Channel-forming activity of syringopeptin 25 A in mercury-supported phospholipid monolayers and negatively charged bilayers

Interactions of the cationic lipodepsipeptide syringopeptin 25 A (SP25A) with mercury-supported dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioeleoylphosphatidic acid (DOPA) self-assembled monolayers (SAMs) were investigated by AC voltammetry in 0.1 M KCl at pH 3, 5.4 and 6.8. SP25A targets and penetrates the DOPS SAM much more effectively than the other SAMs not only at pH 6.8, where the DOPS SAM is negatively charged, but also at pH 3, where it is positively charged just as SP25A. Similar investigations at tethered bilayer lipid membranes (tBLMs) consisting of a thiolipid called DPTL anchored to mercury, with a DOPS, DOPA or DOPC distal monolayer on top of it, showed that, at physiological transmembrane potentials, SP25A forms ion channels spanning the tBLM only if DOPS is the distal monolayer. The distinguishing chemical feature of the DOPS SAM is the ionic interaction between the protonated amino group of a DOPS molecule and the carboxylate group of an adjacent phospholipid molecule. Under the reasonable assumption that SP25A preferentially interacts with this ion pair, the selective lipodepsipeptide antimicrobial activity against Gram-positive bacteria may be tentatively explained by its affinity for similar protonated amino-carboxylate pairs, which are expected to be present in the peptide moieties of peptidoglycan strands

Human acylpeptide hydrolase. Studies on its thiol groups and mechanism of action.

The presence of a cysteine residue(s) near the active site of acylpeptide hydrolase was suggested by inactivation of the enzyme with sulfhydryl-modifying agents and by the substantial protection against inactivation afforded by the competitive inhibitor acetylmethionine. 5,5'-dithiobis-(2-nitrobenzoate) titrations of the native and the denatured enzyme together with analysis for cysteic acid after performic acid oxidation showed that the enzyme contained 12 free SH groups and three disulfide bonds/monomer. Chemical modification with radiolabeled iodoacetamide led to the labeling of Cys-30 and Cys-64 suggesting that one or both of these Cys residues are close to the active site. Modification of one or both of them probably inhibits the enzyme either because of a distortion of the active site or because the adducts present a barrier to the efficient diffusion of substrates into and products out of the active site. Studies on the mechanism of action of acylpeptide hydrolase have employed p-nitrophenyl-N-propyl carbamate as a potent active site-directed inhibitor. Enzyme inactivation, which follows pseudo first-order kinetics, is diminished by the competitive inhibitor acetylmethionine. The inhibited enzyme slowly regains activity at a rate that is increased in the presence of the nucleophile hydroxylamine. A general mechanism involving an acyl-enzyme intermediate is supported by evidence for the formation of acetyl-alanyl hydroxamate during hydrolysis of acetyl-alanine p-nitroanilide in the presence of hydroxylamine. The effect on Vmax and Km during this reaction indicate that hydrolysis of the acyl-enzyme intermediate is rate-limiting

The Streptomyces coelicolor small ORF trpM stimulates growth and morphological development and exerts opposite effects on actinorhodin and calcium-dependent antibiotic production

In actinomycetes, antibiotic production is often associated with a morpho-physiological differentiation program that is regulated by complex molecular and metabolic networks. Many aspects of these regulatory circuits have been already elucidated and many others still deserve further investigations. In this regard, the possible role of many small open reading frames (smORFs) in actinomycete morpho-physiological differentiation is still elusive. In Streptomyces coelicolor, inactivation of the smORF trpM (SCO2038) – whose product modulates L-tryptophan biosynthesis – impairs production of antibiotics and morphological differentiation. Indeed, it was demonstrated that TrpM is able to interact with PepA (SCO2179), a putative cytosol aminopeptidase playing a key role in antibiotic production and sporulation. In this work, a S. coelicolor trpM knock-in (Sco-trpMKI) mutant strain was generated by cloning trpM into overexpressing vector to further investigate the role of trpM in actinomycete growth and morpho-physiological differentiation. Results highlighted that trpM: (i) stimulates growth and actinorhodin (ACT) production; (ii) decreases calcium-dependent antibiotic (CDA) production; (iii) has no effect on undecylprodigiosin production. Metabolic pathways influenced by trpM knock- in were investigated by combining two-difference in gel electrophoresis/nanoliquid chromatography coupled to electrospray linear ion trap tandem mass spectrometry (2D- DIGE/nanoLC-ESI-LIT-MS/MS) and by LC-ESI-MS/MS procedures, respectively. These analyses demonstrated that over-expression of trpM causes an over-representation of factors involved in protein synthesis and nucleotide metabolism as well as a down-representation of proteins involved in central carbon and amino acid metabolism. At the metabolic level, this corresponded to a differential accumulation pattern of different amino acids – including aromatic ones but tryptophan – and central carbon intermediates. PepA was also down-represented in Sco-trpMKI. The latter was produced as recombinant His-tagged protein and was originally proven having the predicted aminopeptidase activity. Altogether, these results highlight the stimulatory effect of trpM in S. coelicolor growth and ACT biosynthesis, which are elicited through the modulation of various metabolic pathways and PepA representation, further confirming the complexity of regulatory networks that control antibiotic production in actinomycetes

