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Graphical abstract, An improved procedure for solid phase coupling of Boc-aminooxyacetic acid to peptides is described. By avoiding base-containing activation mixtures which cause over-acylation, it practically suppresses this unwanted side reaction and leads to near quantitative yields of Aoa-peptides, useful as glycoprobe precursors in glycomic studies.

Synthetic analogues of the bradykinin potentiating nonapeptide BPP 9α indicate significantly different structural requirements for potentiation of the bradykinin (BK)-induced smooth muscle contraction (GPI) and the inhibition of isolated somatic angiotensin I-converting enzyme (ACE). The results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation. Our results indicate a stimulation of inositol phosphates (IP n ) formation independently from the B 2 receptor. Furthermore, the results with La 3+ support the role of extracellular Ca 2+ and its influx through corresponding channels. The missing effect of calyculin on the GPI disproves the role of phosphatases in the potentiating action. These experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. The chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. Thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor.

The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired.

Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2′-deoxyuridine-5′-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix–loop–helix domain on the protein surface was identified as the putative RNA-binding site., We used crystallography and site-specific cross-linking to study interacting regions of the human thymidylate synthase and its cognate mRNA, which form a complex that represses translation of the enzyme and is implicated in resistance of tumours to antimetabolite chemotherapy.

Graphical abstract, The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.

The carboxypeptidase activity occurring in hog intestinal mucosa is apparently due to two distinct enzymes which may be responsible for the release of basic COOH-terminal amino acids from short peptides. The plasma membrane-bound carboxypeptidase activity which occurs at neutral optimum pH levels was found to be enhanced by CoCl(2) and inhibited by guanidinoethylmercaptosuccinic acid, o-phenanthroline, ethylenediamine tetraacetic acid and cadmium acetate; whereas the soluble carboxypeptidase activity which occurs at an optimum pH level of 5.0 was not activated by CoCl(2) and only slightly inhibited by o-phenanthroline, ethylenediamine tetraacetic acid, NiCl(2) and CdCl(2). The latter activity was presumably due to lysosomal cathepsin B, which is known to be present in the soluble fraction of hog intestinal mucosa. Although the membrane-bound enzyme was evenly distributed along the small intestine, it was not anchored in the phospholipidic bilayer via a glycosyl-phosphatidylinositol moiety, as carboxypeptidase M from human placenta is. The enzyme was not solubilized by phosphatidylinositol-specific phospholipase C, but was solubilized to practically the same extent by several detergents. The purified trypsin-solubilized form is a glycoprotein with a molecular mass of 200 kDa, as determined by performing SDS-PAGE and gel filtration, which differs considerably from the molecular mass of human placental carboxypeptidase M (62 kDa). It was found to cleave lysyl bonds more rapidly than arginyl bonds, which is not so in the case of carboxypeptidase M, and immunoblotting analysis provided further evidence that hog intestinal and human placental membrane-bound carboxypeptidases do not bear much resemblance to each other. Since the latter enzyme has been called carboxypeptidase M, it is suggested that the former might be carboxypeptidase D, the recently described new member of the carboxypeptide B-type family.

Highlights • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • Energy metabolism was the most significantly altered pathway in BDV Hu-H1-infected OL cells. • The Raf/MEK/ERK signaling cascade was significantly perturbed in BDV Hu-H1-infected OL cells. • BDV Hu-H1caused constitutive activation of the ERK1/2 pathway, but cell proliferation was down-regulated at the same time. • BDV Hu-H1 manages to down-regulate cell proliferation, in the presence of activated but not translocated ERK–RSK complex., Borna disease virus (BDV) is a neurotropic, non-cytolytic RNA virus which replicates in the cell nucleus targeting mainly hippocampal neurons, but also astroglial and oligodendroglial cells in the brain. BDV is associated with a large spectrum of neuropsychiatric pathologies in animals. Its relationship to human neuropsychiatric illness still remains controversial. We could recently demonstrate that human BDV strain Hu-H1 promoted apoptosis and inhibited cell proliferation in a human oligodendroglial cell line (OL cells) whereas laboratory BDV strain V acted contrariwise. Here, differential protein expression between BDV Hu-H1-infected OL cells and non-infected OL cells was assessed through a proteomics approach, using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry. A total of 63 differential host proteins were identified in BDV Hu-H1-infected OL cells compared to non-infected OL cells. We found that most changes referred to alterations related to the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, the tricarboxylic acid (TCA) cycle, and glycolysis /gluconeogenesis. By manual querying, two differential proteins were found to be associated with mitogen-activated protein kinase (MAPK) signal transduction. Five key signaling proteins of this pathway (i.e., p-Raf, p-MEK, p-ERK1/2, p-RSK, and p-MSK) were selected for Western blotting validation. p-ERK1/2 and p-RSK were found to be significantly up-regulated, and p-MSK was found to be significantly down-regulated in BDV Hu-H1-infected OL cells compared to non-infected OL cell. Although BDV Hu-H1 constitutively activated the ERK–RSK pathway, host cell proliferation and nuclear translocation of activated pERK in BDV Hu-H1-infected OL cells were impaired. These findings indicate that BDV Hu-H1 infection of human oligodendroglial cells significantly perturbs host energy metabolism, activates the downstream ERK–RSK complex of the Raf/MEK/ERK signaling cascade, and disturbs host cell proliferation possibly through impaired nuclear translocation of pERK, a finding which warrants further research.

