Heinrich Heide

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L

Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

APOOL Is a Cardiolipin-Binding Constituent of the Mitofilin/MINOS Protein Complex Determining Cristae Morphology in Mammalian Mitochondria

Mitochondrial cristae morphology is highly variable and altered under numerous pathological conditions. The protein complexes involved are largely unknown or only insufficiently characterized. Using complexome profiling we identified apolipoprotein O (APOO) and apolipoprotein O-like protein (APOOL) as putative components of the Mitofilin/MINOS protein complex which was recently implicated in determining cristae morphology. We show that APOOL is a mitochondrial membrane protein facing the intermembrane space. It specifically binds to cardiolipin in vitro but not to the precursor lipid phosphatidylglycerol. Overexpression of APOOL led to fragmentation of mitochondria, a reduced basal oxygen consumption rate, and altered cristae morphology. Downregulation of APOOL impaired mitochondrial respiration and caused major alterations in cristae morphology. We further show that APOOL physically interacts with several subunits of the MINOS complex, namely Mitofilin, MINOS1, and SAMM50. We conclude that APOOL is a cardiolipin-binding component of the Mitofilin/MINOS protein complex determining cristae morphology in mammalian mitochondria. Our findings further assign an intracellular role to a member of the apolipoprotein family in mammals.

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

MicroRNA-223 Antagonizes Angiogenesis by Targeting β1 Integrin and Preventing Growth Factor Signaling in Endothelial Cells

Rationale: Endothelial cells in situ are largely quiescent, and their isolation and culture are associated with the switch to a proliferative phenotype. Objective: To identify antiangiogenic microRNAs expressed by native endothelial cells that are altered after isolation and culture, as well as the protein targets that regulate responses to growth factors. Methods and Results: Profiling studies revealed that miR-223 was highly expressed in freshly isolated human, murine, and porcine endothelial cells, but those levels decreased in culture. In primary cultures of endothelial cells, vascular endothelial cell growth factor and basic fibroblast growth factor further decreased miR-223 expression. The overexpression of precursor-miR-223 did not affect basal endothelial cell proliferation but abrogated vascular endothelial cell growth factor–induced and basic fibroblast growth factor–induced proliferation, as well as migration and sprouting. Inhibition of miR-223 in vivo using specific antagomirs potentiated postnatal retinal angiogenesis in wild-type mice, whereas recovery of perfusion after femoral artery ligation and endothelial sprouting from aortic rings from adult miR-223−/y animals were enhanced. MiR-223 overexpression had no effect on the growth factor–induced activation of ERK1/2 but inhibited the vascular endothelial cell growth factor–induced and basic fibroblast growth factor–induced phosphorylation of their receptors and activation of Akt. &bgr;1 integrin was identified as a target of miR-223 and its downregulation reproduced the defects in growth factor receptor phosphorylation and Akt signaling seen after miR-223 overexpression. Reintroduction of &bgr;1 integrin into miR-223–ovexpressing cells was sufficient to rescue growth factor signaling and angiogenesis. Conclusions: These results indicate that miR-223 is an antiangiogenic microRNA that prevents endothelial cell proliferation at least partly by targeting &bgr;1 integrin.

Functional Dissection of the Proton Pumping Modules of Mitochondrial Complex I

A catalytically active subcomplex of respiratory chain complex I lacks 14 of its 42 subunits yet retains half of its proton-pumping capacity, indicating that its membrane arm has two pump modules.

