Acetylation controls Notch3 stability and function in T-cell leukemia.

Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R(1692-1731) mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R(1692-1731) mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.

Redox proteomics in aging rat brain: involvement of mitochondrial reduced glutathione status and mitochondrial protein oxidation in the aging process.

Increasing evidence supports the notion that increased oxidative stress is a fundamental cause in the aging process and in neurodegenerative diseases. As a result, a decline in cognitive function is generally associated with brain aging. Reactive oxygen species (ROS) are highly reactive intermediates, which can modify proteins, nucleic acids, and polyunsaturated fatty acids, leading to neuronal damage. Because proteins are major components of biological systems and play key roles in a variety of cellular functions, oxidative damage to proteins represents a primary event observed in aging and age-related neurodegenerative disorders. In the present study, with a redox proteomics approach, we identified mitochondrial oxidatively modified proteins as a function of brain aging, specifically in those brain regions, such as cortex and hippocampus, that are commonly affected by the aging process. In all brain regions examined, many of the identified proteins were energy-related, such as pyruvate kinase, ATP synthase, aldolase, creatine kinase, and α-enolase. These alterations were associated with significant changes in both cytosolic and mitochondrial redox status in all brain regions analyzed. Our finding is in line with current literature postulating that free radical damage and decreased energy production are characteristic hallmarks of the aging process. In additon, our results further contribute to identifying common pathological pathways involved both in aging and in neurodegenerative disease development.

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry.

The conversion of the cellular prion protein (PrP(C)) into a misfolded isoform (PrP(TSE)) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC-MS method that allowed to demonstrate that in the brain of 109(Met/Ile) heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrP(TSE) with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.

TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity.

The vgf gene has been identified as an energy homeostasis regulator. Vgf encodes a 617-aa precursor protein that is processed to yield an incompletely characterized panel of neuropeptides. Until now, it was an unproved assumption that VGF-derived peptides could regulate metabolism. Here, a VGF peptide designated TLQP-21 was identified in rat brain extracts by means of immunoprecipitation, microcapillary liquid chromatography-tandem MS, and database searching algorithms. Chronic intracerebroventricular (i.c.v.) injection of TLQP-21 (15 mug/day for 14 days) increased resting energy expenditure (EE) and rectal temperature in mice. These effects were paralleled by increased epinephrine and up-regulation of brown adipose tissue beta2-AR (beta2 adrenergic receptor) and white adipose tissue (WAT) PPAR-delta (peroxisome proliferator-activated receptor delta), beta3-AR, and UCP1 (uncoupling protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic and anorexigenic neuropeptides was unchanged. Furthermore, in mice that were fed a high-fat diet for 14 days, TLQP-21 prevented the increase in body and WAT weight as well as hormonal changes that are associated with a high-fat regimen. Biochemical and molecular analyses suggest that TLQP-21 exerts its effects by stimulating autonomic activation of adrenal medulla and adipose tissues. In conclusion, we present here the identification in the CNS of a previously uncharacterized VGF-derived peptide and prove that its chronic i.c.v. infusion effected an increase in EE and limited the early phase of diet-induced obesity.

Identification of the pathological prion protein allotypes in scrapie-infected heterozygous bank voles (Clethrionomys glareolus) by high-performance liquid chromatography-mass spectrometry.

Cerebral formation of the pathological isoform of the prion protein (PrP) is a crucial molecular event in prion diseases. The bank vole (Clethrionomys glareolus) is a rodent species highly susceptible to natural scrapie. The PrP gene of bank vole is polymorphic (Met/Ile) at codon 109. Here we show that homozygous 109Met/Met voles have incubation times shorter than heterozygous 109Met/Ile voles after experimental challenge with three different scrapie isolates. An HPLC-MS/MS method was optimized and applied to investigate whether in heterozygous animals both PrP allotypes are able to undergo pathological conversion. The results demonstrate that both allotypes of the prion protein participate to pathological deposition.

Creutzfeldt-Jakob disease associated with the R208H mutation in the prion protein gene.

The authors investigated a patient who died of apparent sporadic Creutzfeldt-Jakob disease (CJD) but carried a R208H substitution in the prion protein (PrP). The patient phenotype was indistinguishable from typical sporadic CJD (i.e., MM1 subtype). In addition, pathologic PrP, PrP(Sc), originated from both the normal and the mutated PRNP allele and had the same characteristics as PrP(Sc) type 1. The authors propose that the R208H mutation influences disease susceptibility without significantly affecting PrP(Sc) properties or disease phenotype.

