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Upregulation of glycolysis and downregulation of mitochondrial oxidative phosphorylation, termed as the Warburg effect, are characteristic of tumor cells1,2. Restriction of pyruvate flux into the mitochondrial matrix is one of the major mechanisms underlying this phenomenon3. Warburg-type metabolism is beneficial for rapidly proliferating cells, however its function remains unclear. Moreover, it is unknown what the metabolic consequences of activation of mitochondrial respiration in Warburg-type cancer cells are. Here we created a chemogenetic instrument, Grubraw, that generates pyruvate directly in the mitochondrial matrix bypassing restricted pyruvate influx. In cancer cells, Grubraw-driven pyruvate synthesis in the matrix increased mitochondrial membrane potential, oxygen consumption rate, and the amounts of TCA cycle intermediates. In a mouse model of human melanoma xenografts, chemogenetic activation of mitochondria caused a decrease in tumor growth rate. Surprisingly, cancer cells actively exported pyruvate generated by Grubraw in the mitochondria into the extracellular medium. Our results demonstrate that cells with Warburg-type metabolism use a previously unknown mechanism of carbon flux control to dispose of excessive mitochondrial pyruvate.

A file containing supplementary figures 1-8 and the supplementary table S1.

Supplementary Tables S4 to S8

Supplementary Figures S1-S10

Supplementary Materials & Methods, Supplementary Tables S1-S3.

A file containing RPPA and RNAseq datasets, pathway analyses and prediction calculations presented in the main figures.

Pancreatic ductal adenocarcinoma (PDAC) displays a dismal prognosis due to late diagnosis and high chemoresistance incidence. For advanced disease stages or patients with comorbidities, treatment options are limited to gemcitabine alone or in combination with other drugs. While gemcitabine resistance has been widely attributed to the levels of one of its targets, RRM1, the molecular consequences of gemcitabine resistance in PDAC remain largely elusive. Here we sought to identify genomic, epigenomic, and transcriptomic events associated with gemcitabine resistance in PDAC and their potential clinical relevance. We found that gemcitabine-resistant cells displayed a coamplification of the adjacent RRM1 and STIM1 genes. Interestingly, RRM1, but not STIM1, was required for gemcitabine resistance, while high STIM1 levels caused an increase in cytosolic calcium concentration. Higher STIM1-dependent calcium influx led to an impaired endoplasmic reticulum stress response and a heightened nuclear factor of activated T-cell activity. Importantly, these findings were confirmed in patient and patient-derived xenograft samples. Taken together, our study uncovers previously unknown biologically relevant molecular properties of gemcitabine-resistant tumors, revealing an undescribed function of STIM1 as a rheostat directing the effects of calcium signaling and controlling epigenetic cell fate determination. It further reveals the potential benefit of targeting STIM1-controlled calcium signaling and its downstream effectors in PDAC.Significance:Gemcitabine-resistant and some naïve tumors coamplify RRM1 and STIM1, which elicit gemcitabine resistance and induce a calcium signaling shift, promoting ER stress resistance and activation of NFAT signaling.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24 h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

Melanoma is the deadliest of skin cancers and has a high tendency to metastasize to distant organs. Calcium and metabolic signals contribute to melanoma invasiveness; however, the underlying molecular details are elusive. The MCU complex is a major route for calcium into the mitochondrial matrix but whether MCU affects melanoma pathobiology was not understood. Here, we show that MCUA expression correlates with melanoma patient survival and is decreased in BRAF kinase inhibitor-resistant melanomas. Knockdown (KD) of MCUA suppresses melanoma cell growth and stimulates migration and invasion. In melanoma xenografts, MCUA_KD reduces tumor volumes but promotes lung metastases. Proteomic analyses and protein microarrays identify pathways that link MCUA and melanoma cell phenotype and suggest a major role for redox regulation. Antioxidants enhance melanoma cell migration, while prooxidants diminish the MCUA_KD-induced invasive phenotype. Furthermore, MCUA_KD increases melanoma cell resistance to immunotherapies and ferroptosis. Collectively, we demonstrate that MCUA controls melanoma aggressive behavior and therapeutic sensitivity. Manipulations of mitochondrial calcium and redox homeostasis, in combination with current therapies, should be considered in treating advanced melanoma.

