Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post‐natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.     

Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)     

Iron mobilization from ferritin by chelating agents

The release of iron from horse spleen ferritin by the chelating agents desferrioxamine B, rhodotorulic acid, 2,3-dihydroxybenzoate, 2,2′-bipyridyl and pyridine-2-aldehyde-2-pyridyl hydrazone (Paphy) has been studied in vitro. Ferritin prepared by classical procedures involving thermal denaturation releases its iron less effectively than ferritin isolated by a modified procedure that avoids this step. Desferrioxamine B and rhodotorulic acid are the most effective in releasing iron from both preparations of ferritin. When FMN is added, iron release by desferrioxamine B, rhodotorulic acid, and 2,3-dihydroxybenzoate was effectively blocked, whereas both bipyridyl and Paphy showed a marked simulation. A substantial increase in iron release was also observed for bipyridyl and Paphy with ascorbate; a less important increase was noted for rhodotorulic acid. EDTA exerted a marked inhibition of iron release from ferritin with rhodotorulic acid, 2,3-dihydroxybenzoate, bipyridyl, and Paphy. The effects of citrate and oxalate on iron release by the chelators was small. The effect of the concentration of flavin on iron release from ferritin by bipyridyl and desferrioxamine B have been studied. Desferrioxamine is unable to mobilize FeII from ferritin following reduction by reduced FMN, whereas bipyridyl can rapidly complex the ferrous iron. The results are discussed in the context of our current concepts of storage iron mobilization in the treatment of iron overload.     

A survey of alkaloids in the genera Harpalyce and Brongniartia (Fabaceae—Brongniartieae)

The presence of alkaloids in six species of Brongniartia and three species of Harpalyce is reported. This survey revealed remarkable qualitative differences in the alkaloid profiles of these two genera. B. discolor, B. lupinoides, B. sousae and B. intermedia showed a typical -pyridone pattern, with cytisine, anagyrine and baptifoline as major alkaloids. In leaves of the first three species ormosanine-type alkaloids occurred additionally. B. flava and B. vazquezii are devoid of -pyridones, but accumulate lupanine, hydroxylated lupanines and ester alkaloids. All three species of Harpalyce were similar in accumulating -pyridones, but H. formosa differed from H. brasiliana and H. pringlei in the presence of epilupinine. In general the alkaloid profiles of Brongniartia and Harpalyce show similarities to those of the Australian genera Hovea, Lamprolobium, Plagiocarpus and Templetonia and support therefore the actual concept of the enlarged tribe Brongniartieae.     

The effect of ADF/cofilin and profilin on the dynamics of monomeric actin

The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomains 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.     

Substantial Metabolic Activity of Human Brown Adipose Tissue during Warm Conditions and Cold-Induced Lipolysis of Local Triglycerides

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2,5-Diketo-gluconic acid reductase from Corynebacterium glutamicum: Characterization of stability,catalytic properties and inhibition mechanism for use in vitamin C synthesis

2,5-Diketo-d-gluconic acid (2,5-DKG) reductase is an NADPH-dependent, monomeric aldo-keto reductase (AKR) which catalyzes the reduction of 2,5-DKG to 2-keto-l-gulonic acid (2-KLG) – the immediate precursor of vitamin C. The reaction catalyzed by 2,5-DKG reductase is attractive to bypass several chemical steps and produce vitamin C biocatalytically. In a screening of 22 bacterial strains, nine 2,5-DKG reductase producing bacterial strains were found. The gene of Corynebacterium glutamicum 2,5-DKG reductase was cloned and overexpressed in Escherichia coli. By batch fermentation 409 mg L^?1 of 2,5-DKG reductase with a C-terminal His₆-tag were obtained. The purified 2,5-DKG reductase was characterized in detail. The enzyme is most active in a pH range from 5.0 to 8.0 and its stability is high at temperatures below 35 °C. Catalytic constants for 2,5-DKG and NADPH were determined and a weak inhibition by the product 2-KLG was found. 2,5-DKG reductase activity is strongly inhibited by the common process ions Mg²⁺, Ca²⁺, SO₄^3? and Cl^?, which suggests that these should be avoided in the process. The inhibition mechanism for Cl^? was elucidated. It is a competitive inhibitor with respect to NADPH and a noncompetitive inhibitior with respect to 2,5-DKG.     

One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min

This article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimal process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads in 15 min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81 ng ml⁻¹ with a limit of detection of 0.4 ng ml⁻¹ and an analytical sensitivity of 0.7 ng ml⁻¹. It detects CRP spiked in diluted human whole blood and serum with high analytical precision, as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.     

Synthesis and AcidBase Properties of an Imidazole-Containing Nucleotide Analog, 1-(2'-Deoxy-β-D-ribofuranosyl)imidazole 5'-Monophosphate (dImMP(2-) )

Deletion of the substituted pyrimidine ring in purine-2'-deoxynucleoside 5'-monophosphates leads to the artificial nucleotide analog dImMP(2-) . This analog can be incorporated into DNA to yield, upon addition of Ag(+) ions, a molecular wire. Here, we measured the acidity constants of H(2) (dImMP)(±) having one proton at N(3) and one at the PO$\rm{{_{3}^{2-}}}$ group by potentiometric pH titrations in aqueous solution. The micro acidity constants show that N(3) is somewhat more basic than PO$\rm{{_{3}^{2-}}}$ and, consequently, the (H??dImMP)(-) tautomer with the proton at N(3) dominates to ca. 75%. The calculated micro acidity constants are confirmed by (31) P- and (1) H-NMR chemical shifts. The assembled data allow many quantitative comparisons, e.g., the N(3)-protonated and thus positively charged imidazole residue facilitates deprotonation of the P(O)(2) (OH)(-) group by 0.3?pK units. Information on the intrinsic site basicities also allows predictions about metal-ion binding; e.g., Mg(2+) and Mn(2+) will primarily coordinate to the phosphate group, whereas Ni(2+) and Cu(2+) will preferably bind to N(3). Macrochelate formation for these metal ions is also predicted. The micro acidity constant for N(3)H(+) deprotonation in the (H???dImMP?H)(±) species (pk(a) 6.46) and the M(n+) -binding properties are of relevance for understanding the behavior of dImMP units present in DNA hairpins and metalated duplexes.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.