Human salivary peptides derived from histatins.

Human saliva from a healthy donor was subjected to fractionation by gel chromatography and six pools were collected and analysed by MALDI-TOF-MS and HPLC-ESI-MS. Three peptides, corresponding to 888.3, 687.3, and 524.1 amu and SNYLYDN, YLYDN, and LYDN sequences (determined by automated Edman sequencing), were isolated from pool 4. YLYDN was not previously described in human saliva. The peptides show the common C-terminal sequence of histatin 3 and histatin 1. To exclude the possibility that the three peptides were an artifact of the purification procedure, nine samples of human saliva were collected from healthy donors, immediately acidified with 0.2% TFA, and analysed by RP-HPLC-ESI-MS. The three peptides were detected in all the analyzed samples. SNYLYDN was always found at a concentration higher than that of YLYDN and LYDN. A correlation analysis performed on quantitative data indicated that the three peptides derive only from histatin 3. Other already known histatins also were searched for in the chromatogram. Histatins 1, 2, 3, 5, 6, 7, 8, and 10 were observed, although not in all samples analyzed, whereas other minor histatins were not detected.

Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor.

The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, > 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF-processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C-terminal rat VGF nonapeptide, thus indicating that they included the C-terminus of proVGF. Ectopic expression of the neuroendocrine-specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site-directed mutagenesis was used to identify the KRKRKK(488) motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR(555) site. By the combined use of high-resolution separation techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C-terminal region of proVGF.

Contryphan-Vn: a novel peptide from the venom of the Mediterranean snail Conus ventricosus.

The isolation, purification, and biochemical characterization of the novel peptide Contryphan-Vn, extracted from the venom of the Mediterranean marine snail Conus ventricosus, is reported. Contryphan-Vn is the first Conus peptide described from a vermivorous species and the first purified from the venom of the single Mediterranean Conus species. The amino acid sequence of Contryphan-Vn is As with other contryphans, Contryphan-Vn contains a d-tryptophan residue, is amidated at the C-terminus, and maintains the five-residue intercystine loop size. However, Contryphan-Vn differs from the known contryphans by the insertion of the Asp residue at position 2, by the lack of hydroxylation of Pro(4), and, remarkably, by the presence of the basic residue Lys(6) within the intercystine loop. Although the biological function(s) of contryphans is still unknown, these characteristics suggest distinct molecular target(s) and/or function(s) for Contryphan-Vn.

Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen.

BACKGROUND: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. METHODS: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. RESULTS: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF(3)/Gn(GF)XF(3) (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. CONCLUSION: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.

Effects of temporins on molecular dynamics and membrane permeabilization in lipid vesicles.

Temporins are a novel family of small (10-13 residues) cationic antimicrobial peptides recently isolated from the skin of the European red frog Rana temporaria. Although recently acquired evidence shows that temporins have the potential to kill bacteria by permeabilizing the cytoplasmic membrane, the molecular mechanisms of membrane selectivity and permeabilization are largely unknown. In this study, it was found that temporins cause the release of fluorescent markers entrapped in phosphatidylcholine liposomes in a manner that depends significantly on the size of the solute. Temporins were also shown to lack a detergent-like effect on lipid vesicles, indicating that marker leakage caused by these peptides is not due to total membrane disruption but to perturbation of bilayer organization on a local scale. Binding of temporins to liposomes did lead to a small increase in lipid hydrocarbon chain mobility, as revealed by EPR spectroscopy of nitroxide-labeled fatty acids incorporated in the bilayer. Reference experiments were conducted using the bee venom peptide melittin, whose properties and behavior in natural and model membrane systems are well known. Our findings for temporins are discussed in relation to the models proposed to date to account for the action of antimicrobial peptides on membranes.

Cysteinyldopaenkephalins: synthesis, characterization and binding to bovine brain opioid receptors.

The reaction of opioid peptides with mushroom tyrosinase in the presence of an excess of a thiol compound gives rise to cysteinyldopaenkephalins (CDEnks). The major product is represented by the 5-S-CDEnk (80%) and the minor one by the isomer 2-S-CDEnk (20%). The adducts between leucine-enkephalin (Leu-enk) and cysteine have been isolated by high performance liquid chromatography (HPLC) and identified by amino acid analysis and electrospray ion mass spectrometry. 5-S-CDEnk is able to bind to opioid receptors in bovine brain membranes. Its binding affinity is higher for delta than for mu receptors and about 8-fold lesser than that exploited by Leu-enk. In the presence of the peroxidase/H(2)O(2) system, CDEnks can be converted into the corresponding pheo-opiomelanins.