Pancreatic ductal adenocarcinoma (PDAC) displays a dismal prognosis due to late diagnosis and high chemoresistance incidence. For advanced disease stages or patients with comorbidities, treatment options are limited to gemcitabine alone or in combination with other drugs. While gemcitabine resistance has been widely attributed to the levels of one of its targets, RRM1, the molecular consequences of gemcitabine resistance in PDAC remain largely elusive. Here we sought to identify genomic, epigenomic, and transcriptomic events associated with gemcitabine resistance in PDAC and their potential clinical relevance. We found that gemcitabine-resistant cells displayed a coamplification of the adjacent RRM1 and STIM1 genes. Interestingly, RRM1, but not STIM1, was required for gemcitabine resistance, while high STIM1 levels caused an increase in cytosolic calcium concentration. Higher STIM1-dependent calcium influx led to an impaired endoplasmic reticulum stress response and a heightened nuclear factor of activated T-cell activity. Importantly, these findings were confirmed in patient and patient-derived xenograft samples. Taken together, our study uncovers previously unknown biologically relevant molecular properties of gemcitabine-resistant tumors, revealing an undescribed function of STIM1 as a rheostat directing the effects of calcium signaling and controlling epigenetic cell fate determination. It further reveals the potential benefit of targeting STIM1-controlled calcium signaling and its downstream effectors in PDAC.Significance:Gemcitabine-resistant and some naïve tumors coamplify RRM1 and STIM1, which elicit gemcitabine resistance and induce a calcium signaling shift, promoting ER stress resistance and activation of NFAT signaling.

Redox signaling and cardiac function are tightly linked. Still, it is largely unknown which protein targets are affected by H2O2 in cardiomyocytes that underly the impaired inotropic effects in oxidative stress. Here, we combined a new chemogenetic mouse model (HyPer-DAO mice) and a redox proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we prove that increased endogenous production of H2O2 in cardiomyocytes leads to a reversibly impaired cardiac contractility in vivo. We identified the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch and link its modification to mitochondrial metabolism and glutathione synthesis. Microsecond molecular dynamics simulations combined with experiments using cysteine-gene-edited point mutated cells revealed that IDH3γ Cys148 and 284 are critically involved in H2O2-dependent regulation of IDH3 activity. The oxidative modification of IDH3γ regulates the competition between energy production by TCA cycle flux and cellular anti-oxidant defense by glutathione synthesis.

It is exactly a century since polarography was developed, which is seen as a predecessor of all voltammetric techniques. As cyclic voltammetry (CV) is the most prominent member in the family of voltammetric techniques for mechanistic studies, the so-called “pulse voltammetric techniques” emerged as simple and viable alternatives to CV for mechanistic characterizations and analytical application, as well as for kinetic and thermodynamic evaluations. The theories and practical application of pulse voltammetric techniques were largely developed by several women electrochemists. In this short overview, we outline some of the major achievements of five women electrochemists who contributed immensely to the theoretical and practical application of pulse voltammetric technique. Since the theory and application of pulse voltammetric techniques largely relies on the works of Janet Osteryoung, Sebojka Komorsky Lovric, Angela Molina, Anna Brainina, and Oliveira Brett, we give in this review a short historical overview of the major accomplishments of these five exceptional women electrochemists.

