The mitochondrial-cytosolic pathway of FeS cluster assembly in Arabidopsis thaliana

The aim of this work was to unravel the molecular basis of the connection of mitochondrial and cytosolic FeS assembly. Following on from previous work showing that the mitochondrial ABC-transporter ATM3 is required for cytosolic FeS assembly (Bernard et al., 2009), one part of my project was to provide in-vivo evidence for the substrate of ATM3. I found that the mitochondrial glutathione pool in atm3 seedlings is shifted towards oxidation, indicating accumulation of oxidised glutathione. Furthermore I showed that ATM3 genetically interacts with two enzymes involved in persulfide metabolism (ETHE1, NFS1). This complemented invitro studies by Dr. Schaedler (Schaedler et al., 2014) who showed that oxidised glutathione is a preferred substrate and glutathione carrying additional S0 can be transported by yeast Atm1. To gather further insight into biosynthesis of the ATM3 substrate I investigated the role of a cytosolic and a mitochondrial glutaredoxin (GRXS17, GRXS15). Mutants had minor but specific effects on FeS enzymes and I concluded that the glutaredoxins are not generally involved in de-novo cluster assembly. Another approach was to identify unknown components of mitochondrial-cytosolic persulfide transport. I characterised two mutants with phenotypic resemblance to atm3 mutants. For one, I located a point mutation in the sequence of ATM3 leading to an amino acid exchange in the 6th transmembrane domain and showed that the ATM3 protein was lacking. The second line was a mutant of CNX2, a mitochondrial enzyme necessary for generation of the first MoCo intermediate cPMP. I found ATM3 protein breakdown in cnx2-2 and in a mutant of CNX5 which is involved in MoCo assembly and t-RNA thiomodification. ATM3 was previously suggested to export cPMP (Teschner et al., 2010). My findings give new evidence for a link between ATM3 and MoCo assembly

LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of Leishmania mexicana

BACKGROUND: Leishmania parasites undergo profound morphological and biochemical changes while passing through their life cycle. Protein kinases have been shown to be involved in the differentiation from the extracellular flagellated promastigotes to the intracellular "non-flagellated" amastigotes and vice versa. Moreover, these enzymes are likely involved in the regulation of the proliferation of the different life stages. RESULTS: Here, we characterize LmxMPK4, a mitogen-activated protein (MAP) kinase homologue from Leishmania mexicana. The kinase reveals all sequence motifs for classification as a MAP kinase. LmxMPK4 proved to be active as a recombinant protein. The kinase is expressed in promastigotes and amastigotes. It was impossible to generate homozygous gene deletion mutants for LmxMPK4 in promastigotes. Moreover, amastigotes bearing only an episomal copy of the gene stably retained LmxMPK4 over a prolonged period without antibiotic pressure in infected mice. CONCLUSION: LmxMPK4 is essential for promastigotes and amastigotes of Leishmania. It shows significant amino acid sequence divergence to mammalian MAP kinases. Thus, LmxMPK4 is a promising new drug target

Skin-derived stem cells for wound treatment using cultured epidermal autografts : clinical applications and challenges

The human skin fulfills important barrier, sensory, and immune functions—all of which contribute significantly to health and organism integrity. Widespread skin damage requires immediate treatment and coverage because massive skin loss fosters the invasion of pathogens, causes critical fluid loss, and may ultimately lead to death. Since the skin is a highly immunocompetent organ, autologous transplants are the only viable approach to permanently close a widespread skin wound. Despite the development of tissue-saving autologous transplantation techniques such as mesh and Meek grafts, treatment options for extensive skin damage remain severely limited. Yet, the skin is also a rich source of stem and progenitor cells. These cells promote wound healing under physiological conditions and are potential sources for tissue engineering approaches aiming to augment transplantable tissue by generating cultured epidermal autografts (CEAs). Here, we review autologous tissue engineering strategies as well as transplantation products based on skin-derived stem cells. We further provide an overview of clinical trial activities in the field and discuss relevant translational and clinical challenges associated with the use of these products

Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling

Organisms need to balance sufficient uptake of iron (Fe) with possible toxicity. In plant roots, a regulon of uptake genes is transcriptionally activated under Fe deficiency, but it is unknown how this response is inactivated when Fe becomes available. Here we describe the function of 2 partially redundant E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2, in Arabidopsis thaliana and provide evidence that they target the transcription factor FIT, a key regulator of Fe uptake, for degradation. The btsl double mutant failed to effectively down-regulate the transcription of genes controlled by FIT, and accumulated toxic levels of Fe in roots and leaves. The C-terminal domains of BTSL1 and BTSL2 exhibited E3 ligase activity, and interacted with FIT but not its dimeric partner bHLH39. The BTSL proteins were able to poly-ubiquitinate FIT in vitro and promote FIT degradation in vivo. Thus, posttranslational control of FIT is critical to prevent excess Fe uptake

Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling

Organisms need to balance sufficient uptake of iron (Fe) with possible toxicity. In plant roots, a regulon of uptake genes is transcriptionally activated under Fe deficiency, but it is unknown how this response is inactivated when Fe becomes available. Here we describe the function of 2 partially redundant E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2, in Arabidopsis thaliana and provide evidence that they target the transcription factor FIT, a key regulator of Fe uptake, for degradation. The btsl double mutant failed to effectively down-regulate the transcription of genes controlled by FIT, and accumulated toxic levels of Fe in roots and leaves. The C-terminal domains of BTSL1 and BTSL2 exhibited E3 ligase activity, and interacted with FIT but not its dimeric partner bHLH39. The BTSL proteins were able to poly-ubiquitinate FIT in vitro and promote FIT degradation in vivo. Thus, posttranslational control of FIT is critical to prevent excess Fe uptake

Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria

Mitochondria play a key role in the biosynthesis of two metal cofactors, iron-sulfur (FeS) clusters and molybdenum cofactor (Moco). The two pathways intersect at several points, but a scarcity of mutants has hindered studies to better understand these links. We screened a collection of sirtinol-resistant Arabidopsis thaliana mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of ATM3 , encoding the ATP-binding cassette Transporter of the Mitochondria 3 (systematic name ABCB25), confirming the previously reported role of ATM3 in both FeS cluster and Moco biosynthesis. We also identified a mutant allele in CNX2, Cofactor of Nitrate reductase and Xanthine dehydrogenase 2 , encoding GTP 3′,8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single nucleotide polymorphism in cnx2-2 leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. cnx2-2 plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a cnx2-1 knockout mutant, which could only survive with ammonia as nitrogen source. Measurement of cyclic pyranopterin monophosphate (cPMP) levels by LC-MS/MS showed that this Moco intermediate was below the limit of detection in both cnx2-1 and cnx2-2 , and accumulated more than 10-fold in seedlings mutated in the downstream gene CNX5 . Interestingly, atm3-1 mutants had less cPMP than wild type, correlating with previous reports of a similar decrease in nitrate reductase activity. Taken together, our data functionally characterise CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis

Molecular alterations in the cerebellum of sporadic Creutzfeldt-Jakob disease subtypes with DJ-1 as a key regulator of oxidative stress

Cerebellar damage and granular and Purkinje cell loss in sporadic Creutzfeldt-Jakob disease (sCJD) highlight a critical involvement of the cerebellum during symptomatic progression of the disease. In this project, global proteomic alterations in the cerebellum of brain from the two most prevalent subtypes (MM1 and VV2) of sCJD were studied. Twodimensional gel electrophoresis (2DE) coupled mass spectrometric identification revealed 40 proteins in MM1 and 43 proteins in VV2 subtype to be differentially expressed. Of those, 12 proteins showed common differential expression in their expression between two subtypes. Differentially expressed proteins mainly belonged to (i) cell cycle, gene expression and cell death; (ii) cellular stress response/ oxidativestress(OS)and(iii)signaltransductionandsynaptic functions, related molecular functions. We verified 10 differentially expressed proteins at transcriptional and translational level aswell. Interestingly, protein deglycase DJ-1(an antioxidative protein) showed an increase in its messenger RNA (mRNA) expression in both MM1 and VV2 subtypes but protein expression only in VV2 subtype in cerebellum of sCJD patients. Nuclear translocalization of DJ-1 confirmed its expressional alteration due to OS in sCJD. Downstream experiments showed the activation of nuclear factor erythroid-2 related factor 2 (Nrf2)/antioxidative response element (ARE) pathway. DJ-1 protein concentration was significantly increased during the clinical phase in cerebrospinal fluid of sCJD patients and also at presymptomatic and symptomatic stages in cerebellum of humanized PrP transgenic mice inoculated with sCJD (MM1 and VV2) brain. These results suggest the implication of oxidative stress during the pathophysiology of sCJD

TREM2 expression in the brain and biological fluids in prion diseases

Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune cell surface receptor that regulates microglial function and is involved in the pathophysiology of several neurodegenerative diseases. Its soluble form (sTREM2) results from shedding of the TREM2 ectodomain. The role of TREM2 in prion diseases, a group of rapidly progressive dementias remains to be elucidated. In the present study, we analysed the expression of TREM2 and its main sheddase ADAM10 in the brain of sporadic Creutzfeldt-Jakob disease (sCJD) patients and evaluated the role of CSF and plasma sTREM2 as a potential diagnostic marker of prion disease. Our data indicate that, compared to controls, TREM2 is increased in sCJD patient brains at the mRNA and protein levels in a regional and subtype dependent fashion, and expressed in a subpopulation of microglia. In contrast, ADAM10 is increased at the protein, but not the mRNA level, with a restricted neuronal expression. Elevated CSF sTREM2 is found in sCJD, genetic CJD with mutations E200K and V210I in the prion protein gene (PRNP), and iatrogenic CJD, as compared to healthy controls (HC) (AUC = 0.78-0.90) and neurological controls (AUC = 0.73-0.85), while CSF sTREM2 is unchanged in fatal familial insomnia. sTREM2 in the CSF of cases with Alzheimer's disease, and multiple sclerosis was not significantly altered in our series. CSF sTREM2 concentrations in sCJD are PRNP codon 129 and subtype-related, correlate with CSF 14-3-3 positivity, total-tau and YKL-40, and increase with disease progression. In plasma, sTREM2 is increased in sCJD compared with HC (AUC = 0.80), displaying positive correlations with plasma total-tau, neurofilament light, and YKL-40. We conclude that comparative study of TREM2 in brain and biological fluids of prion diseases reveals TREM2 to be altered in human prion diseases with a potential value in target engagement, patient stratification, and disease monitoring