The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity

Significance Complex I is the largest membrane protein complex of the respiratory chain. In addition to 14 central subunits conserved from bacteria to man, the mitochondrial enzyme comprises some 30 so-called accessory subunits of largely unknown structure and function. We show that accessory subunit NB4M [human ortholog NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYR protein superfamily, anchors an acyl carrier protein to complex I, thus forming a structurally well-defined subdomain essential for complex I activity. Our study offers structural insights into the functional interplay of several central and accessory subunits that seem to be specifically involved in controlling the catalytic activity of mitochondrial complex I. This also provides the structural basis for human complex I inactivation in HIV-linked LYRM6 deficiency. Mitochondrial complex I is the largest and most complicated enzyme of the oxidative phosphorylation system. It comprises a number of so-called accessory subunits of largely unknown structure and function. Here we studied subunit NB4M [NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYRM family of proteins. Chromosomal deletion of the corresponding gene in the yeast Yarrowia lipolytica caused concomitant loss of the mitochondrial acyl carrier protein subunit ACPM1 from the enzyme complex and paralyzed ubiquinone reductase activity. Exchanging the LYR motif and an associated conserved phenylalanine by alanines in subunit NB4M also abolished the activity and binding of subunit ACPM1. We show, by single-particle electron microscopy and structural modeling, that subunits NB4M and ACPM1 form a subdomain that protrudes from the peripheral arm in the vicinity of central subunit domains known to be involved in controlling the catalytic activity of complex I.

The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity

A thioredoxin-like chloroplast protein of the fructosebisphosphatase-stimulating f-type, but with an unusually high molecular mass of 28 kDa has previously been identified and purified to homogeneity in a fractionation scheme for resolution of the acid- and heat-stable, regular-size (12kDa) thioredoxins of the unicellular green algae, Scenedesmus obliquus. An apparently analogous protein of 26 kDa was described in a cyanobacterium, Anabaena sp., but no such large thioredoxin species f exists in the thioredoxin profiles of higher plants. The structure of the 28 kDa protein, which had been envisaged to represent a precursor, or fusion product of the two more specialized, common chloroplast thioredoxins f and m has now been determined by amino acid sequencing. Although it exhibits virtually all the properties and enzyme-modulating activities of a thioredoxin proper this algal protein, surprisingly, does not belong to the thioredoxin family of small redox proteins but is identical with OEE (oxygen evolving enhancer) protein 1, an auxiliary component of the photosystem II manganese cluster. Extracts of Chlorella vulgaris and Chlamydomonas reinhardtii also contain heat-stable protein fractions of 23-26 kDa capable of specifically stimulating chloroplast fructosebisphosphatase in vitro. In contrast, OEE protein 1 from spinach is not able to modulate FbPase or NADP malate dehydrogenase from spinach chloroplasts. A dual function of the OEE protein in algal photosynthesis is envisaged.

Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes

Platelets from patients with diabetes are hyperreactive and demonstrate increased adhesiveness, aggregation, degranulation, and thrombus formation, processes that contribute to the accelerated development of vascular disease. Part of the problem seems to be dysregulated platelet Ca(2+) signaling and the activation of calpains, which are Ca(2+)-activated proteases that result in the limited proteolysis of substrate proteins and subsequent alterations in signaling. In the present study, we report that the activation of μ- and m-calpain in patients with type 2 diabetes has profound effects on the platelet proteome and have identified septin-5 and the integrin-linked kinase (ILK) as novel calpain substrates. The calpain-dependent cleavage of septin-5 disturbed its association with syntaxin-4 and promoted the secretion of α-granule contents, including TGF-β and CCL5. Calpain was also released by platelets and cleaved CCL5 to generate a variant with enhanced activity. Calpain activation also disrupted the ILK-PINCH-Parvin complex and altered platelet adhesion and spreading. In diabetic mice, calpain inhibition reversed the effects of diabetes on platelet protein cleavage, decreased circulating CCL5 levels, reduced platelet-leukocyte aggregate formation, and improved platelet function. The results of the present study indicate that diabetes-induced platelet dysfunction is mediated largely by calpain activation and suggest that calpain inhibition may be an effective way of preserving platelet function and eventually decelerating atherothrombosis development.

The Polarity Protein Scrib Is Essential for Directed Endothelial Cell Migration

Rationale: Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells. Objective: We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis. Methods and Results: Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin &agr;5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin &agr;5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin &agr;5 whereas surface expression of integrin &agr;V was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin &agr;5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin &agr;5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin &agr;5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP)s843 transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels. Conclusions: Scrib is a novel regulator of integrin &agr;5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis.

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex i

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III2 + IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.