adenosyl methionine-dependent methyltransferases (MTases). Several MTases require a cofactor for their metabolic stability and enzymatic activity. TRMT112 is a small evolutionary conserved protein that acts as a co-factor and activator for different MTases involved in rRNA, tRNA and protein methylation. Using a SILAC screen, we pulled down seven methyltransferases-N6AMT1, WBSCR22, METTL5, ALKBH8, THUMPD2, THUMPD3 and TRMT11-as interaction partners of TRMT112. We showed that TRMT112 stabilises all seven MTases in cells. TRMT112 and MTases exhibit a strong mutual feedback loop when expressed together in cells. TRMT112 interacts with its partners in a similar way; however, single amino acid mutations on the surface of TRMT112 reveal several differences as well. In summary, mammalian TRMT112 can be considered as a central "hub" protein that regulates the activity of at least seven methyltransferases.

DMSO as new, counterintuitive excipient for freeze-drying human keratinocytes.

DMSO is widely used as powerful cryoprotectant for the storage and transport of frozen cells. Beyond this established application of DMSO, we could now show that it has also promising lyoprotectant effects in the field of lyophilisation of therapeutic cells. Freeze-drying of HaCaT keratinocytes in 10% HES, 5% HE and in presence of DMSO led to an increase in cell membrane integrity from 25.3 ± 2.7 % without DMSO to 41.4 ± 4.3 % with 2% DMSO, as determined by trypan blue exclusion. Interruption of the lyophilisation cycle at different sampling points showed a rapid decrease of cell membrane integrity below a critical residual moisture content. DMSO was able to stabilise cell membranes below this moisture level up to a final residual moisture content of less than 1%. Furthermore, DMSO increased the total protein content of cells after freeze-drying and subsequent SDS PAGE analysis indicated that certain abundant proteins were better preserved with the use of DMSO. Owed to its low vapour pressure, a significant part of DMSO is not removed during freeze-drying and remains as plasticiser in the lyophilised cake. However, a Tg above 60°C for 2% DMSO indicates that samples can still be stored at temperatures of 2-8°C. Also, no macroscopic or microscopic collapse can be observed by SEM or BET measurements and DMSO addition leads even to more elegant cakes with reduced cake cracking. With a better preservation of cell membranes and cellular structures, DMSO can contribute to the still unsolved problem of freeze-drying cells of higher complexity.

Acute Intermittent Porphyria: An Overview of Therapy Developments and Future Perspectives Focusing on Stabilisation of HMBS and Proteostasis Regulators.

Acute intermittent porphyria (AIP) is an autosomal dominant inherited disease with low clinical penetrance, caused by mutations in the hydroxymethylbilane synthase (HMBS) gene, which encodes the third enzyme in the haem biosynthesis pathway. In susceptible HMBS mutation carriers, triggering factors such as hormonal changes and commonly used drugs induce an overproduction and accumulation of toxic haem precursors in the liver. Clinically, this presents as acute attacks characterised by severe abdominal pain and a wide array of neurological and psychiatric symptoms, and, in the long-term setting, the development of primary liver cancer, hypertension and kidney failure. Treatment options are few, and therapies preventing the development of symptomatic disease and long-term complications are non-existent. Here, we provide an overview of the disorder and treatments already in use in clinical practice, in addition to other therapies under development or in the pipeline. We also introduce the pathomechanistic effects of HMBS mutations, and present and discuss emerging therapeutic options based on HMBS stabilisation and the regulation of proteostasis. These are novel mechanistic therapeutic approaches with the potential of prophylactic correction of the disease by totally or partially recovering the enzyme functionality. The present scenario appears promising for upcoming patient-tailored interventions in AIP.

Dicoumarol, an NQO1 inhibitor, blocks cccDNA transcription by promoting degradation of HBx.

BACKGROUND & AIMS: Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. METHODS: We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model. RESULTS: Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. CONCLUSIONS: Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability. LAY SUMMARY: Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.

Unravelling enhancement of antibody fragment stability - Role of format structure and cysteine modification.

Antibody-based diagnostics and therapeutics have huge commercial value. However, applications of antibodies are often limited by instability, particularly for recombinant antibody formats. This paper describes the conversion of a single-chain variable fragment (scFv) antibody to a single-chain antibody fragment (scAb) with notably improved stability characteristics. This scAb retains antigen-binding activity (i) at high temperature (up to 60 °C), (ii) in guanidine hydrochloride (GdnHCl, up to 1 M), and (iii) when stored at 37 °C for 6 months. However, limited improvement was observed when the original scFv was converted to a larger fragment antigen-binding (Fab) format. Certain Cys-to-Ala mutations in the third complementarity determining region of the antibody heavy chain (CDRH3) also led to stability improvements. Our findings indicate that the stability of an antibody derivative depends on its format and on the positions of cysteines in the CDRs.

Novel Xylene-Linked Maltoside Amphiphiles (XMAs) for Membrane Protein Stabilisation.

Membrane proteins are key functional players in biological systems. These biomacromolecules contain both hydrophilic and hydrophobic regions and thus amphipathic molecules are necessary to extract membrane proteins from their native lipid environments and stabilise them in aqueous solutions. Conventional detergents are commonly used for membrane protein manipulation, but membrane proteins surrounded by these agents often undergo denaturation and aggregation. In this study, a novel class of maltoside-bearing amphiphiles, with a xylene linker in the central region, designated xylene-linked maltoside amphiphiles (XMAs) was developed. When these novel agents were evaluated with a number of membrane proteins, it was found that XMA-4 and XMA-5 have particularly favourable efficacy with respect to membrane protein stabilisation, indicating that these agents hold significant potential for membrane protein structural study.

Carrageenan analysis. Part 1: Characterisation of the carrageenan test material and stability in swine-adapted infant formula.

A method was developed and validated in support of a 28-day feeding study of swine-adapted infant formula stabilised with carrageenan administered to neonatal piglets. Carrageenan concentrations in the test formulations were 0, 300, 1000 and 2250 mg kg(-1) formula. Extraction of carrageenan from swine-adapted infant formula was achieved by breaking carrageenan-protein cross-linkages using saturated sodium chloride, followed by separation of the non-gelling carrageenan fraction via centrifugation. The extraction of carrageenan from formula was successful with respect to consistent recovery of the non-gelling carrageenan fraction from both test and control formula samples. Molecular weight analysis (Mw) of the recovered carrageenan fractions from the test and control formula samples confirmed that the carrageenan used to manufacture the formula was not degraded during the infant formula production process and subsequent storage for 4 months covering the 28-day piglet dietary feeding study. Carrageenan has excellent stability in infant formulations.