Background Melanoma, the deadliest of skin cancers, has a high propensity to form brain metastases that are associated with a markedly worsened prognosis. In spite of recent therapeutic advances, melanoma brain lesions remain a clinical challenge, biomarkers predicting brain dissemination are not clear and differences with other metastatic sites are poorly understood. Methods We examined a genetically diverse panel of human-derived melanoma brain metastasis (MBM) and extracranial cell lines using targeted sequencing, a Reverse Phase Protein Array, protein expression analyses, and functional studies in vitro and in vivo. Results Brain-specific genetic alterations were not detected; however, MBM cells in vitro displayed lower proliferation rates and MBM-specific protein expression patterns associated with proliferation, DNA damage, adhesion, and migration. MBM lines displayed higher levels of RAC1 expression, involving a distinct RAC1-PAK1-JNK1 signaling network. RAC1 knockdown or treatment with small molecule inhibitors contributed to a less aggressive MBM phenotype in vitro, while RAC1 knockdown in vivo led to reduced tumor volumes and delayed tumor appearance. Proliferation, adhesion, and migration were higher in MBM vs nonMBM lines in the presence of insulin or brain-derived factors and were affected by RAC1 levels. Conclusions Our findings indicate that despite their genetic variability, MBM engage specific molecular processes such as RAC1 signaling to adapt to the brain microenvironment and this can be used for the molecular characterization and treatment of brain metastases.

Salinity poses a serious threat to global agriculture and human food security. A better understanding of plant adaptation to salt stress is, therefore, mandatory. In the non-photosynthetic cells of the root, salinity perturbs oxidative balance in mitochondria, leading to cell death. In parallel, plastids accumulate the jasmonate precursor cis (+)12-Oxo-Phyto-Dienoic Acid (OPDA) that is then translocated to peroxisomes and has been identified as promoting factor for salt-induced cell death as well. In the current study, we probed for a potential interaction between these three organelles that are primarily dealing with oxidative metabolism. We made use of two tools: (i) Rice OPDA Reductase 7 (OsOPR7), an enzyme localised in peroxisomes converting OPDA into the precursors of the stress hormone JA-Ile. (ii) A Trojan Peptoid, Plant PeptoQ, which can specifically target to mitochondria and scavenge excessive superoxide accumulating in response to salt stress. We show that overexpression of OsOPR7 as GFP fusion in tobacco (Nicotiana tabacum L. cv. Bright Yellow 2, BY-2) cells, as well as a pretreatment with Plant PeptoQ can mitigate salt stress with respect to numerous aspects including proliferation, expansion, ionic balance, redox homeostasis, and mortality. This mitigation correlates with a more robust oxidative balance, evident from a higher activity of superoxide dismutase (SOD), lower levels of superoxide and lipid peroxidation damage, and a conspicuous and specific upregulation of mitochondrial SOD transcripts. Although both, Plant PeptoQ and ectopic OsOPR7, were acting in parallel and mostly additive, there are two specific differences: (i) OsOPR7 is strictly localised to the peroxisomes, while Plant PeptoQ found in mitochondria. (ii) Plant PeptoQ activates transcripts of NAC, a factor involved in retrograde signalling from mitochondria to the nucleus, while these transcripts are suppressed significantly in the cells overexpressing OsOPR7. The fact that overexpression of a peroxisomal enzyme shifting the jasmonate pathway from the cell-death signal OPDA towards JA-Ile, a hormone linked with salt adaptation, is accompanied by more robust redox homeostasis in a different organelle, the mitochondrion, indicates that cross-talk between peroxisome and mitochondrion is a crucial factor for efficient adaptation to salt stress.

Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria-ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induction of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1⍺ covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1⍺ interaction requires the C-terminal active site of ERO1⍺ and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1⍺ complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca

Different types of cells, such as endothelial cells, tumor-associated fibroblasts, pericytes, and immune cells, release extracellular vesicles (EVs) in the tumor microenvironment. The components of EVs include proteins, DNA, RNA, and microRNA. One of the most important functions of EVs is the transfer of aforementioned bioactive molecules, which in cancer cells may affect tumor growth, progression, angiogenesis, and metastatic spread. Furthermore, EVs affect the presentation of antigens to immune cells via the transfer of nucleic acids, peptides, and proteins to recipient cells. Recent studies have also explored the potential application of EVs in cancer treatment. This review summarizes the mechanisms by which EVs regulate melanoma development, progression, and their potentials to be applied in therapy. We initially describe vesicle components; discuss their effects on proliferation, anti-melanoma immunity, and drug resistance; and finally focus on the effects of EV-derived microRNAs on melanoma pathobiology. This work aims to facilitate our understanding of the influence of EVs on melanoma biology and initiate ideas for the development of novel therapeutic strategies.

