Reduction of actin-related protein complex 2/3 in fetal Down syndrome brain

Down syndrome (DS) patients present with morphological abnormalities in brain development, leading to mental retardation. Given the importance of actin cytoskeleton to form the basis of various cell functions, the regulation of actin system is crucial during brain development. We therefore aimed to study the expression levels of actin binding proteins in fetal DS and control cortex. We evaluated the levels of eight actin binding proteins using the proteomic approach of two-dimensional gel electrophoresis with subsequent mass spectroscopical identification of protein spots. In fetal DS brain we found a significant reduction of the actin-related protein complex 2/3 (Arp2/3) 20 kDa subunit and the coronin-like protein p57, which are involved in actin filament cross-linking and nucleation and capping of actin filaments. We conclude that deficient levels of these proteins may, at least partially, be involved in the dysgenesis of the brain in DS.     

Expression of cystathionine beta-synthase, pyridoxal kinase, and ES1 protein homolog (mitochondrial precursor) in fetal Down syndrome brain

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized by somatic anomalies and mental retardation. The phenotype of DS is thought to result from overexpression of genes encoded on chromosome 21. Although several studies reported mRNA levels of genes localized on chromosome 21, mRNA data cannot be simply extrapolated to protein levels. Furthermore, most protein data have been generated using immunochemical methods. In this study we investigated expression of three proteins (cystathionine beta-synthase (CBS), pyridoxal kinase (PDXK), ES1 protein homolog, mitochondrial precursor (ES1)) whose genes are encoded on chromosome 21 in fetal DS (n = 8; mean gestational age of 19.8 +/- 2.0 weeks) and controls (n = 7; mean gestational age of 18.8 +/- 2.2 weeks) brains (cortex) using proteomic technologies. Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied. Subsequent quantitative analysis of CBS and PDXK revealed levels comparable between DS and controls. By contrast, ES1 was two-fold elevated (P < 0.01) in fetal DS brain. This protein shows significant homology with the E. coli SCRP-27A/ELBB and zebrafish ES1 protein and contains a potential targeting sequence to mitochondria in its N-terminal region. Based on the assumption that structural similarities reflect functional relationship, it may be speculated that ES1 is serving a basic function in mitochondria. Although no function of the human ES1 protein is known yet, ES1 may be a candidate protein involved in the pathogenesis of the brain deficit in DS.     

Molecular changes in fetal Down syndrome brain

Trisomy of human chromosome 21 is a major cause of mental retardation and other phenotypic abnormalities collectively known as Down syndrome. Down syndrome is associated with developmental failure followed by processes of neurodegeneration that are known to supervene later in life. Despite a widespread interest in Down syndrome, the cause of developmental failure is unclear. The brain of a child with Down syndrome develops differently from that of a normal one, although characteristic morphological differences have not been noted in prenatal life. On the other hand, a review of the existing literature indicates that there are a series of biochemical alterations occurring in fetal Down syndrome brain that could serve as substrate for morphological changes. We propose that these biochemical alterations represent and/or precede morphological changes. This review attempts to dissect these molecular changes and to explain how they may lead to mental retardation.     

Manifold reduction of moesin in fetal Down syndrome brain

Moesin is a member of the ERM family and is involved in plasma membrane-actin cytoskeleton cross-linking, resulting cell adhesion, shape, and motility. Because moesin was shown to be highly expressed in growth cones and moesin/radixin suppression led to impaired structure and function of this key element in brain development, we tested the ERM family, ezrin, radixin, and moesin, in fetal Down syndrome (DS) cortex at the early second trimester. We applied two-dimensional gel electrophoresis with subsequent MALDI detection and identification of protein spots followed by quantification with specific software. Moesin was shown to be significantly and manifold reduced in fetal DS brain, whereas reduction of ezrin and radixin did not reach statistical significance. We therefore propose the involvement of moesin in developmental impairment of DS brain, including deteriorated arborisation, neuritic outgrowth, and neuronal migration. Furthermore, decreased moesin is the second F-actin bundling protein, besides drebrin, that is manifold reduced in fetal DS brain.     

