Sebastian Franken

Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization

NeuN (neuronal nuclei) is an antigen used widely in research and diagnostics to identify postmitotic neurons. The present study aims at an initial understanding of the molecular nature and functional significance of this as yet ill‐defined antigen. Using isoelectric focusing, both the 46‐ and 48‐kDa isoforms of NeuN can be separated in multiple spots spanning a pH range of 8–10.5, suggesting that they might be phosphorylated. Enzymatic dephosphorylation abolishes NeuN immunoreactivity, confirming that NeuN is indeed a phosphoprotein, and establishing that binding of the defining antibody depends on its state of phosphorylation. Combined biochemical and immunohistochemical analysis show that both the 46‐ and the 48‐kDa NeuN isoforms can be localized to the cell nucleus as well as in the neuronal cytoplasm. Their relative concentration in these compartments is distinct, however, with the 48‐kDa isoform being the predominant isoform in the cytoplasm. Within the nucleus, NeuN is found preferentially in areas of low chromatin density and virtually excluded from areas containing densely packed DNA. The present identification of multiple differentially phosphorylated isoforms of NeuN, together with recent reports on the dependence of NeuN immunoreactivity levels on a variety of physiologic or pathologic signals, suggests a previously unappreciated level of complexity in the regulation of this enigmatic, neuron‐specific antigen.

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

Protein aggregation negatively affects key enzymes of major metabolic pathways in mitochondria. The main mitochondrial chaperones Hsp70 and Hsp60 have a limited protective effect against aggregation. In contrast, the ATP-dependent matrix protease Pim1/LON significantly reduces aggregate levels by preventing the accumulation of damaged polypeptides.

The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies.

Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of polypeptides named after HDGF, which was identified by its mitogenic activity towards fibroblasts. In the present study, we describe a hitherto unknown HRP, termed HRP-4. The cDNA of bovine HRP-4 (bHRP-4) predicts a polypeptide of 235 amino acids. Northern- and Western-blot analyses of various bovine tissues demonstrated that HRP-4 is only expressed in the testis. Recombinantly produced bHRP-4 and murine HDGF (mHDGF) histidine-tagged polypeptides display growth-factor activity for cultured primary human fibroblasts at an optimum concentration of 1 ng/ml in serum-free medium. The growth-factor activity declines with increasing concentrations to reach background levels at 1 microg/ml. The expression of the fusion proteins, bHRP-4-green fluorescent protein and mHDGF-green fluorescent protein, in HEK-293 cells demonstrates nuclear localization of the proteins. bHRP-4 and mHDGF bind to the glycosaminoglycans heparin and heparan sulphate, but not to chondroitin sulphate. Affinity constants determined for these interactions are between 6 and 42 nM. Comparison of the bHRP-4 amino acid sequence with HRP-1-3 and p52/75/lens epithelium-derived growth factor (LEDGF) shows that these proteins share a conserved N-terminal part of 91 amino acids but have C-termini of different lengths and charge. This demonstrates the modular structure of these proteins and allows its classification into three groups based on charge, size and sequence comparison. HRP-4, HRP-1 and HDGF are small acidic proteins, HRP-3 is a small basic protein, and HRP-2 and p52/75/LEDGF are larger basic proteins.

The role of protein quality control in mitochondrial protein homeostasis under oxidative stress

Mitochondria contribute significantly to the cellular production of ROS. The deleterious effects of increased ROS levels have been implicated in a wide variety of pathological reactions. Apart from a direct detoxification of ROS molecules, protein quality control mechanisms are thought to protect protein functions in the presence of elevated ROS levels. The reactivities of molecular chaperones and proteases remove damaged polypeptides, maintaining enzyme activities, thereby contributing to cellular survival both under normal and stress conditions. We characterized the impact of oxidative stress on mitochondrial protein homeostasis by performing a proteomic analysis of isolated yeast mitochondria, determining the changes in protein abundance after ROS treatments. We identified a set of mitochondrial proteins as substrates of ROS‐dependent proteolysis. Enzymes containing oxidation‐sensitive prosthetic groups like iron/sulfur clusters represented major targets of stress‐dependent degradation. We found that several proteins involved in ROS detoxification were also affected. We identified the ATP‐dependent protease Pim1/LON as a major factor in the degradation of ROS‐modified soluble polypeptides localized in the matrix compartment. As Pim1/LON expression was induced significantly under ROS treatment, we propose that this protease system performs a crucial protective function under oxidative stress conditions.

Binding of selenoprotein P to heparin: characterization with surface plasmon resonance.

