Robert J. Cotter

Morphology and Toxicity of Aβ-(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of Alzheimer's Disease

In the course of analyzing the chemical composition of Alzheimers disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Aβ peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 M guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic Aβ-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Aβ-(1-40/42) were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal killing. It is possible that these potentially pathogenic Aβ-(1-40/42) dimers and trimers from Alzheimers disease amyloid represent the soluble oligomers of Aβ recently described in Alzheimers disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).

Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response

At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)–lipid A with poor Toll-like receptor 4 (TLR4)–stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.

The Sirtuins Hst3 and Hst4p Preserve Genome Integrity by Controlling Histone H3 Lysine 56 Deacetylation

BACKGROUND Acetylation of histone H3 lysine 56 (K56Ac) occurs transiently in newly synthesized H3 during passage through S phase and is removed in G2. However, the physiologic roles and effectors of K56Ac turnover are unknown. RESULTS The sirtuins Hst3p and, to a lesser extent, Hst4p maintain low levels of K56Ac outside of S phase. In hst3 hst4 mutants, K56 hyperacetylation nears 100%. Residues corresponding to the nicotinamide binding pocket of Sir2p are essential for Hst3p function, and H3 K56 deacetylation is inhibited by nicotinamide in vivo. Rapid inactivation of Hst3/Hst4p prior to S phase elevates K56Ac to 50% in G2, suggesting that K56-acetylated nucleosomes are assembled genome-wide during replication. Inducible expression of Hst3p in G1 or G2 triggers deacetylation of mature chromatin. Cells lacking Hst3/Hst4p exhibit many phenotypes: spontaneous DNA damage, chromosome loss, thermosensitivity, and acute sensitivity to genotoxic agents. These phenotypes are suppressed by mutation of histone H3 K56 into a nonacetylatable residue or by loss of K56Ac in cells lacking the histone chaperone Asf1. CONCLUSIONS Our results underscore the critical importance of Hst3/Hst4p in controlling histone H3 K56Ac and thereby maintaining chromosome integrity.

An Inner Membrane Enzyme in Salmonellaand Escherichia coli That Transfers 4-Amino-4-deoxy-l-arabinose to Lipid A INDUCTION IN POLYMYXIN-RESISTANT MUTANTS AND ROLE OF A NOVEL LIPID-LINKED DONOR

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance inEscherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or twol-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on theS. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, oryfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IVA, a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesizel-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132–43144), ArnT utilizes the novel lipid undecaprenyl phosphate-α-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L- arabinose, phosphoethanolamine and palmitate

Two-thirds of the lipid A in wild-typeEscherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4′. The remaining lipid A contains a monophosphate substituent at position 4′ and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998)J. Biol. Chem. 273, 12466–12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 °C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-l-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.

Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PmrA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation

Lipid A of Salmonella typhimurium can be resolved into multiple molecular species. Many of these substances are more polar than the predominant hexa-acylated lipid A 1,4′-bisphosphate of Escherichia coli K-12. By using new isolation methods, we have purified six lipid A subtypes (St1 to St6) from wild type S. typhimurium. We demonstrate that these lipid A variants are covalently modified with one or two 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties. Each lipid A species with a defined set of polar modifications can be further derivatized with a palmitoyl moiety and/or a 2-hydroxymyristoyl residue in place of the secondary myristoyl chain at position 3′. The unexpected finding that St5 and St6 contain two l-Ara4N residues accounts for the anomalous structures of lipid A precursors seen in S. typhimurium mutants defective in 3-deoxy-d-manno-octulosonic acid biosynthesis in which only the 1-phosphate group is modified with thel-Ara4N moiety (Strain, S. M., Armitage, I. M., Anderson, L., Takayama, K., Quershi, N., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 16089–16098). Phosphoethanolamine (pEtN)-modified lipid A species are much less abundant than l-Ara4N containing forms in wild typeS. typhimurium grown in broth but accumulate to high levels when l-Ara4N synthesis is blocked inpmrA C pmrE− andpmrA C pmrF− mutants. Purification and analysis of selected compounds demonstrate that one or two pEtN moieties may be present. Our findings show that S. typhimurium contains versatile enzymes capable of modifying both the 1- and 4′-phosphates of lipid A with l-Ara4N and/or pEtN groups. PmrA null mutants of S. typhimurium produce lipid A species without any pEtN or l-Ara4N substituents. However, PmrA is not needed for the incorporation of 2-hydroxymyristate or palmitate.

Time-resolved laser desorption mass spectrometry. I. Desorption of preformed ions

Abstract A laser desorption time-of-flight (LD-TOF) mass spectrometer has been used to study the time-resolved desorption of nonvolatile organic salts following a laser pulse. An electron beam, pulsed prior to the pulse which draws the ions from the source, can be used to examine the neutral species desorbed by the laser. Experiments on tetraalkylammonium halides indicate desorption rates for neutral decomposition products vastly different from those for intact ions and neutral clusters, so that the method can be used to resolve competing thermal processes.

Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens.

The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.

Oxygen Requirement for the Biosynthesis of theS-2-Hydroxymyristate Moiety in Salmonella typhimurium Lipid A FUNCTION OF LpxO, A NEW Fe2+/α-KETOGLUTARATE-DEPENDENT DIOXYGENASE HOMOLOGUE

Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium andPseudomonas aeruginosa, may contain secondaryS-2-hydroxyacyl chains. S. typhimurium has recently been shown to synthesize itsS-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O2. Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of 18O2confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/asparaginyl β-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhimuriumaspartyl/asparaginyl β-hydroxylase homologue (designatedlpxO) was cloned into pBluescriptSK and expressed inEscherichia coli K-12, which does not containlpxO. Analysis of the resulting construct revealed thatlpxO expression is sufficient to induce O2-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO very likely is a novel Fe2+/α-ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends. We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase.

Elevated Aβ42 in Skeletal Muscle of Alzheimer Disease Patients Suggests Peripheral Alterations of AβPP Metabolism

The levels of amyloid-β40 (Aβ40) and Aβ42 peptides were quantified in temporalis muscles and brain of neuropathologically diagnosed Alzheimer disease (AD) and of nondemented individuals. This was achieved by using a novel analytical approach consisting of a combination of fast-performance liquid chromatographic (FPLC) size exclusion chromatography developed under denaturing conditions and europium immunoassay on the 4.0- to 4.5-kd fractions. In the temporalis muscles of the AD and nondemented control groups, the average values for Aβ42 were 15.7 ng/g and 10.2 ng/g (P = 0.010), and for Aβ40 they were 37.8 ng/g and 29.8 ng/g (P = 0.067), respectively. Multiple regression analyses of the AD and control combined populations indicated that 1) muscle Aβ40 and muscle Aβ42 levels were correlated with each other (P < 0.001), 2) muscle Aβ40 levels were positively correlated with age (P = 0.036), and 3) muscle Aβ42 levels were positively correlated with Braak stage (P = 0.042). Other forms of the Aβ peptide were discovered by mass spectrometry, revealing the presence of Aβ starting at residues 1, 6, 7, 9, 10, and 11 and ending at residues 40, 42, 44, 45, and 46. It is possible that in AD the skeletal muscle may contribute to the elevated plasma pool of Aβ and thus indirectly to the amyloid deposits of the brain parenchyma and cerebral blood vessels. The increased levels of Aβ in the temporalis muscles of AD patients suggest that alterations in AβPP and Aβ metabolism may be manifested in peripheral tissues.