Geranylgeranylated-SCFFBXO10 Regulates Selective Outer Mitochondrial Membrane Proteostasis and Function.

E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external cues to regulate protein homeostasis. However, the regulation of membrane protein ubiquitination by E3s within distinct cell membrane compartments or organelles is not well understood. We show that FBXO10, the interchangeable component of the SKP1/CUL1/F-box ubiquitin ligase complex (SCF-E3), undergoes lipid-modification with geranylgeranyl isoprenoid at Cysteine953 (C953), facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide does not contain a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and the interaction with two cytosolic factors, PDE6δ (a prenyl group-binding protein), and HSP90 (a mitochondrial chaperone) orchestrate specific OMM targeting of prenyl-FBXO10 across diverse membrane compartments. The geranylgeranylation-deficient FBXO10(C953S) mutant redistributes away from the OMM, leading to impaired mitochondrial ATP production, decreased mitochondrial membrane potential, and increased mitochondrial fragmentation. Phosphoglycerate mutase 5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative mass spectrometry analyses of enriched mitochondria (LFQ-MS/MS), leveraging the redistribution of FBXO10(C953S). FBXO10, but not FBXO10(C953S), promoted polyubiquitylation and degradation of PGAM5. Examination of the role of this pathway in a physiological context revealed that the loss of FBXO10 or expression of prenylation-deficient-FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human iPSCs and murine myoblasts. Our studies identify a mechanism for selective E3-ligase mediated regulation of mitochondrial membrane proteostasis and metabolic health, potentially amenable to therapeutic intervention.

Detection of membrane-anchoring lipid modifications of proteins in cells by radioactive metabolic labeling.

Prenylation and palmitoylation are two major lipid modifications of cellular proteins that anchor proteins to cell membranes. Here, we present a protocol for detecting these modifications in cellular proteins by radioactive metabolic labeling. We describe steps for metabolic labeling of cells, cell harvesting for carrying out immunoprecipitations, subjecting immunocomplexes to SDS-PAGE, and transferring them to polyvinylidine flouride (PVDF) membranes. We then detail detection of labeled target proteins by exposing PVDF membranes to phosphor screens and using a phosphor imager machine. For complete details of this protocol, please refer to Liang et al.1.

Ubiquitin proteasome system in immune regulation and therapeutics.

The ubiquitin proteasome system (UPS) is a proteolytic machinery for the degradation of protein substrates that are post-translationally conjugated with ubiquitin polymers through the enzymatic action of ubiquitin ligases, in a process termed ubiquitylation. Ubiquitylation of substrates precedes their proteolysis via proteasomes, a hierarchical feature of UPS. E3-ubiquitin ligases recruit protein substrates providing specificity for ubiquitylation. Innate and adaptive immune system networks are regulated by ubiquitylation and substrate degradation via E3-ligases/UPS. Deregulation of E3-ligases/UPS components in immune cells is involved in the development of lymphomas, neurodevelopmental abnormalities, and cancers. Targeting E3-ligases for therapeutic intervention provides opportunities to mitigate the unintended broad effects of 26S proteasome inhibition. Recently, bifunctional moieties such as PROTACs and molecular glues have been developed to re-purpose E3-ligases for targeted degradation of unwanted aberrant proteins, with a potential for clinical use. Here, we summarize the involvement of E3-ligases/UPS components in immune-related diseases with perspectives.

Encounters for foot and ankle pain in UK primary care: a population-based cohort study of CPRD data.

BACKGROUND: Older patients who have foot pain report variation in access to services to manage their foot health. To plan services it is essential to understand the scale and burden of foot pain that exists for GPs. AIM: To provide UK-wide population-level data of the frequency of foot and/or ankle pain encounters recorded in general practice. DESIGN AND SETTING: Population-based cohort design study using data drawn from the UK Clinical Practice Research Datalink (CPRD) from January 2010 to December 2013. METHOD: All CPRD data were collected prospectively by participating GPs. The primary outcome was prevalence of GP encounters for foot and/or ankle pain, stratified by age, sex, and different subgroups of causes. RESULTS: A foot and/or ankle pain encounter was recorded for 346 067 patients, and there was a total of 567 095 recorded encounters (mean per person 1.6, standard deviation [SD] 1.3). The prevalence of recorded encounters of foot and/or ankle pain was 2980 per 100 000 (3%). The number of patients with a recorded encounter of foot and/or ankle pain was 1820 per 100 000 (1.8%). Foot and/or ankle pain encounters were reported across all age groups (54.4% females), with those aged 71-80 years placing the greatest burden on GPs. The most common specified referrals were to orthopaedics (n = 36 881) and physiotherapy (n = 33 987), followed by podiatry (n = 25 980). CONCLUSION: The burden of foot and/or ankle pain encounters recorded by GPs is not insubstantial, and spans all ages, with a high proportion of referrals to orthopaedics. The authors recommend further exploration of 'first-contact practitioners' for foot and/or ankle pain in general practice to alleviate the burden on GPs.

Exploring repositioning movements in sitting with 'at risk' groups using accelerometry and interface pressure mapping technologies.