ObjectivesNon-alcoholic fatty liver disease (NAFLD) can persist in the stage of simple hepatic steatosis or progress to steatohepatitis (NASH) with an increased risk for cirrhosis and cancer. We examined the mechanisms controlling the progression to severe NASH in order to develop future treatment strategies for this disease.DesignNFATc1 activation and regulation was examined in livers from patients with NAFLD, cultured and primary hepatocytes and in transgenic mice with differential hepatocyte-specific expression of the transcription factor (Alb-cre, NFATc1c.a. andNFATc1Δ/Δ). Animals were fed with high-fat western diet (WD) alone or in combination with tauroursodeoxycholic acid (TUDCA), a candidate drug for NAFLD treatment. NFATc1-dependent ER stress-responses, NLRP3 inflammasome activation and disease progression were assessed both in vitro and in vivo.ResultsNFATc1 expression was weak in healthy livers but strongly induced in advanced NAFLD stages, where it correlates with liver enzyme values as well as hepatic inflammation and fibrosis. Moreover, high-fat WD increased NFATc1 expression, nuclear localisation and activation to promote NAFLD progression, whereas hepatocyte-specific depletion of the transcription factor can prevent mice from disease acceleration. Mechanistically, NFATc1 drives liver cell damage and inflammation through ER stress sensing and activation of the PERK-CHOP unfolded protein response (UPR). Finally, NFATc1-induced disease progression towards NASH can be blocked by TUDCA administration.ConclusionNFATc1 stimulates NAFLD progression through chronic ER stress sensing and subsequent activation of terminal UPR signalling in hepatocytes. Interfering with ER stress-responses, for example, by TUDCA, protects fatty livers from progression towards manifest NASH.

Ischemic cerebral stroke is one of the leading causes of death and disability in humans. However, molecular processes underlying the development of this pathology remain poorly understood. There are major gaps in our understanding of metabolic changes that occur in the brain tissue during the early stages of ischemia and reperfusion. In particular, it is generally accepted that both ischemia (I) and reperfusion (R) generate reactive oxygen species (ROS) that cause oxidative stress which is one of the main drivers of the pathology, although ROS generation during I/R was never demonstrated in vivo due to the lack of suitable methods. In the present study, we record for the first time the dynamics of intracellular pH and H2O2 during I/R in cultured neurons and during experimental stroke in rats using the latest generation of genetically encoded biosensors SypHer3s and HyPer7. We detect a buildup of powerful acidosis in the brain tissue that overlaps with the ischemic core from the first seconds of pathogenesis. At the same time, no significant H2O2 generation was found in the acute phase of ischemia/reperfusion. HyPer7 oxidation in the brain was detected only 24 h later. Comparison of in vivo experiments with studies on cultured neurons under I/R demonstrates that the dynamics of metabolic processes in these models significantly differ, suggesting that a cell culture is a poor predictor of metabolic events in vivo., Highlights • The dynamics of stroke-induced changes in intracellular pH and H2O2 was recorded. • Ischemia leads to immediate acidification of the affected part of the brain. • Only slight increase in H2O2 in the brain tissue occurs during ischemia/reperfusion. • The pronounced increase in H2O2 in the brain occurs next day after I/R. • Neurons show different I/R-induced pH and H2O2 dynamics in vitro and in vivo.

Mitochondria are hubs for calcium and redox signaling; they take up and release calcium depending on their metabolic needs and generate superoxide radicals and hydrogen peroxide (H2O2) during ATP production. Moreover, mitochondria play a central role in hydrogen sulfide (H2S) metabolism. At low concentrations, H2O2 and H2S serve as redox signaling molecules, but their overproduction can cause oxidative stress and cell damage. As an important regulator of mitochondrial bioenergetics, calcium transport needs to be precisely coordinated; a task executed by a number of proteins residing in the outer membrane and inner membrane and in the mitochondrial intramembrane space. In mitochondria, calcium controls redox signaling, however, the redox regulation of calcium transporters is less well understood. Here, we summarize the latest findings on mitochondrial calcium- and redox signaling and focus on describing the role of oxidative thiol modifications in the control of the mitochondrial calcium transport machinery.