Turnover of mitochondrial inner membrane proteins in hepatoma monolayer cultures

The degradation rates of inner mitochondrial membrane proteins were examined in serum-deprived hepatoma cultures. In those nonproliferating cells, the degradation of composite mitochondrial proteins was a first order process with a half-life of 34 h. The half-lives of specific inner mitochondrial membrane polypeptides were determined by examining the 3H/35S of isolated polypeptides from cells given [3H]methionine and [35S]methionine pulses, respectively, before and after a 2-day chase period. The 33 most abundant polypeptides resolved on a bidirectional polyacrylamide gel system showed half-lives ranging from 20 to 100+ h. The 15 polypeptides translated on mitochondrial ribosomes in the presence of inhibitory concentrations of cycloheximide also displayed heterogeneous rates of degradation (t1/2 = 35-100+ h). None of the isolated adenosine triphosphatase (coupling factor F1) or immunoprecipitated cytochrome c oxidase subunits were significantly turned over during the case period. Five of eight cytochrome b-c1 complex subunits, however, were turned over significantly more rapidly (t1/2 = 39-42 h) than the other three (t1/2 = 94+ h). The results demonstrate heterogeneous degradation rates for inner membrane polypeptides, extending in some cases to those within the same respiratory complex.     

A gel-based proteomic method reveals several protein pathway abnormalities in fetal Down syndrome brain

A large series of protein pathway components have been shown to be dysregulated in Down syndrome (DS) brain. No information about pathomechanisms linked to the trisomic state can be obtained from adult DS brain, however, as neurodegeneration occurs from the fourth decade. The aim of the study was to search for protein dysregulation in fetal DS brain before neurodegenerative changes are observed. Proteins were extracted from fetal DS and control frontal cortex, run on 2-DE, followed by quantification of protein spots with subsequent nano-ESI-LC-MS/MS analysis using an ion trap. Aberrant expression of proteins tropomodulin-2, tubulin alpha 1A chain, and alpha-internexin may indicate disturbed synaptic plasticity; fatty acid binding protein 7 suggests impaired maintenance of neuroepithelial cells; and creatine kinase B may reflect defective energy metabolism. RNA binding protein 4B derangement may represent impaired splicing, altered retrotransposon gag domain-containing protein 1 levels may be pointing to altered retrotransposition, and level changes of the potassium-chloride transporter solute carrier family 12 member 7 may lead to impaired ion fluxes with electrophysiological consequences. Taken together, aberrant protein levels from several pathways in fetal DS are challenging as well as fertilizing the area of research and providing the basis for additional neurochemical and functional studies.     

Supramolecular organization of protein complexes in the mitochondrial inner membrane

The liquid state model that envisions respiratory chain complexes diffusing freely in the membrane is increasingly challenged by reports of supramolecular organization of the complexes in the mitochondrial inner membrane. Supercomplexes of complex III with complex I and/or IV can be isolated after solubilisation with mild detergents like digitonin. Electron microscopic studies have shown that these have a distinct architecture and are not random aggregates. A 3D reconstruction of a I1III2IV1 supercomplex shows that the ubiquinone and cytochrome c binding sites of the individual complexes are facing each other, suggesting a role in substrate channelling. Formation of supercomplexes plays a role in the assembly and stability of the complexes, suggesting that the supercomplexes are the functional state of the respiratory chain. Furthermore, a supramolecular organisation of ATP synthases has been observed in mitochondria, where ATP synthase is organised in dimer rows. Dimers can be isolated by mild detergent extraction and recent electron microscopic studies have shown that the membrane domains of the two partners in the dimer are at an angle to each other, indicating that in vivo the dimers would cause the membrane to bend. The suggested role in crista formation is supported by the observation of rows of ATP synthase dimers in the most curved parts of the cristae. Together these observations show that the mitochondrial inner membrane is highly organised and that the molecular events leading to ATP synthesis are carefully coordinated.     

Different import pathways through the mitochondrial intermembrane space for inner membrane proteins.

Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space.     

Reconstitution of the protein insertion machinery of the mitochondrial inner membrane.

We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes. The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane. The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix. The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex.     

ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952-14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.     

Manifold decrease of sialic acid synthase in fetal Down syndrome brain

Summary. Background: Down syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. A large series of biochemical defects have been observed in fetal and adult DS brain that help in unraveling the molecular mechanisms underlying mental retardation. Aims: As sialylation of glycoconjugates plays an important role in brain development, this study aimed to look at the sialic acid metabolism by measuring sialic acid synthase (SAS; N-acetylneuraminate synthase) in early second trimester fetal control and DS brain. Results: In this regard, protein profiling was performed by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass-spectrometry followed by database search and subsequent quantification of spot using specific software. SAS, the enzyme catalyzing synthesis of N-acetyl-neuraminic acid (syn: sialic acid) was represented as a single spot and found to be significantly and manifold reduced (P < 0.01) in cortex of fetuses with DS (control vs. DS, 0.052 ± 0.025 vs. 0.012 ± 0.006). Conclusion: The intriguing finding of the manifold decrease of SAS in DS fetal cerebral cortex as early as in the second trimester of pregnancy may help to explain the brain deficit observed in DS. Decreased SAS may well lead to altered sialic acid metabolism, required for brain development and, more specifically, for sialylation of key brain proteins, including neuronal cell adhesion molecule and myelin associated glycoprotein.     