BACKGROUND: Despite high quality guidelines underpinning pressure ulcer care (NPUAP/EPUAP/PPPIA, 2014), pressure ulceration still poses a significant financial impact on health care services in treatment and staff costs as well as having a profound effect on the health and quality of life of individuals experiencing them. Repositioning is a key preventative technique recommended by occupational therapists and other health care professionals. The frequency and quality of repositioning movements performed by individuals, however, can be difficult to determine. This paper explores the use of technology in monitoring repositioning movements in sitting. OBJECTIVE: To explore the outputs of technologies such as interface pressure mapping systems and accelerometers in enabling the therapist to accurately monitor seated behaviour and enhance practice through targeted interventions to prevent sitting acquired pressure ulceration. METHOD: Reviewing the findings of two recent research studies with 'at risk' cohorts (spinal cord injury; elderly orthopaedic), using accelerometry and seated interface pressures, this paper will highlight how useful this technology is in clinical practice to monitor weight shifts and repositioning behaviours. RESULT: Both studies illustrated that the majority of individuals did not adhere to the frequency or magnitude of movements currently recommended to redistribute seating interface pressures. When repositioning was performed it was ineffective in reducing seated pressures. CONCLUSION: In an era of personalised medicine, technology has an important role to play in providing the service user, caregivers and healthcare staff with important biofeedback information about seated behaviours, particularly those that minimise the risk of developing sitting acquired pressure ulcers. This information can augment occupational therapists' clinical decision-making in maximising active pressure ulcer prevention.

Investigation of interfacial properties of pure and mixed poloxamers for surfactant-mediated shear protection of mammalian cells.

The Poloxamer family of surfactants are commonly used in the biopharmaceutical industry as cell culture media additives to protect cells from the turbulent environment of sparged bioreactors. Despite the widespread use of poloxamers in cell culture, their performance as cell protectants varies depending on their physical structure, molecular weight, and batch-to-batch composition. In this study, the interfacial properties of Poloxamer 188 (P188), Poloxamer 407 (P407), and a mixture of P188 and P407 were characterized to investigate the mechanism of surfactant-mediated shear protection of mammalian cells. The foam stability and equilibrium surface tension of these surfactant systems correlated with their ability to mitigate physical damage to cells in a turbulent environment. We demonstrate that while P188 can function as highly effective shear protectant, the presence of a surface-active contaminant can greatly hinder its protective characteristics. P407 was found to function as such an interfacially active "impurity," disrupting shear protection when mixed with P188 by preferentially adsorbing to the gas-liquid and membrane-liquid interface. Addition of surface-active impurities altered the interfacial properties of the surfactant system and could be detected using an equilibrium surface tension assay. The mechanism of disruption by P407 was determined to be independent of cell-to-bubble attachment, suggesting that poloxamer adsorption to and subsequent reinforcement of the cell membrane may play a key role in protecting cells in high shear environments. This investigation contributes to our understanding of the mechanism of surfactant-mediated shear protection of cells and demonstrates that a surface tension assay can be utilized as a screening tool to ensure that poloxamer lots are free of surface active impurities.

National variation in the composition of rheumatology multidisciplinary teams: a cross-sectional study.

The objective of this study is to describe the composition of multidisciplinary teams (MDT) working within rheumatology departments across the UK. All rheumatology departments in the United Kingdom (UK) were invited to participate in a national electronic survey between February 2014 and April 2015 as a part of a national audit for the management of rheumatoid and early inflammatory arthritis commissioned by Healthcare Quality Improvement Partnership. Rheumatology departments were asked to report their MDT composition; defined as a rheumatologist (consultant or specialist trainee), specialist nurse, occupational therapist physiotherapist, and podiatrist. The data were collected as Whole Time Equivalent (WTE) of each professional group at each department adjusted to 100,000 population. The data were grouped according to British Society for Rheumatology regions to study regional variations. The survey was completed by 164/167 departments (98% response rate). All departments reported an MDT comprising a rheumatologist (consultant or specialist trainee) and almost all included a specialist nurse but only 28 (17%) of the departments had MDTs comprising all the professional groups. There was a high degree of regional variation in the provision of Allied Health Professionals (physiotherapists, occupational therapists, and podiatrists) in the UK. MDT care is recommended for the management of inflammatory arthritis, but few UK rheumatology departments have a full complement of healthcare professionals within their MDT. There is a high degree of regional variation in the composition and staffing levels of the rheumatology MDT across the UK; the impact of which warrants further investigation.

In-line monitoring of esterification using a miniaturised mid-infrared spectrometer.

A miniaturised mid-infrared (MIR) spectrometer attached directly to an in-line immersion probe with an attenuated total reflectance crystal has been used to monitor the progression of the batch reaction of crotonic acid and 2-butanol in toluene. Univariate calibration, using the signal at 1188 cm(-1) in the 2nd derivative spectrum, gave accurate (average error, 1.6%) and precise (average relative standard deviation, 5.2%) estimation of 2-butyl crotonate concentrations in the range 0.08-0.49 mol dm(-3). Calibration by partial least squares was of no additional benefit in this application. The performance of in-line MIR spectrometry was comparable to that of an off-line reference gas chromatography method and superior to that of other in-line process analysis techniques (near-infrared, Raman or UV-visible spectrometries).