Christoph Weise

GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Aβ42

Processing of the amyloid precursor protein (APP) by β‐ and γ‐secretases leads to the generation of amyloid‐β (Aβ) peptides with varying lengths. Particularly Aβ42 contributes to cytotoxicity and amyloid accumulation in Alzheimers disease (AD). However, the precise molecular mechanism of Aβ42 generation has remained unclear. Here, we show that an amino‐acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Aβ species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Aβ42, leave the level of Aβ40 unaffected, but increase Aβ38 and shorter Aβ species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that γ‐secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Aβ42 production.

Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).

To identify possible ligands of the orphan somatostatin‐like receptor 1 (SLC‐1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC‐1 and G protein‐gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N‐terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK‐mediated currents but also by the induction of Ca2+ dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the Gi‐ or Gq‐mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.

Reverse physiology in drosophila: identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family.

By using degenerate oligonucleotide primers deduced from the conserved regions of the mammalian somatostatin receptors, a novel G‐protein‐coupled receptor from Drosophila melanogaster has been isolated exhibiting structural similarities to mammalian somatostatin/galanin/opioid receptors. To identify the bioactive ligand, a ‘reverse physiology’ strategy was used whereby orphan Drosophila receptor‐expressing frog oocytes were screened against potential ligands. Agonistic activity was electrophysiologically recorded as inward potassium currents mediated through co‐expressed G‐protein‐gated inwardly rectifying potassium channels (GIRK). Using this approach a novel peptide was purified from Drosophila head extracts. Mass spectrometry revealed an octapeptide of 925 Da with a sequence Ser‐Arg‐Pro‐Tyr‐Ser‐Phe‐Gly‐Leu‐NH2 reminiscent of insect allatostatin peptides known to control diverse functions such as juvenile hormone synthesis during metamorphosis or visceral muscle contractions. Picomolar concentrations of the synthesized octapeptide activated the cognate receptor response mediated through GIRK1, indicating that we have isolated the 394‐amino‐acid Drosophila allatostatin receptor which is coupled to the Gi/Go class of G proteins.

A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF UDP-N-ACETYL-GLUCOSAMINE 2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE

N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC5.1.3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC2.7.1.60) in rat liver (Hinderlich, S., Stäsche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272, 24313–24318). In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme. An open reading frame of 2166 base pairs encodes 722 amino acids with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both epimerase and kinase activity. Distribution of UDP-GlcNAc 2-epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both specific enzyme activities. Secreting organs (liver, salivary glands, and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-epimerase/ManNAc kinase, whereas significant levels of these activities were absent from other organs (lung, kidney, spleen, brain, heart, skeletal muscle, and testis). Northern blot analysis revealed no UDP-GlcNAc 2-epimerase/ManNAc kinase mRNA in the non-secreting tissues.

Isolated Apolipophorin III from Galleria mellonella Stimulates the Immune Reactions of This Insect.

Apolipophorin III (apoLp-III) was isolated from the haemolymph of last instar larvae of Galleria mellonella. The ultraviolet (u.v.) spectrum and the N-terminal amino acid sequence reveal high similarities with the apoLp-III from Manduca sexta. The protein is heat-stable. The molecular mass of apoLp-III was determined to be 18 077 Da using mass spectrometry. The heat treatment (90 degrees C, 30 min) resulted in a pI shift from 6.6 for the non-heated to 6.1 for the heat-treated apoLp-III without change in the molecular mass, indicating that a conformational change might have been caused by the heat treatment, rather than covalent alterations. Intrahaemocoelic injection of pure apoLp-III into last instar G. mellonella larvae is followed by a dose-dependent increase of antibacterial activity in cell-free haemolymph of treated larvae 24 h after injection. Furthermore, pure apoLp-III enhances the phagocytic activity of isolated haemocytes in vitro. The newly discovered role of apoLp-III in inducing immune-related functions in insects is discussed in regard to the known features of this molecule in lipid metabolism. Arylphorin, another heat-stable protein in G. mellonella haemolymph, was likewise isolated in this study. The protein was identified by N-terminal protein sequencing, the sequence obtained exactly matches the known sequence data for this protein. Copyright 1997 Elsevier Science Ltd. All rights reserved

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31 932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP‐ribose) polymerase 1, however, NMNAT was not modified by poly(ADP‐ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [γ‐32P]ATP. We propose that NMNATs activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.

Regulation of glutamate dehydrogenase by reversible ADP‐ribosylation in mitochondria

Mitochondrial ADP‐ribosylation leads to modification of two proteins of ∼26 and 53 kDa. The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine‐specific ADP‐ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N‐terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [14C]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP‐ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP‐ribose from NAD+ onto the acceptor site. ADP‐ ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP‐ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP‐ribosylated GDH was reactivated by an Mg2+‐dependent mitochondrial ADP‐ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP‐ribosylation.

Homophilic Interactions of the Amyloid Precursor Protein (APP) Ectodomain Are Regulated by the Loop Region and Affect β-Secretase Cleavage of APP

We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDS-labile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited cross-linking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of α-secretory APP increased, soluble β-cleaved APP levels decreased. Because Aβ40 and Aβ42 decreased to similar levels as soluble β-cleaved APP, we conclude either that β-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.

Role of Amyloid-β Glycine 33 in Oligomerization, Toxicity, and Neuronal Plasticity

The aggregation of the amyloid-β (Aβ) peptide plays a pivotal role in the pathogenesis of Alzheimers disease, as soluble oligomers are intimately linked to neuronal toxicity and inhibition of hippocampal long-term potentiation (LTP). In the C-terminal region of Aβ there are three consecutive GxxxG dimerization motifs, which we could previously demonstrate to play a critical role in the generation of Aβ. Here, we show that glycine 33 (G33) of the central GxxxG interaction motif within the hydrophobic Aβ sequence is important for the aggregation dynamics of the peptide. Aβ peptides with alanine or isoleucine substitutions of G33 displayed an increased propensity to form higher oligomers, which we could attribute to conformational changes. Importantly, the oligomers of G33 variants were much less toxic than Aβ42 wild type (WT), in vitro and in vivo. Also, whereas Aβ42 WT is known to inhibit LTP, Aβ42 G33 variants had lost the potential to inhibit LTP. Our findings reveal that conformational changes induced by G33 substitutions unlink toxicity and oligomerization of Aβ on the molecular level and suggest that G33 is the key amino acid in the toxic activity of Aβ. Thus, a specific toxic conformation of Aβ exists, which represents a promising target for therapeutic interventions.

Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

Cisplatin (cisPt), Pt(NH3)2Cl2, is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1’s metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.