Unrestrictive identification of post-translational modifications in Hevea brasiliensis latex.

The natural rubber latex extracted from the bark of Hevea brasiliensis plays various important roles in modern society. Post-translational modifications (PTMs) of the latex proteins are important for the stability and functionality of the proteins. In this study, latex proteins were acquired from the C-serum, lutoids, and rubber particle layers of latex without using prior enrichment steps; they were fragmented using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron-transfer dissociation (ETD) activation methods. PEAKS 7 were used to search for unspecified PTMs, followed by analysis through PTM prediction tools to crosscheck both results. There were 73 peptides in 47 proteins from H. brasiliensis protein sequences derived from UniProtKB were identified and predicted to be post-translationally modified. The peptides with PTMs identified include phosphorylation, lysine acetylation, N-terminal acetylation, hydroxylation, and ubiquitination. Most of the PTMs discovered have yet to be reported in UniProt, which would provide great assistance in the research of the functional properties of H. brasiliensis latex proteins, as well as being useful biomarkers. The data are available via the MassIVE repository with identifier MSV000082419.

Identification of amino acid residues responsible for taurocyamine binding in mitochondrial taurocyamine kinase from Arenicola brasiliensis.

In order to investigate the residues associated with binding of the substrate taurocyamine in Arenicola mitochondrial taurocyamine kinase (TK), we performed Ala-scanning of the amino acid sequence HTKTV at positions 67-71 on the GS loop, and determined apparent K(m) and V(max) (appK(m) and appV(max), respectively) of the mutant forms for the substrates taurocyamine and glycocyamine. The appK(m) values for taurocyamine of the K69A, T70A and V71A mutants were significantly increased as compared with wild-type, suggesting that these residues are associated with taurocyamine binding. Of special interest is a property of V71A mutant: its catalytic efficiency for glycocyamine was twice that for taurocyamine, indicating that the V71A mutant acts like a glycocyamine kinase, rather than a TK. The role of the amino acid residue K95 of Arenicola MiTK was also examined. K95 was replaced with R, H, Y, I, A and E. K95R, K95H and K95I have a 3-fold higher affinity for taurocyamine, and activity was largely lost in K95E. On the other hand, the K95Y mutant showed a rather unique feature; namely, an increase in substrate concentration caused a decrease in initial velocity of the reaction (substrate inhibition). This is the first report on the key amino acid residues responsible for taurocyamine binding in mitochondrial TK.

Accumulation of aspartic acid421- and glutamic acid391-cleaved tau in neurofibrillary tangles correlates with progression in Alzheimer disease.

Truncations of tau protein at aspartic acid421 (D421) and glutamic acid391 (E391) residues are associated with neurofibrillary tangles (NFTs) in the brains of Alzheimer disease (AD) patients. Using immunohistochemistry with antibodies to D421- and E391-truncated tau (Tau-C3 and MN423, respectively), we correlated the presence of NFTs composed of these truncated tau proteins with clinical and neuropathologic parameters in 17 AD and 23 non-AD control brains. The densities of NFTs composed of D421- or E391-truncated tau correlated with clinical dementia index and Braak staging in AD. Glutamic acid391 tau truncation was prominent in the entorhinal cortex, whereas D421 truncation was prominent in the subiculum, suggesting that NFTs composed of either D421- or E391-truncated tau may be formed mutually exclusively in these areas. Both truncations were associated with the prevalence of the apolipoprotein E epsilon4 allele. By double labeling, intact tau in NFTs was commonly associated with D421-cleaved tau but not with E391-truncated tau; D421-cleaved tau was never associated with E391-truncated tau. These results indicate that tau is not randomly proteolyzed at different domains, and that proteolysis occurs sequentially from the C-terminus to inner regions of tau in AD progression. Identification of NFTs composed of tau at different stages of truncation may facilitate assessment of neurofibrillary pathology in AD.

A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic.

Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.

Differences in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins.

The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikreins were assayed with the bradykinin-containing synthetic peptides Abz-MTEMARRPPGFSPFRSVTVQNH2 (where Abz stands for o-aminobenzoyl) and Abz-MTSVIRRPPGFSPFRAPRV-NH2, which correspond to fragments Met374-Gln393 and Met375-Val393 of mouse and rat LMWKs (low-molecular-mass kininogens) with the addition of Abz. Bradykinin was released from these peptides by the mK1- and rK1-mediated hydrolysis of Arg-Arg and Arg-Ser (or Arg-Ala) peptide bonds. However, owing to preferential hydrolysis of Phe-Arg compared with the Arg-Ala bond in the peptide derived from rat LMWK, hK1 released bradykinin only from the mouse LMWK fragment and preferentially released des-[Arg9]bradykinin from the rat LMWK fragment (Abz-MTSVIRRPPGFSPFRAPRV-NH2). The formation of these hydrolysis products was examined in more detail by determining the kinetic parameters for the hydrolysis of synthetic, internally quenched fluorescent peptides containing six N- or C-terminal amino acids of bradykinin added to the five downstream or upstream residues of mouse and rat kininogens respectively. One of these peptides, Abz-GFSPFRAPRVQ-EDDnp (where EDDnp stands for ethylenediamine 2,4-dinitrophenyl), was preferentially hydrolysed at the Phe-Arg bond, confirming the potential des-[Arg9]bradykinin-releasing activity of hK1 on rat kininogen. The proline residue that is two residues upstream of bradykinin in rat kininogen is, in part, responsible for this pattern of hydrolysis, since the peptide Abz-GFSPFRASRVQ-EDDnp was preferentially cleaved at the Arg-Ala bond by hK1. Since this peptidase accepts the arginine or phenylalanine residue at its S1 subsite, this preference seems to be determined by the prime site of the substrates. These findings also suggested that the effects observed in rats overexpressing hK1 should consider the activation of B1 receptors by des-[Arg9]bradykinin. For further comparison, two short internally quenched fluorescent peptides that bind to hK1 with affinity in the nM range and some inhibitors described previously for hK1 were also assayed with mK1 and rK1.

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants.

To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists.

Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity.

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1 receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.

A ligação de cálcio à troponina C analisada por diálise de fluxo e por espectroscopia de fluorescência / Calcium binding to troponin C analized by flow dialysis and by fluorescence spectroscopy

A troponina C é o componente do complexo heterotrimérico troponina ao qual o Ca²+ se associa. Pearlstone et al. (Biochemistry 31, 6545, (1992)) utilizaram o cDNA da troponina C de músculo esquelético de galinha (sTnC) para construir um mutante onde a fenilalanina da posição 29 foi substituída por triptofano (mutante F29W). Esse mutante permitiu que a ligação de Ca²+ aos sítios regulatórios amino terminais fosse acompanhada através de técnicas fluorescentes. Entretanto, algumas propriedades da sTnC foram alteradas pela mutação (Li et al. (1995) Biochemistry 34, 8330). No presente estudo, ensaios de ligação direta de Ca²+ bem como titulações de Ca²+ seguidas por fluorescência foram usadas para melhor se entenderem...