The inner membrane protein Mdm33 controls mitochondrial morphology in yeast

Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.     

A chloride- and calcium-dependent glutamate-binding protein from rat brain. Identification as a ubiquitous constituent of the inner mitochondrial membrane

We have recently solubilized and enriched a chloride- and calcium-dependent glutamate-binding protein from rat brain (Brose, N., Halpain, S., Suchanek, C., and Jahn, R. (1989) J. Biol. Chem. 264, 9619-9625). The partially purified protein fraction, containing two major protein components of 51,000 Da and 105,000 Da, was used to generate a rabbit antiserum. This serum quantitatively precipitated the binding activity from membrane extracts. Small amounts of the antiserum inhibited glutamate binding when chloride was absent from the incubation medium. Three protein bands were labeled by the serum on immunoblots. From the affinity purified antibody fractions contained in the serum, only the antibodies directed against a 51,000-Da protein were able to immunoprecipitate the binding activity, indicating that this protein is an essential component of the binding site. A survey of a variety of rat tissues by immunoblot analysis revealed a ubiquitous distribution of the protein. After subcellular fractionation of liver and brain, the 51,000-Da protein copurified with mitochondrial markers. Furthermore, exclusive labeling of mitochondria was observed by light and electron microscopy immunocytochemistry. Subfractionation of purified liver mitochondria resulted in a selective association of the protein with inner mitochondrial membranes. Pharmacological characterization of glutamate binding to liver mitochondrial membranes revealed a pattern almost identical to that of the chloride- and calcium-dependent glutamate-binding site in rat brain.     

Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores

Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of 20 nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F₀F₁-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared.     

Insertion into the mitochondrial inner membrane of a polytopic protein, the nuclear-encoded Oxa1p.

Oxa1p, a nuclear-encoded protein of the mitochondrial inner membrane with five predicted transmembrane (TM) segments is synthesized as a precursor (pOxa1p) with an N-terminal presequence. It becomes imported in a process requiring the membrane potential, matrix ATP, mt-Hsp70 and the mitochondrial processing peptidase (MPP). After processing, the negatively charged N-terminus of Oxa1p (approximately 90 amino acid residues) is translocated back across the inner membrane into the intermembrane space and thereby attains its native N(out)-C(in) orientation. This export event is dependent on the membrane potential. Chimeric preproteins containing N-terminal stretches of increasing lengths of Oxa1p fused on mouse dehydrofolate reductase (DHFR) were imported into isolated mitochondria. In each case, their DHFR moieties crossed the inner membrane into the matrix. Thus Oxa1p apparently does not contain a stop transfer signal. Instead the TM segments are inserted into the membrane from the matrix side in a pairwise fashion. The sorting pathway of pOxa1p is suggested to combine the pathways of general import into the matrix with a bacterial-type export process. We postulate that at least two different sorting pathways exist in mitochondria for polytopic inner membrane proteins, the evolutionarily novel pathway for members of the ADP/ATP carrier family and a conserved Oxa1p-type pathway.     

Taz1, an outer mitochondrial membrane protein, affects stability and assembly of inner membrane protein complexes: implications for Barth Syndrome

The Saccharomyces cerevisiae Taz1 protein is the orthologue of human Tafazzin, a protein that when inactive causes Barth Syndrome (BTHS), a severe inherited X-linked disease. Taz1 is a mitochondrial acyltransferase involved in the remodeling of cardiolipin. We show that Taz1 is an outer mitochondrial membrane protein exposed to the intermembrane space (IMS). Transport of Taz1 into mitochondria depends on the receptor Tom5 of the translocase of the outer membrane (TOM complex) and the small Tim proteins of the IMS, but is independent of the sorting and assembly complex (SAM). TAZ1 deletion in yeast leads to growth defects on nonfermentable carbon sources, indicative of a defect in respiration. Because cardiolipin has been proposed to stabilize supercomplexes of the respiratory chain complexes III and IV, we assess supercomplexes in taz1delta mitochondria and show that these are destabilized in taz1Delta mitochondria. This leads to a selective release of a complex IV monomer from the III2IV2 supercomplex. In addition, assembly analyses of newly imported subunits into complex IV show that incorporation of the complex IV monomer into supercomplexes is affected in taz1Delta mitochondria. We conclude that inactivation of Taz1 affects both assembly and stability of respiratory chain complexes in the inner membrane of mitochondria.