APE1 polymorphic variants cause persistent genomic stress and affect cancer cell proliferation

Apurinic/apyrimidinic endonuclease 1 (APE1) is the main mammalian AP-endonuclease responsible for the repair of endogenous DNA damage through the base excision repair (BER) pathway. Molecular epidemiological studies have identified several genetic variants associated with human diseases, but a well-defined functional connection between mutations in APE1 and disease development is lacking. In order to understand the biological consequences of APE1 genetic mutations, we examined the molecular and cellular consequences of the selective expression of four non-synonymous APE1 variants (L104R, R237C, D148E and D283G) in human cells. We found that D283G, L104R and R237C variants have reduced endonuclease activity and impaired ability to associate with XRCC1 and DNA polymerase \u3b2, which are enzymes acting downstream of APE1 in the BER pathway. Complementation experiments performed in cells, where endogenous APE1 had been silenced by shRNA, showed that the expression of these variants resulted in increased phosphorylation of histone H2Ax and augmented levels of poly(ADP-ribosyl)ated (PAR) proteins. Persistent activation of DNA damage response markers was accompanied by growth defects likely due to combined apoptotic and autophagic processes. These phenotypes were observed in the absence of exogenous stressors, suggesting that chronic replication stress elicited by the BER defect may lead to a chronic activation of the DNA damage response. Hence, our data reinforce the concept that non-synonymous APE1 variants present in the human population may act as cancer susceptibility alleles

A Novel White Laccase from Pleurotus ostreatus

Abstract Two laccase isoenzymes (POXA1 and POXA2) produced by Pleurotus ostreatus were purified and fully characterized. POXA1 and POXA2 are monomeric glycoproteins with 3 and 9% carbohydrate content, molecular masses of about 61 and 67 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, of about 54 and 59 kDa by gel filtration in native conditions, and of 61 kDa by matrix-assisted laser desorption ionization mass spectrometry (only for POXA1) and pI values of 6.7 and 4.0, respectively. The N terminus and three tryptic peptides of POXA1 have been sequenced, revealing clear homology with laccases from other microorganisms, whereas POXA2 showed a blocked N terminus. The stability of POXA2 as a function of temperature was particularly low, whereas POXA1 showed remarkable high stability with respect to both pH and temperature. Both enzymes oxidize syringaldazine and ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) together with a variety of different substituted phenols and aromatic amines with the concomitant reduction of oxygen, but POXA1 is unable to oxidize guaiacol. Both enzymes were strongly inhibited by sodium azide and thioglycolic acid but not by EDTA. UV/visible absorption spectra, atomic adsorption, and polarographic data indicated the presence of 4 copper atoms/mol of POXA2 but only one copper, two zinc, and one iron atoms were found/mol of POXA1. The neutral pI and the anomalous metal content of POXA1 laccase render this enzyme unique in its structural characteristics. The lack of typical absorbance at 600 nm allows its classification as a "white" laccase

Protective role of carbonic anhydrases III and VII in cellular defense mechanisms upon redox unbalance