Maize polyamine oxidase: primary structure from protein and cDNA sequencing.

The first complete amino acid sequence of a flavin-containing polyamine oxidase was solved by a combined approach of nucleotide and peptide sequence analysis. A cDNA of 1737 bp, isolated from maize seedlings by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends strategies, was cloned and its sequence determined. This cDNA contains information for a polypeptide chain of 500 amino acids. Its amino-terminal sequence shows the typical features of secretion signal peptides. The primary structure of the mature protein was independently confirmed by extensive amino acid sequencing. Structural relationships with flavin-containing monoamine oxidases are also discussed.

A peptide derivative (1-70 fragment) of protein SV-IV accelerates human blood coagulation in vitro by selective inhibition of the heparin-induced antithrombin III activation process.

The effect of two peptide derivatives of the rat SV-IV (SV-IV/A: 1-70 fragment; SV-IV/B: 71-90 fragment) on human blood coagulation was investigated. The SV-IV/A fragment was found to possess the same procoagulant activity of the native protein, whereas SV-IV/B retained only a very small fraction of the activity. The results obtained strongly suggest that the procoagulant activity of SV-IV/A is due, like SV-IV, to a selective inhibition of the antithrombin III (AT III) activation process induced by heparin, an essential cofactor of AT III. The main data supporting this hypothesis are the following: 1) the concentration of active serum AT III decreases when SV-IV/A is present in the clotting system; 2) the plasma treatment with SV-IV/A reduces the concentration of AT III, but not that of other plasma serine protease inhibitors; 3) the recalcification time (RT) of the plasma treated with a rabbit anti-AT III polyclonal antiserum is not modified by SV-IV/A; 4) the presence of SV-IV/A in a reaction mixture containing pure fibrinogen, alpha-thrombin, heparin, and AT III neutralizes the thrombin inhibition induced by AT III; 5) the concentration of the thrombin-AT III complexes, occurring in sera obtained from CaCl2-coagulated plasma, is markedly reduced in the presence of SV-IV/A; 6) appropriate concentrations of heparin neutralize the inhibitory effect of either SV-IV/A or SV-IV on the AT III activity in vitro.

Mavicyanin, a stellacyanin-like protein from zucchini peelings: primary structure and comparison with other cupredoxins.

The complete amino-acid sequence of mavicyanin, a small blue copper-containing glycoprotein isolated from zucchini peelings, is presented. The sequence of this cupredoxin was deduced from analysis of peptides obtained after cleavage of the protein with trypsin or Asp-N endoproteinase. Mavicyanin consists of a single polypeptide chain of 108 amino-acid residues. Accurate molecular weight determination by electrospray mass spectrometry (12 752 Da) indicates a mass difference of approx. 1005 Da with respect to the mass of the protein, as determined on the basis of the amino-acid sequence (11747 Da). This difference was tentatively assigned to the carbohydrate moiety, not yet characterized, attached to the protein via an N-linkage to Asn-58 and O-linkages to unidentified Ser/Thr residues. The comparison of the primary structure of mavicyanin with those of other cupredoxins shows that three copper ligands (His-44, Cys-57 and His-90) are conserved, while a glutamine residue (Gln-95), as in stellacyanin, is possibly the fourth ligand. An amino-acid sequence alignment of mavicyanin with copper proteins currently identified as phytocyanins is also proposed, showing same invariant residues in key positions related to the maintenance of the beta-barrel fold and to the active site.

Isolation and characterization of the d1 domain of Pseudomonas aeruginosa nitrite reductase.

Proteolitic digestion of nitrite reductase from Pseudomonas aeruginosa allows to obtain and purify a domain containing only the d1 heme and constituted by two noncovalently bound peptides. This d1 domain catayzes oxygen consumption, and binds carbon monoxide with a kinetic constant slightly higher than the parental dimeric holoenzyme. The capacity to oxidize the physiological substrate, cytochrome c551, is lost, even when the proteolytic c heme domain is added to this reaction mixture. This finding suggests that the two domains do not have a significant affinity for each other, and are kept together only by being part of the same polypeptide.

Amino acid sequence of chicken Cu, Zn-containing superoxide dismutase and identification of glutathionyl adducts at exposed cysteine residues.