Peroxisomes communicate with other cellular compartments by transfer of various metabolites. However, whether peroxisomes are sites for calcium handling and exchange has remained contentious. Here we generated sensors for assessment of peroxisomal calcium and applied them for single cell-based calcium imaging in HeLa cells and cardiomyocytes. We found that peroxisomes in HeLa cells take up calcium upon depletion of intracellular calcium stores and upon calcium influx across the plasma membrane. Further, we show that peroxisomes of neonatal rat cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes can take up calcium in a controlled manner. Our results indicate that peroxisomal and cytosolic calcium signals are tightly interconnected. Hence, peroxisomes may play an important role in shaping cellular calcium dynamics by serving as buffers or sources of intracellular calcium.

CTL and NK cells recognize and eliminate cancer cells. However, immune evasion, down regulation of immune function by the tumor microenvironment, or resistance of cancer cells are a major problem. While CTL and NK cells are both important to eliminate cancer, most studies address them individually. In a new experimental human model, we analysed combined primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, MART-1-specific CTL or NK cells eliminated SK-Mel-5 cells within 24 hours indicating that SK-Mel-5 cells are initially not resistant. However, at lower effector-to-target ratios, which resemble conditions of the immune contexture in human cancer, a significant number of SK-Mel-5 cells survived. Whereas CTL pre-exposure induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, NK cell pre-exposure induced resistance in surviving SK-Mel-5 cells to NK cells but not to MART-1 specific CTL. In contrast, there was even a slight enhancement of CTL cytotoxicity against SK-Mel-5 cells following NK cell pre-exposure. In all other combinations, resistance to subsequent cytotoxicity was higher, if melanoma cells were pre-exposed to larger numbers of CTL or NK cells. Increases in human leukocyte antigen class I expression correlated with resistance to NK cells, while reduction in MART-1 antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. This study quantifies the interdependence of CTL and NK cell cytotoxicity and may guide strategies for efficient CTL-NK cell anti-melanoma therapies.Key points summaryCytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. CTL and NK work in parallel, but most studies address them individually.In a new human experimental model, antigen-specific CTL and NK cell cytotoxicity interdependence against melanoma is shown.Whereas high numbers of antigen-specific CTL and NK cells eliminate all melanoma cells, lower, more physiological numbers induce resistance, in case secondary CTL or NK cell exposure follow initial CTL cell exposure or if secondary NK cell exposure follows initial NK cell exposure; only if secondary CTL exposure follows initial NK cell exposure no resistance of melanoma but even a slight enhancement of cytotoxicity was observed.Alterations in HLA-I expression correlated with resistance to NK cells, while reduction in antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous antigen.The results should help to better understand and optimize immune therapies against cancer.Graphical abstract

Hydrogen peroxide (H(2)O(2)) is a key redox intermediate generated within cells. Existing probes for H(2)O(2) have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are either not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast and ultrasensitive ratiometric H(2)O(2) probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H(2)O(2) diffusion from the mitochondrial matrix, to find a functional output of H(2)O(2) gradients in polarized cells, and to prove the existence of H(2)O(2) gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H(2)O(2) imaging in various biological contexts.

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential-driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulfide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2–SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis., Graphical abstract Image 1, Highlights • Loss of COA6 affects respiratory chain complexes IV and I. • Decreased membrane potential-driven protein import due to loss of COA6. • Cysteine residues in the CX3CXNH motif of SCO2 mediate COA6 interaction. • COA6 acts as thiol reductase for copper metallochaperones during CuA biogenesis.