Under oxidative stress conditions, several constitutive cellular defense systems are activated, which involve both enzymatic systems and molecules with antioxidant properties such as glutathione and vitamins. In addition, proteins containing reactive sulfhydryl groups may eventually undergo reversible redox modifications whose products act as protective shields able to avoid further permanent molecular oxidative damage either in stressful conditions or under pathological circumstances. After the recovery of normal redox conditions, the reduced state of protein sulfhydryl groups is restored. In this context, carbonic anhydrases (CAs) III and VII, which are human metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate and proton, have been identified to play an antioxidant role in cells where oxidative damage occurs. Both proteins are mainly localized in tissues characterized by a high rate of oxygen consumption, and contain on their molecular surface two reactive cysteine residues eventually undergoing S-glutathionylation. Here, we will provide an overview on the molecular and functional features of these proteins highlighting their implications into molecular processes occurring during oxidative stress conditions

Growth Factor Receptor-bound Protein 2 Interaction with the Tyrosine-phosphorylated Tail of Amyloid β Precursor Protein Is Mediated by Its Src Homology 2 Domain

The sequential processing of the familial disease gene product amyloid beta precursor protein (AbetaPP) by beta- and gamma-secretases generates amyloid beta, which is considered to be the pathogenic factor of Alzheimer's disease, and the AID peptide (AbetaPP intracellular domain). The AID peptide acts as a positive regulator of apoptosis and modulates transcription and calcium release. To gain clues about the molecular mechanisms regulating the function of AbetaPP and AID, proteins interacting with the AID region of AbetaPP have been isolated using the yeast two-hybrid system. Recent evidence indicates that AbetaPP undergoes post-translational modification events in the AID region and that phosphorylation might regulate its affinity for interacting proteins. To test this possibility and to uncover AbetaPP-binding partners whose interaction depends on AbetaPP phosphorylation, we used a proteomic approach. Here we describe a protein, growth factor receptor-bound protein 2 (Grb2), that specifically binds AbetaPP, phosphorylated in Tyr(682). Furthermore, we show that this interaction is direct and that Grb2 binds to phospho-AbetaPP via its Src homology 2 region. Together with the evidence that Grb2 is in complex with AbetaPP in human brains and that these complexes are augmented in brains from Alzheimer's cases, our data indicate that Grb2 may mediate some biological and possibly pathological AbetaPP-AID function

Tetrapandins, a New Class of Scorpion Toxins That Specifically Inhibit Store-operated Calcium Entry in Human Embryonic Kidney-293 Cells

Venoms from 14 snakes and four scorpions were screened for inhibitory activities toward store-operated Ca2+ entry (SOCE) in human embryonic kidney-293 cells. An inhibitory activity was found in venom from the African scorpion Pandinus imperator. The active agent of this venom was purified by gel filtration and reverse-phase high pressure liquid chromatography methods. Sequence information on the purified fraction, by automatic Edman degradation and mass spectrometry analysis, identified the activity as being contained in two tetrapeptides, which we have named tetrapandins. We demonstrate that synthesized tetrapandins have inhibitory activity for SOCE in human embryonic kidney-293 cells while having no effect on either thapsigargin- or carbachol-stimulated release of Ca2+ stores. These toxins should be extremely useful in future studies to determine downstream events regulated by SOCE as well as to determine whether multiple pathways exist for thapsigargin-stimulated Ca2+ entry

Platelet-derived Growth Factor Induces the β-γ-Secretase-mediated Cleavage of Alzheimer's Amyloid Precursor Protein through a Src-Rac-dependent Pathway

The beta-amyloid peptide (Abeta) present in the senile plaques of Alzheimer's disease derives from the cleavage of a membrane protein, named APP, driven by two enzymes, known as beta- and gamma-secretases. The mechanisms regulating this cleavage are not understood. We have developed an experimental system to identify possible extracellular signals able to trigger the cleavage of an APP-Gal4 fusion protein, which is detected by measuring the expression of the CAT gene transcribed under the control of the Gal4 transcription factor, which is released from the membrane upon the cleavage of APP-Gal4. By using this assay, we purified a protein contained in the C6 cell-conditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the gamma-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the activation of a pathway downstream of the PDGF-receptor, which includes the non-receptor tyrosine kinase Src and the small G-protein Rac1. These findings are confirmed by the observation that a constitutively active form of Src increases Abeta generation and that, in cells stably expressing APP, the generation of A is strongly decreased by the Src tyrosine kinase inhibitor PP2