Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

Tumor burden and clonality in multiple intestinal neoplasia mouse/normal mouse aggregation chimeras

Aggregation chimeras were formed between C57BL/6 mice heterozygous for the Apcmin (Min) mutation and wild-type SWR mice, that differ in their Pla2g2a status, a modifier of Apcmin, and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-Apcmin/+ epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (Pla2g2a wild-type) tissue on adenoma formation in the C57BL/6J-Apcmin/+ epithelium. This suggests that the main genetic modifiers of the Min phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-Apcmin/+-derived epithelium, confirming that the germ-line mutation of the mApc gene is necessary to initiate tumorigenesis in this model system, and that the mApc gene acts in a cell autonomous fashion.

Desynchronization of cells on the developmental path triggers the formation of spiral waves of cAMP during Dictyostelium aggregation

Whereas it is relatively easy to account for the formation of concentric (target) waves of cAMP in the course of Dictyostelium discoideum aggregation after starvation, the origin of spiral waves remains obscure. We investigate a physiologically plausible mechanism for the spontaneous formation of spiral waves of cAMP in D. discoideum. The scenario relies on the developmental path associated with the continuous changes in the activity of enzymes such as adenylate cyclase and phosphodiesterase observed during the hours that follow starvation. These changes bring the cells successively from a nonexcitable state to an excitable state in which they relay suprathreshold cAMP pulses, and then to autonomous oscillations of cAMP, before the system returns to an excitable state. By analyzing a model for cAMP signaling based on receptor desensitization, we show that the desynchronization of cells on this developmental path triggers the formation of fully developed spirals of cAMP. Developmental paths that do not correspond to the sequence of dynamic transitions no relay-relay-oscillations-relay are less able or fail to give rise to the formation of spirals.

Folding and aggregation of designed proteins

Protein aggregation is studied by following the simultaneous folding of two designed identical 20-letter amino acid chains within the framework of a lattice model and using Monte Carlo simulations. It is found that protein aggregation is determined by elementary structures (partially folded intermediates) controlled by local contacts among some of the most strongly interacting amino acids and formed at an early stage in the folding process.

Aggregation and disaggregation of senile plaques in Alzheimer disease

We quantitatively analyzed, using laser scanning confocal microscopy, the three-dimensional structure of individual senile plaques in Alzheimer disease. We carried out the quantitative analysis using statistical methods to gain insights about the processes that govern Aβ peptide deposition. Our results show that plaques are complex porous structures with characteristic pore sizes. We interpret plaque morphology in the context of a new dynamical model based on competing aggregation and disaggregation processes in kinetic steady-state equilibrium with an additional diffusion process allowing Aβ deposits to diffuse over the surface of plaques.

The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a β-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

A transient expansion of the native state precedes aggregation of recombinant human interferon-γ

Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-γ (rhIFN-γ) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-γ aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-γ and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-γ native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-γ against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Identification of Darlin, a Dictyostelium Protein with Armadillo-like Repeats that Binds to Small GTPases and Is Important for the Proper Aggregation of Developing Cells

We purified from Dictyostelium lysates an 88-kDa protein that bound to a subset of small GTPases, including racE, racC, cdc42Hs, and TC4ran, but did not bind to R-ras or rabB. Cloning of the gene encoding this 88-kDa protein revealed that it contained multiple armadillo-like repeats most closely related to the mammalian GTP exchange factor smgGDS. We named this protein darlin (Dictyostelium armadillo-like protein). Disruption of the gene encoding darlin demonstrated that this protein is not essential for cytokinesis, pinocytosis, phagocytosis, or development. However, the ability of darlin null cells to aggregate in response to starvation is severely affected. When starved under liquid medium, the mutant cells were unable to form aggregation centers and streams, possibly because of a defect in cAMP relay signaling. This defect was not due to an inability of the darlin mutants to activate adenylate cyclase in response to G protein stimulation. These results suggest that the darlin protein is involved in a signaling pathway that may modulate the chemotactic response during early development.