Abstract The binding of selenoprotein P to glycosaminoglycans using heparin as a model compound was studied by surface plasmon resonance. It was found that heparin contains two binding sites for selenoprotein P, a highaffinity, lowcapacity site (K[tilde operator]1n) and a lowaffinity, highcapacity site (K [tilde operator] 140n). Binding at both sites is sensitive to pH and ionic strength, and the highaffinity site is abolished by histidine carbethoxylation with diethylpyrocarbonate. The pH and salt dependence of binding suggests electrostatic interactions with heparin. The concentrations of selenoprotein P in plasma ([tilde operator]50n) are sufficiently high to facilitate binding of selenoprotein P to proteoglycans on the vascular endothelium, and this may contribute to the formation of a protective barrier against oxidants such as peroxynitrite or hydroperoxides.

Specific downregulation and mistargeting of the lipid raft-associated protein MAL in a glycolipid storage disorder

Metachromatic leukodystrophy (MLD) is a lysosomal lipid storage disease caused by arylsulfatase A deficiency. In MLD patients the sphingolipid sulfatide increasingly accumulates leading to progressive demyelination. We have analysed arylsulfatase A-deficient mice, a MLD mouse model, and we show that accumulation of sulfatide is not restricted to the lysosomal compartment but also occurs in myelin itself. Although, this sulfatide storage did not affect the overall composition of most myelin proteins, it specifically caused a severe reduction of MAL. This demonstrates a regulatory link between sulfatide accumulation and MAL expression and indicates the existence of regulatory mechanisms between lipid and myelin protein synthesis in oligodendrocytes. In addition, in cultured renal epithelial cells, sulfatide accumulation diverts MAL to the late endosomal/lysosomal compartment and thus also affects the intracellular distribution of MAL. The specific reduction and mistargeting of MAL protein as a reaction to sulfatide overload may contribute to the pathogenic mechanisms in metachromatic leukodystrophy.

Expression patterns and different subcellular localization of the growth factors HDGF (hepatoma-derived growth factor) and HRP-3 (HDGF-related protein-3) suggest functions in addition to their mitogenic activity.

HDGF (hepatoma-derived growth factor) and the HRPs (HDGF-related proteins) comprise a family of six proteins which display high identity in their N-terminus, but differ at the C-terminus. Here we investigate the patterns of expression of HDGF and HRP-3, by generating antisera specifically recognizing each growth factor. Whereas HRP-3 protein is expressed only in brain, HDGF can be found in a broad range of tissues, with highest levels in brain, testis, lung and spleen. The expression of HDGF and HRP-3 was found to be regulated during brain development, with highest levels around birth, followed by a decline until postnatal day 9. Interestingly, expression of HRP-3 increases again in adult brain. In situ hybridization and immunohistochemistry of cerebellar, cerebral and hippocampal brain slices showed that expression of both growth factors is not limited to areas of high proliferative activity. Both mRNAs and proteins are expressed in neuronal as well as glial cells. Immunocytochemistry of cultured neocortical neurons revealed that HDGF and HRP-3 can be found in the nucleus as well as the cytoplasm. HDGF is restricted to the neuronal soma, whereas HRP-3 can also be found in neurites. Thus the expression of HDGF and HRP-3 in differentiated cells, post-mitotic neurons and primary cultures of rat neocortex points to functions in brain that might not be limited to proliferation. In addition, their simultaneous expression in the same cell and their different subcellular localization in cultured neurons suggest different functions of HDGF and HRP-3 within single cells.

Collapsin Response Mediator Proteins of Neonatal Rat Brain Interact with Chondroitin Sulfate

Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix.

Metachromatic leukodystrophy: consequences of sulphatide accumulation

Metachromatic leukodystrophy is a lysosomal lipid storage disorder. It is caused by mutations in the gene for arylsulphatase A, an enzyme involved in the degradation of the sphingolipid 3′‐O‐sulphogalactosylceramide (sulphatide). This membrane lipid can be found in various cell types, but in particularly high concentrations in the myelin of the nervous system. Patients suffer from progressive, finally lethal, demyelination due to accumulation of sulphatide. In the nervous system, lipid storage not only affects oligodendrocytes but also neurons and, in addition, leads to astrogliosis and activation of microglia. At the cellular level, lysosomal sulphatide storage also affects the lipid composition of myelin itself and has consequences for the amount and localization of particular myelin membrane‐associated proteins. Here we review data, largely based on an arylsulphatase A knock‐out mouse model of metachromatic leukodystrophy.

Disruption of the gene encoding 3β‐hydroxysterol Δ14‐reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis

Tm7sf2 gene encodes 3β‐hydroxysterol Δ14‐reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Δ14‐unsaturated sterol intermediates during the conversion of lanosterol to cholesterol. The C‐terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Δ14‐reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3β‐hydroxysterol Δ14‐reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3β‐hydroxysterol Δ14‐reductase activity were detectable in liver microsomes of Tm7sf2(−/−) mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2(−/−) mice are able to perform cholesterol biosynthesis. Lamin B receptor 3β‐hydroxysterol Δ14‐reductase activity determined in liver nuclei showed comparable values in wild‐type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2(−/−) mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell‐cycle progression are downregulated in the liver of Tm7sf2(−/−) mice, whereas genes involved in xenobiotic metabolism are upregulated.