While the long-term physiological adaptation of the neuromuscular system to changed functional demands is usually reflected by unilateral skeletal muscle transitions, the progressive degeneration of distinct motor neuron populations is often associated with more complex changes in the abundance and/or isoform expression pattern of contractile proteins and metabolic enzymes. In order to evaluate these intricate effects of primary motor neuronopathy on the skeletal muscle proteome, label-free MS was employed to study global alterations in the WR (wobbler) mouse model of progressive neurodegeneration. In motor neuron disease, fibre-type specification and the metabolic weighting of bioenergetic pathways appear to be strongly influenced by both a differing degree of a subtype-specific vulnerability of neuromuscular synapses and compensatory mechanisms of fibre-type shifting. Proteomic profiling confirmed this pathobiochemical complexity of disease-induced changes and showed distinct alterations in 72 protein species, including a variety of fibre-type-specific isoforms of contractile proteins, metabolic enzymes, metabolite transporters and ion-regulatory proteins, as well as changes in molecular chaperones and various structural proteins. Increases in slow myosin light chains and the troponin complex and a decrease in fast MBP (myosin-binding protein) probably reflect the initial preferential loss of the fast type of neuromuscular synapses in motor neuron disease., The systematic biochemical analysis of muscle from the wobbler mouse model of motor neuron disease suggests that the loss of neuromuscular synapses causes complex changes in the protein profile of contractile tissues, affecting especially the contractile apparatus and energy metabolism.

IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.

The Eph (erythropoietin-producing hepatocellular carcinoma) B receptors are important in a variety of cellular processes through their roles in cell-to-cell contact and signalling; their up-regulation and down-regulation has been shown to have implications in a variety of cancers. A greater understanding of the similarities and differences within this small, highly conserved family of tyrosine kinases will be essential to the identification of effective therapeutic opportunities for disease intervention. In this study, we have developed a route to production of multi-milligram quantities of highly purified, homogeneous, recombinant protein for the kinase domain of these human receptors in Escherichia coli. Analyses of these isolated catalytic fragments have revealed stark contrasts in their amenability to recombinant expression and their physical properties: e.g., a >16°C variance in thermal stability, a 3-fold difference in catalytic activity and disparities in their inhibitor binding profiles. We find EphB3 to be an outlier in terms of both its intrinsic stability, and more importantly its ligand-binding properties. Our findings have led us to speculate about both their biological significance and potential routes for generating EphB isozyme-selective small-molecule inhibitors. Our comprehensive methodologies provide a template for similar in-depth studies of other kinase superfamily members.

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe(225)) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL(3)) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe(229) and Tyr(192) residues were the main cleavage sites. Interestingly, the Phe(225) residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL(3); however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL(3) at only the N-terminus, especially at Phe(33). CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe(225) and Phe(229) residues newly exposed by chymase, but did not cleave Tyr(192). These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).

The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins.

The disulfide bridges of human complement component C3b, derived from C3 by removal of the 77-residue C3a, have been determined. The 10 bridges are Cys537-Cys794, Cys605-Cys640, Cys851-Cys1491, Cys1079-Cys1136, Cys1336-Cys1467, Cys1367-Cys1436, Cys1484-Cys1489, Cys1496-Cys1568, Cys1515-Cys1639, and Cys1615-Cys1624. Including the 3 bridges in C3a (Cys670-Cys698, Cys672-Cys705, and Cys685-Cys706) previously determined by high-resolution X-ray crystallography [Hoppe-Seyler's Z. Physiol. Chem. 361 (1980) 1389-1399] all disulfide bridges of C3 are localized. C3 and the strongly related C4 and C5 are members of the alpha 2-macroglobulin superfamily. The predicted bridge patterns of C4 and C5 are discussed and compared with that of alpha 2-macroglobulin.