Selective disruption of protein aggregation by cyclodextrin dimers

β-Cyclodextrin (CD) dimers (n = 11) were synthesized and tested against eight enzymes, seven of which were dimeric or tetrameric, for inhibitor activity. Initial screening showed that only l-lactate dehydrogenase and citrate synthase were inhibited but only by two specific CD dimers in which two β-CDs were linked on the secondary face by a pyridine-2,6-dicarboxylic group. Further investigation suggested that these CD dimers inhibit the activity of l-lactate dehydrogenase and citrate synthase at least in part by disruption of protein–protein aggregation.

Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in Escherichia coli

We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synthase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we characterized previously. In the process of accomplishing this task, we developed a novel PCR procedure, recombinant PCR, which is very useful in joining two overlapping DNA fragments that do not have a common or unique restriction site. The full-length cDNA was cloned in pMAL-c2 for heterologous expression in Escherichia coli as a maltose-binding protein fusion. The recombinant protein was purified by using amylose-resin affinity and hydroxylapatite chromatography. As expected from the coding capacity of the cDNA expressed, the chimeric recombinant protein has a molecular weight of 310,000 and reacts with antibodies against both human FAS and maltose-binding protein. The maltose-binding protein-human FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified from HepG2 cells. Like the native HepG2 FAS, the products of MBP-hFAS are mainly palmitic acid (>90%) and minimal amounts of stearic and arachidic acids. Similarly, a human FAS cDNA encoding domain I (β-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, and β-hydroxyacyl dehydratase) was cloned and expressed in E. coli using pMAL-c2. The expressed fusion protein, MBP-hFAS domain I, was purified to apparent homogeneity (Mr 190,000) and exhibited the activities of the acetyl/malonyl transacylases and the β-hydroxyacyl dehydratase. In addition, a human FAS cDNA encoding domains II and III (enoyl and β-ketoacyl reductases, acyl carrier protein, and thioesterase) was cloned in pET-32b(+) and expressed in E. coli as a fusion protein with thioredoxin and six in-frame histidine residues. The recombinant fusion protein, thioredoxin-human FAS domains II and III, that was purified from E. coli had a molecular weight of 159,000 and exhibited the activities of the enoyl and β-ketoacyl reductases and the thioesterase. Both the MBP and the thioredoxin-His-tags do not appear to interfere with the catalytic activity of human FAS or its partial activities.

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein. A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons. Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear. To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein. The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation. Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation. Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation. However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments. More dramatically, the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.

Huntingtin aggregation monitored by dynamic light scattering

An initial stage of fibrillogenesis in solutions of glutathione S-transferase-huntingtin (GST-HD) fusion proteins has been studied by using dynamic light scattering. Two GST-HD systems with poly-l-glutamine (polyGln) extensions of different lengths (20 and 51 residues) have been examined. For both systems, kinetics of z-average translation diffusion coefficients (Dapp) and their angular dependence have been obtained. Our data reveal that aggregation does occur in both GST-HD51 and GST-HD20 solutions, but that it is much more pronounced in the former. Thus, our approach provides a powerful tool for the quantitative assay of GST-HD fibrillogenesis in vitro.

Biosynthesis of the Pseudomonas polyketide coronafacic acid requires monofunctional and multifunctional polyketide synthase proteins

Coronafacic acid (CFA) is the polyketide component of the phytotoxin coronatine, a virulence factor of the plant pathogen Pseudomonas syringae. Our current knowledge of polyketide biosynthesis largely is based on the analysis of polyketide synthases (PKSs) in actinomycetes and other Gram-positive bacteria. Consequently, the cloning and characterization of the CFA biosynthetic gene cluster will contribute significantly to our knowledge of polyketide synthesis in Pseudomonas. In this report, we describe two genes in the CFA biosynthetic gene cluster that encode PKSs that are structurally and functionally similar to the multifunctional modular PKSs, which catalyze the synthesis of macrolide antibiotics. The CFA PKS genes were overproduced in Escherichia coli and shown to cross-react with antisera made to a modular PKS involved in erythromycin synthesis. A scheme for CFA biosynthesis is presented that incorporates the activities of all proteins in the CFA PKS. In this report a gene cluster encoding a pseudomonad polyketide has been completely sequenced and the deduced gene functions have been used to develop a biosynthetic scheme.

Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein–protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.