SUMMARY Mitochondrial Ca2+ (Ca2+m) uptake is mediated by an inner membrane Ca2+ channel called the uniporter. Ca2+ uptake is driven by the considerable voltage present across the inner membrane (ΔΨm) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca2+ concentration is maintained 5–6 orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca2+]m under basal conditions. In its absence, mitochondria become constitutively loaded with Ca2+, triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca2+ threshold for Ca2+m uptake without affecting the kinetic properties of MCU-mediated Ca2+ uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca2+m uptake that is essential to prevent [Ca2+]m overload and associated stress.

Ca2+ flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca2+ signals and activation of cell death pathways1–11. Mitochondrial Ca2+ uptake occurs at regions of close apposition with intracellular Ca2+ release sites 12–14, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca2+ selective ion channel (MiCa15) called the uniporter16–18 whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore19, 20. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca2+ uptake in HeLa cells 21. Here we identified CCDC90A, hereafter referred to as MCUR1 (Mitochondrial Calcium Uniporter Regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca2+ uptake. MCUR1 binds to MCU and regulates ruthenium red-sensitive MCU-dependent Ca2+ uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca2+ uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP, and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca2+ uptake and maintenance of normal cellular bioenergetics.

Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A (SrtA) for short peptide sequences (LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme’s poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation.

Dysregulation of mitochondrial Ca 2؉ -dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca 2؉ transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1-mediated Ca 2؉ transport remains unresolved. This study examines LETM1-mediated mitochondrial Ca 2؉ transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf-Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca 2؉ influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in ⌬EF hand, D676A D688K

In dystrophic muscle, an increase in reactive oxygen species (ROS) production and sarcolemmal calcium (Ca 2þ) influx contributes to stretch-induced muscle damage however mechanistic insights into the activation of these pathways is lacking. In mdx myofibers (murine Duchenne muscular dystrophy), we have demonstrated that with mechanical stretch, the microtubule (MT) cytoskeleton is a critical mechano-transduction element for the activation of NADPH oxi-dase2 (Nox2) derived ROS production; a pathway we term X-ROS signaling [1]. Downstream, we showed that X-ROS sensitized stretch activated channels (SACs) to increase sarcolemmal Ca 2þ influx during stretch. The significance of the MT cytoskeleton activation of X-ROS in mdx was revealed when the acute targeting of MT density proffered protection from contraction induced damage. In mammalian cells, the MT network is a dynamic structure in which MT density is determined by the stability of MT filaments. Our initial studies used acute pharmacological stabilization (taxol) or destabilization (colchicine) to establish MT network density as critical for the mechano-activation of X-ROS. We now interrogate critical upstream pathways and use new pharmacological and molecular approaches to explore the role of endogenous modulators of MT stability and how they may contribute to the enhanced X-ROS in dystrophic skeletal muscle.

Knockdown of SLC25A23 decreases mitochondrial Ca2+ uptake, and SLC25A23 interacts with MCU and MICU1, components of mitochondrial Ca2+ uniporter. Expression of SLC25A23 EF-hand-domain mutants has a dominant-negative phenotype of reduced mitochondrial Ca2+ uptake. It also attenuates basal ROS and oxidant-induced ATP decline and cell death.

Nitric oxide and other reactive nitrogen species target multiple sites in the mitochondria to impact cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose dependent progressive increase in NO-dependent DAF fluorescence that corresponded to mitochondrial membrane potential loss, and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially-derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.

Salmonella lacking the TCA enzyme aconitase trigger NLRP3 inflammasome activation in infected macrophages, leading to elevated inflammatory responses and reduced virulence.