Controlled delivery of molecules interfering specifically with target activities in a cell of interest can be a powerful tool for experimental manipulation, because it can be administered at a defined time point and does not require genetic transformation, which in some systems is difficult and time consuming. Peptides as versatile tools that can be tailored for binding numerous binding partners, are of special interest. However, their passage through membranes, their intracellular targeting, and their sensitivity to proteases is limiting. The use of peptoids, where cationic amino-acid side chains are linked to nitrogen (rather than to carbon) of the peptide bond, can circumvent these limitations, because they are not cleavable by proteases. In the current work, we provide a proof-of-concept that such Trojan Peptoids, the plant PeptoQ, can be used to target a functional cargo (i.e. a rhodamine-labelled peptoid and a coenzyme Q10 derivative) into mitochondria of tobacco BY-2 cells as experimental model. We show that the uptake is specific for mitochondria, rapid, dose-dependent, and requires clathrin-mediated endocytosis, as well as actin filaments, while microtubules seem to be dispensable. Viability of the treated cells is not affected, and they show better survival under salt stress, a condition that perturbs oxidative homeostasis in mitochondria. In congruence with improved homeostasis, we observe that the salt induced accumulation of superoxide is mitigated and even inverted by pretreatment with PeptoQ. Using double labelling with appropriate fluorescent markers, we show that targeting of this Trojan Peptoid to the mitochondria is not based on a passage through the plasma membrane (as thought hitherto), but on import via endocytotic vesicles and subsequent accumulation in the mitochondrial intermembrane space, from where it can enter the matrix, e.g. when the permeability of the inner membrane is increased under salt stress.

Aims: H2O2 is produced by all eukaryotic cells under physiological and pathological conditions. Due to its enormous relevance for cell signaling at low concentrations and antipathogenic function at high concentrations, precise quantification of extracellular local hydrogen peroxide concentrations ([H2O2]) originating from single cells is required. Results: Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [H2O2] kinetics originating from single primary human monocytes (MCs) ex vivo. For the electrochemical techniques square wave voltammetry, cyclic and linear scan voltammetry, and chronoamperometry, detection limits for [H2O2] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [H2O2] 5–8 μm above a single MC increased by 3.4 nM/s within the first 10 min before reaching a plateau. After extracellular addition of H2O2 to an unstimulated MC, the local [H2O2] decreased on average by 4.2 nM/s due to degradation processes of the cell. Using the scanning mode of the setup, we found that H2O2 is evenly distributed around the producing cell and can still be detected up to 30 μm away from the cell. The electrochemical single-cell measurements were validated in MC populations using electron spin resonance spectroscopy and the Amplex® UltraRed assay. Innovation and Conclusion: We demonstrate a highly sensitive, spatially, and temporally resolved electrochemical approach to monitor dynamics of production and degradation processes for H2O2 separately. Local extracellular [H2O2] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 29, 501–517.

To fulfill its role in protein biogenesis, the endoplasmic reticulum (ER) depends on the Hsp70-type molecular chaperone BiP, which requires a constant ATP supply. However, the carrier that catalyzes ATP uptake into the ER was unknown. Here, we report that our screen of gene expression datasets for member(s) of the family of solute carriers that are co-expressed with BiP and are ER membrane proteins identifies SLC35B1 as a potential candidate. Heterologous expression of SLC35B1 in E. coli reveals that SLC35B1 is highly specific for ATP and ADP and acts in antiport mode. Moreover, depletion of SLC35B1 from HeLa cells reduces ER ATP levels and, as a consequence, BiP activity. Thus, human SLC35B1 may provide ATP to the ER and was named AXER (ATP/ADP exchanger in the ER membrane). Furthermore, we propose an ER to cytosol low energy response regulatory axis (termed lowER) that appears as central for maintaining ER ATP supply., Endoplasmic reticulum (ER) functioning requires a constant supply of ATP, but the exchanger required for ATP uptake into the ER is unknown. Here, the authors report that SLC35B1, here named AXER, or ATP/ADP exchanger into the ER, can transport ATP into the ER.

Oxidative modifications of cellular building blocks such as proteins, lipids, and DNA have a major impact on cell behavior, fate, and clinical outcome. Reactive oxygen species (ROS) are important factors that influence these redox processes. Calcium ion (Ca