The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

The agent responsible for the recent severe acute respiratory syndrome (SARS) outbreak is a previously unidentified coronavirus. While there is a wealth of epidemiological studies, little if any molecular characterization of SARS coronavirus (SCoV) proteins has been carried out. Here we describe the molecular characterization of SCoV E protein, a critical component of the virus responsible for virion envelope morphogenesis. We conclusively show that SCoV E protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to SCoV. The hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of E protein's pivotal role in viral budding. The molecular understanding of this critical component of SCoV may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity.

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214–226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses.

Highly-purified cathepsin D processed human big endothelin 1–38 into endothelin-like fragments but did not appear to generate endothelin 1–21 under the conditions employed. By contrast, human cathepsin E specifically cleaved human big endothelin into endothelin 1–21 and the C-terminal fragment under identical conditions but did not degrade either product further.

Graphical abstract 1j IC50 (BRAF) = 0.24 μM; IC50 (pERK) = 0.58 μM; GI50 (SRB) = 0.87 μM., V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.

Highlights • Ursolic acid and ursocholanic acid are efficiently encapsulated within PMPC-PDPA polymersomes of spherical or tubular morphology. • PMPC-PDPA polymersomes spherical or tubular morphology are found to enter into parkin-mutant fibroblasts causing no deleterious effects. • ATP levels are increased in parkin-mutant fibroblasts after incubation with ursolic or ursocholanic acid loaded PMPC-PDPA polymersomes. • The efficacy of ursolic acid and ursochocholanic acid loaded PMPC-PDPA nanoparticles is similar to DMSO based formulations of the same compounds., Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a class of steroid-like hydrophobic compounds able to rescue mitochondrial function in parkin-mutant fibroblasts. Whilst these possess therapeutic potential, the size and high hydrophobicity of some may limit their ability to penetrate the blood-brain barrier from systemic circulation, something that could be improved by novel drug formulations. In the present study, the steroid-like compounds Ursolic Acid (UA) and Ursocholanic Acid (UCA) were successfully encapsulated within nanoscopic polymersomes formed by poly(2-(methacryloyloxy)ethyl phosphorylcholine)–poly(2-di-isopropylamino)ethyl methacrylate) (PMPC-PDPA) and separated into spherical and tubular morphologies to assess the effects of nanoparticle mediated delivery on drug efficacy. Following incubation with either morphology, parkin-mutant fibroblasts demonstrated time and concentration dependent increases in intracellular ATP levels, resembling those resulting from treatment with nascent UA and UCA formulated in 0.1% DMSO, as used in the original drug screen. Empty PMPC-PDPA polymersomes did not alter physiological measures related to mitochondrial function or induce cytotoxicity. In combination with other techniques such as ligand functionalisation, PMPC-PDPA nanoparticles of well-defined morphology may prove a promising platform for tailoring the pharmacokinetic profile and organ specific bio-distribution of highly hydrophobic compounds.

Mitochondrial oxidative damage contributes to a wide range of pathologies. One therapeutic strategy to treat these disorders is targeting antioxidants to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation. To date only hydrophobic antioxidants have been targeted to mitochondria; however, extending this approach to hydrophilic antioxidants offers new therapeutic and research opportunities. Here we report the development and characterization of MitoC, a mitochondria-targeted version of the hydrophilic antioxidant ascorbate. We show that MitoC can be taken up by mitochondria, despite the polarity and acidity of ascorbate, by using a sufficiently hydrophobic link to the TPP moiety. MitoC reacts with a range of reactive species, and within mitochondria is rapidly recycled back to the active ascorbate moiety by the glutathione and thioredoxin systems. Because of this accumulation and recycling MitoC is an effective antioxidant against mitochondrial lipid peroxidation and also decreases aconitase inactivation by superoxide. These findings show that the incorporation of TPP function can be used to target polar and acidic compounds to mitochondria, opening up the delivery of a wide range of bioactive compounds. Furthermore, MitoC has therapeutic potential as a new mitochondria-targeted antioxidant, and is a useful tool to explore the role(s) of ascorbate within mitochondria., Graphical abstract, Highlights • A mitochondria-targeted ascorbate, MitoC, has been developed • MitoC is taken up by energized mitochondria and there recycled • Mitochondrial oxidative damage is decreased by MitoC • MitoC is a useful reagent to explore mitochondrial oxidative stress