Luiz Juliano

Amyloid-β binds to the extracellular cysteine-rich domain of frizzled and inhibits wnt/β-catenin signaling

The amyloid-β peptide (Aβ) plays a major role in neuronal dysfunction and neurotoxicity in Alzheimer disease. However, the signal transduction mechanisms involved in Aβ-induced neuronal dysfunction remain to be fully elucidated. A major current unknown is the identity of the protein receptor(s) involved in neuronal Aβ binding. Using phage display of peptide libraries, we have identified a number of peptides that bind Aβ and are homologous to neuronal receptors putatively involved in Aβ interactions. We report here on a cysteine-linked cyclic heptapeptide (denominated cSP5) that binds Aβ with high affinity and is homologous to the extracellular cysteine-rich domain of several members of the Frizzled (Fz) family of Wnt receptors. Based on this homology, we investigated the interaction between Aβ and Fz. The results show that Aβ binds to the Fz cysteine-rich domain at or in close proximity to the Wnt-binding site and inhibits the canonical Wnt signaling pathway. Interestingly, the cSP5 peptide completely blocks Aβ binding to Fz and prevents inhibition of Wnt signaling. These results indicate that the Aβ-binding site in Fz is homologous to cSP5 and that this is a relevant target for Aβ-instigated neurotoxicity. Furthermore, they suggest that blocking the interaction of Aβ with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in Alzheimer disease.

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N α-Boc or-Fmoc derivative of glutamic acid with the α-carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the α-amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.

A multifunctional serine protease primes the malaria parasite for red blood cell invasion

The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin‐like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain‐like proteins. Here, we report that PfSUB1 has a further role in ‘priming’ the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.

Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates

The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. The whole procedure, including neutrophil purification and kinetic measurements, can be done in 4–5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.

Substrate activation of insulin-degrading enzyme (insulysin). A potential target for drug development.

The rate of the insulin-degrading enzyme (IDE)-catalyzed hydrolysis of the fluorogenic substrate 2-aminobenzoyl-GGFLRKHGQ-ethylenediamine-2,4-dinitrophenyl is increased 2–7-fold by other peptide substrates but not by peptide non-substrates. This increased rate is attributed to a decrease in Km with little effect on Vmax. An ∼2.5-fold increase in the rate of amyloid β peptide hydrolysis is produced by dynorphin B-9. However, with insulin as substrate, dynorphin B-9 is inhibitory. Immunoprecipitation of differentially tagged IDE and gel filtration analysis were used to show that IDE exists as a mixture of dimers and tetramers. The equilibrium between dimer and tetramer is concentration-dependent, with the dimer the more active form. Bradykinin shifted the equilibrium toward dimer. Activation of substrate hydrolysis is not seen with a mixed dimer of IDE containing one active subunit and one subunit that is catalytically inactive and deficient in substrate binding. On the other hand, a mixed dimer containing one active subunit and one subunit that is catalytically inactive but binds substrate with normal affinity is activated by peptides. These findings suggest that peptides bind to one subunit of IDE and induce a conformational change that shifts the equilibrium to the more active dimer as well as activates the adjacent subunit. The selective activation of IDE toward amyloid β peptide relative to insulin suggests the potential for development of compounds that increase IDE activity toward amyloid β peptide as a therapeutic intervention for the treatment of Alzheimers disease.

Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins

Five intramolecularly quenched fluorogenic substrates for arginyl hydrolases with the sequence Abz-Phe-Arg-X-Y-EDDnp (X = Arg or Ser; Y = Val, Pro, or Arg) were synthesized by classical solution methods. Kinetics of their hydrolysis by tissue and plasma kallikreins, trypsin, and thrombin characterized Abz-Phe-Arg-Ser-Arg-EDDnp as a specific and sensitive substrate for the continuous assay of tissue kallikreins while Abz-Phe-Arg-Arg-Pro-EDDnp was the best substrate for human plasma kallikrein. The five peptides were poor substrates for trypsin and resistant to thrombin.

Metagenomic Analysis of a Tropical Composting Operation at the São Paulo Zoo Park Reveals Diversity of Biomass Degradation Functions and Organisms

Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the São Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders.

Substrate Specificity of Human Kallikrein 6 SALT AND GLYCOSAMINOGLYCAN ACTIVATION EFFECTS

Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km = 38,667 s–1 mm–1). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Aβ amyloid peptide, reactive center loop of α1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1–4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.

Evidence for the role of neurogenic inflammation components in trypsin-elicited scratching behaviour in mice

We investigated the mechanisms underlying the pruritogenic response induced by trypsin in mice, to assess the relevance of neurogenic inflammation components in this response.

A glimpse on biological activities of tellurium compounds

Tellurium is a rare element which has been regarded as a toxic, non-essential trace element and its biological role is not clearly established to date. Besides of that, the biological effects of elemental tellurium and some of its inorganic and organic derivatives have been studied, leading to a set of interesting and promising applications. As an example, it can be highlighted the uses of alkali-metal tellurites and tellurates in microbiology, the antioxidant effects of organotellurides and diorganoditellurides and the immunomodulatory effects of the non-toxic inorganic tellurane, named AS-101, and the plethora of its uses. Inasmuch, the nascent applications of organic telluranes (organotelluranes) as protease inhibitors and its applications in disease models are the most recent contribution to the scenario of the biological effects and applications of tellurium and its compounds discussed in this manuscript.