The copper, zinc-containing superoxide dismutase electromorphs from chicken erythrocytes have been isolated, their complete amino acid sequence determined and the identity of the protein moieties established. All electromorphs are constituted by a polypeptide chain made of 153 amino acid residues, corresponding to a molecular mass of 15,598 Da. Accurate molecular mass determination by electrospray mass spectrometry of the separated electromorphs unequivocally proved that, in the chicken superoxide dismutase, either one or two cysteine residues/subunit are involved in a mixed disulfide with glutathione. The same post-translational modification has been proven to occur in human superoxide dismutase. A different rate of S-thiolation by endogenous glutathione was also demonstrated to be responsible for charge heterogeneity in cells. Effect of this modification on the catalytic and molecular properties of superoxide dismutases, and possible mechanisms for the S-thiolation process, were also investigated and discussed.

Biological activities of CNBr fragments of a major protein secreted from the rat seminal vesicle epithelium.

Two fragments of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, were produced in vitro by protein cleavage with CNBr at level of the single methionine residue (Met-70) occurring in its polypeptide chain. After their purification by reversed-phase chromatography, SV-IV/A (1-70 fragment) and SV-IV/B (71-90 fragment) were assayed as transglutaminase substrates, and their anti-inflammatory, anti-thrombotic and immunosuppressive properties were evaluated in comparison with native SV-IV. Both fragments retained the SV-IV ability to act as transglutaminase substrates in vitro; fast atom bombardment mass spectrometry analyses of the reaction products pointed to Gln-9 and Gln-86 as acyl donor sites, and to Lys-59, -79 and -80 as acyl acceptor sites. In contrast, only SV-IV/A was shown to possess, like SV-IV, the property of inhibiting both the intensity of the carrageenin-induced rat foot edema and the platelet aggregation induced in vivo by different agents. Finally, the two protein fragments were found to be completely unable to inhibit both the mitogen-induced proliferation of human T cells and the mixed lymphocyte reaction.

In vivo incorporation of copper into the iron-exchangeable and manganese-exchangeable superoxide dismutase from Propionibacterium shermanii. Amino acid sequence and identity of the protein moieties.

Propionibacterium shermanii, an aerotolerant anaerobe, produces an iron-containing or a manganese-containing superoxide dismutase, depending on the metal supplied in the culture medium [Meier, B., Barra, D., Bossa, F., Calabrese, L. & Rotilio, G. (1982) J. Biol. Chem. 257, 13977-13980]. In this study, we demonstrate in vivo incorporation of copper into an active superoxide-dismutase protein when iron and manganese are absent from the growth medium. Superoxide dismutases containing either iron, manganese or copper were isolated from P. shermanii, their complete amino acid sequences were determined and the identity of their protein moieties was established. The polypeptide chain is made up of 201 amino acid residues, corresponding to a molecular mass of 22.6 kDa. From sedimentation equilibrium experiments, the native protein shows a molecular mass of approximately 86 kDa and therefore consists of four identical subunits. The primary structure was compared with the structure of other Fe-superoxide dismutases and Mn-superoxide dismutases, in particular those possessing a strict metal cofactor specificity.

The primary structure of turtle Cu,Zn superoxide dismutase. Structural and functional irrelevance of an insert conferring proteolytic susceptibility.

A copper,zinc superoxide dismutase, has been isolated from the marine turtle Caretta caretta and the complete amino acid sequence obtained. The sequence was determined by isolation and analysis of peptides obtained after cleavage of the carboxymethylated apoenzyme with trypsin or Staphylococcus aureus protease. Turtle superoxide dismutase consists of 166 amino acid residues, which represents the largest number to date for a cytosolic copper,zinc superoxide dismutase. The comparison of this amino acid sequence with that of bovine superoxide dismutase revealed a one-residue C-terminal extension, two single residue insertions and a 12-residue insertion in the N-terminal region, in turtle superoxide dismutase. The new segment consists of a threefold repeating sequence and was found to be the site for selective proteolytic attack by trypsin under native conditions. The biochemical characteristics, the spectroscopic and catalytic properties as well as the thermal stability and the resistance to irreversible denaturation, were carefully examined and were very similar to those of other superoxide dismutases. These results indicate that the presence of the new polypeptide segment does not affect the main folding of the chain and the quaternary structure, nor the functional parameters of turtle superoxide dismutase. The possibility that the new insert constitutes a loop excluded from the protein scaffold providing the framework of the active site is also discussed.