Piero Pucci

Syringopeptins, new phytotoxic lipodepsipeptides of Pseudomonas syringae pv. syringae

The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudopmonas syringae pv. syringae has been determined by a combination of chemical methods, 1H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin‐producing strains afforded 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Val‐Ala‐Ala‐Val‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Dhb‐Ala‐Dab‐Dab‐Tyr, with Tyr acylating a Thr to form a macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue. Evidence was obtained that a third syringomycin‐producing strain and a syringotoxin‐producing strain synthesize 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Ala‐Ala‐Val‐Leu‐Ala‐Ala‐Dhb‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Val‐Ala‐Dab‐Dab‐Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue.

The structure of syringomycins A1, E and G

By a combination of 1D and 2D 1H‐ and 13C‐NMR, FAB‐MS, and chemical and enzymatic reactions carried out at the milligram level, it has been demonstrated that syringomycin E, the major phytotoxic antibiotic produced by Pseudomonas syringae pv. syringae, is a new lipodepsipeptide. Its amino acid sequence is Ser‐Ser‐Dab‐Dab‐Arg‐Phe‐Dhb‐4(Cl)Thr‐3(OH)Asp with the β‐carboxy group of the C‐terminal residue closing a macrocyclic ring on the OH group of the N‐terminal Ser, which in turn is N‐acylated by 3‐hydroxydodecanoic acid. Syringomycins A1 and G, two other metabolites of the same bacterium, differ from syringomycin E only in their fatty acid moieties corresponding, respectively, to 3‐hydroxydecanoic and 3‐hydroxytetradecanoic acid.

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from ID and 2D 1H‐ and 13C‐NMR spectra combined with extensive FAB‐MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodep‐sinonapeptide. Its peptide moiety corresponds to Ser‐Dab‐Gly‐Hse‐Om‐aThr‐Dhb‐(3‐OH)Asp‐(4‐Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N‐terminal Ser, which in turn is N‐acetylated by 3‐hydroxytetradecanoic acid.

Mitochondrial chaperone Trap1 and the calcium binding protein Sorcin interact and protect cells against apoptosis induced by antiblastic agents.

TRAP1, a mitochondrial chaperone (Hsp75) with antioxidant and antiapoptotic functions, is involved in multidrug resistance in human colorectal carcinoma cells. Through a proteomic analysis of TRAP1 coimmunoprecipitation complexes, the Ca(2+)-binding protein Sorcin was identified as a new TRAP1 interactor. This result prompted us to investigate the presence and role of Sorcin in mitochondria from human colon carcinoma cells. Using fluorescence microscopy and Western blot analysis of purified mitochondria and submitochondrial fractions, we showed the mitochondrial localization of an isoform of Sorcin with an electrophoretic motility lower than 20 kDa that specifically interacts with TRAP1. Furthermore, the effects of overexpressing or downregulating Sorcin and/or TRAP1 allowed us to demonstrate a reciprocal regulation between these two proteins and to show that their interaction is required for Sorcin mitochondrial localization and TRAP1 stability. Indeed, the depletion of TRAP1 by short hairpin RNA in colorectal carcinoma cells lowered Sorcin levels in mitochondria, whereas the depletion of Sorcin by small interfering RNA increased TRAP1 degradation. We also report several lines of evidence suggesting that intramitochondrial Sorcin plays a role in TRAP1 cytoprotection. Finally, preliminary evidence that TRAP1 and Sorcin are both implicated in multidrug resistance and are coupregulated in human colorectal carcinomas is provided. These novel findings highlight a new role for Sorcin, suggesting that some of its previously reported cytoprotective functions may be explained by involvement in mitochondrial metabolism through the TRAP1 pathway.

Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

Summary Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.

Novel bioactive lipodepsipeptides from Pseudomonas syringae: The pseudomycins

The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon‐generated mutant of a Pseudomonas syringae wild‐type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25‐A and 25‐B, pseudomycins A, B, C, C′ are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to l‐Ser‐d‐Dab‐l‐Asp‐l‐Lys‐l‐Dab‐l‐aThr‐Z‐Dhb‐l‐Asp(3‐OH) ‐l‐Thr(4‐Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N‐terminal Ser. This is in turn N‐acylated by 3,4‐dihydroxytetradecanoate in pseudomycin A, by 3‐hydroxytetradecanoate in pseudomycin B, by 3,4‐dihydroxyhexadecanoate in pseudomycin C, and by 3‐hydroxyhexadecanoate in pseudomycin C′. Some preliminary data on the biological activity of pseudomycin A are reported.

Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1: Interplay between Two Enzymes

ABSTRACT Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.

Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)—the common precursor of the different series of glycosphingolipids—that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer—vesicular and non-vesicular—through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.

Protein fingerprint by fast atom bombardment mass spectrometry: Characterization of normal and variant human haemoglobins

A procedure using fast atom bombardment mass spectrometry was developed for mapping the proteolytic digest of proteins. The procedure was successfully applied to the tryptic peptides of the human beta-globin chain. Almost all the expected peptides were identified by direct analysis of the peptide mixture on the mass spectrometer. Peptide recognition along the beta-globin chain sequence was easily made on the basis of their molecular weight. The general applicability of this mapping procedure in the analysis of haemoglobinopathies was demonstrated by its use for the structural characterization of a variant beta-globin chain.

Proteomic strategies for the identification of proteinaceous binders in paintings

The identification of proteinaceous components in paintings remains a challenging task for several reasons. In addition to the minute amount of sample available, complex and variable chemical composition of the paints themselves, possible simultaneous presence of several binders and contaminants, and degradation of the original materials due to aging and pollution are complicating factors. We proposed proteomic strategies for the identification of proteins in binders of paintings that can be adapted to overcome the requirements and difficulties presented by specific samples. In particular, we worked on (1) the development of a minimally invasive method based on the direct tryptic cleavage of the sample without protein extraction; (2) the use of microwave to enhance the enzymatic digestion yield, followed by the analysis of the peptide mixtures by nanoLC-MS/MS with electrospray ionization (ESI). Moreover, as an additional tool to tackle the problem of contaminating proteins, we exploited the possibility of generating an exclusion list of the mass signals that in a first run had been fragmented and that the mass spectrometer had to ignore for fragmentation in a subsequent run. The methods, tested on model samples, allowed the identification of milk proteins in a sample from paintings attributed to Cimabue and Giotto, thirteenth-century Italian masters, decorating the vaults of the upper church in the Basilica of St. Francis in Assisi, Italy.