Characteristics of the turnover of uncoupling protein 3 by the ubiquitin proteasome system in isolated mitochondria

AbstractUncoupling protein 3 (UCP3) is implicated in mild uncoupling and the regulation of mitochondrial ROS production. We previously showed that UCP3 turns over rapidly in C2C12 myoblasts, with a half-life of 0.5–4h, and that turnover can be reconstituted in vitro. We show here that rapid degradation of UCP3 in vitro in isolated brown adipose tissue mitochondria required the 26S proteasome, ubiquitin, ATP, succinate to generate a high membrane potential, and a pH of 7.4 or less. Ubiquitin containing lysine-48 was both necessary and sufficient to support UCP3 degradation, implying a requirement for polyubiquitylation at this residue. The 20S proteasome did not support degradation. UCP3 degradation was prevented by simultaneously blocking matrix ATP generation and import, showing that ATP in the mitochondrial matrix was required. Degradation did not appear to require a transmembrane pH gradient, but was very sensitive to membrane potential: degradation was halved when membrane potential decreased 10–20mV from its resting value, and was not significant below about 120mV. We propose that matrix ATP and a high membrane potential are needed for UCP3 to be polyubiquitylated through lysine-48 of ubiquitin and exported to the cytosolic 26S proteasome, where it is de-ubiquitylated and degraded

Total Cellular ATP Production Changes With Primary Substrate in MCF7 Breast Cancer Cells.

Cancer growth is predicted to require substantial rates of substrate catabolism and ATP turnover to drive unrestricted biosynthesis and cell growth. While substrate limitation can dramatically alter cell behavior, the effects of substrate limitation on total cellular ATP production rate is poorly understood. Here, we show that MCF7 breast cancer cells, given different combinations of the common cell culture substrates glucose, glutamine, and pyruvate, display ATP production rates 1.6-fold higher than when cells are limited to each individual substrate. This increase occurred mainly through faster oxidative ATP production, with little to no increase in glycolytic ATP production. In comparison, non-transformed C2C12 myoblast cells show no change in ATP production rate when substrates are limited. In MCF7 cells, glutamine allows unexpected access to oxidative capacity that pyruvate, also a strictly oxidized substrate, does not. Pyruvate, when added with other exogenous substrates, increases substrate-driven oxidative ATP production, by increasing both ATP supply and demand. Overall, we find that MCF7 cells are highly flexible with respect to maintaining total cellular ATP production under different substrate-limited conditions, over an acute (within minutes) timeframe that is unlikely to result from more protracted (hours or more) transcription-driven changes to metabolic enzyme expression. The near-identical ATP production rates maintained by MCF7 and C2C12 cells given single substrates reveal a potential difficulty in using substrate limitation to selectively starve cancer cells of ATP. In contrast, the higher ATP production rate conferred by mixed substrates in MCF7 cells remains a potentially exploitable difference

Role of Sigma-1 Receptors in Neurodegenerative Diseases

Neurodegenerative diseases with distinct genetic etiologies and pathological phenotypes appear to share common mechanisms of neuronal cellular dysfunction, including excitotoxicity, calcium dysregulation, oxidative damage, ER stress and mitochondrial dysfunction. Glial cells, including microglia and astrocytes, play an increasingly recognized role in both the promotion and prevention of neurodegeneration. Sigma receptors, particularly the sigma-1 receptor subtype, which are expressed in both neurons and glia of multiple regions within the central nervous system, are a unique class of intracellular proteins that can modulate many biological mechanisms associated with neurodegeneration. These receptors therefore represent compelling putative targets for pharmacologically treating neurodegenerative disorders. In this review, we provide an overview of the biological mechanisms frequently associated with neurodegeneration, and discuss how sigma-1 receptors may alter these mechanisms to preserve or restore neuronal function. In addition, we speculate on their therapeutic potential in the treatment of various neurodegenerative disorders

Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells

The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusion

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant