Phospholipases A2 isolated from Micrurus lemniscatus coral snake venomBehavioral, electroencephalographic, and neuropathological aspects

The present study evaluated four phospholipase A2 (PLA2) (Mlx-8, Mlx-9, Mlx-11 and Mlx-12) isolated from Micrurus lemniscatus snakevenom (Elapidae). The effects of intrahippocampal administration of these toxins have been determined on behavior, electroencephalography, andneuronal degeneration in rats. These four PLA2 toxins induced motor and EEG alterations in a dose-dependent manner. Behavioral convulsionswere characterized by clonic movements and were often accompanied by EEG alterations. Mlx toxins were convulsive but weakly epileptogenic,since low rates of seizure discharges were observed in EEG records. Neuronal injury seemed to depend on the dose of the toxin used. The highestdoses of toxins caused severe intoxication and death of some animals. The injury of hippocampal cells was characterized by massive neuronal lossand hippocampal gliosis. A high occurrence of compulsive scratching was observed. Moreover, the onset of seizures induced by Mlx toxins wasmarkedly delayed. The similarities between the effects of Mlx PLA2s and those isolated from other Elapidae snakes venoms suggest that their toxicityare probably due to their specific binding to neuronal membranes and to the catalysis of phospholipid hydrolysis, producing lysophospholipidsand fatty acids. These compounds likely disturb the membrane conformation, causing a marked increase in the release of neurotransmitters andconcurrent inhibition of vesicle fission and recycling. These toxins can be a useful tool to investigate properties of endogenous secretory PLA2sand therefore may be important both to study mechanisms involved in neurotransmitter release at nerve terminals and to explain the convulsiveproperties of PLA2s toxins.

Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii revealed from a Duvernoy's (venom) gland transcriptome

We investigated the putative toxins of Philodryas olfersii (Colubridae), a representative of a family of snakes neglected in venom studies despite their growing medical importance. Transcriptomic data of the venom gland complemented by proteomic analysis of the gland secretion revealed the presence of major toxin classes from the Viperidae family, including serine proteases, metalloproteases, C-type lectins, Crisps, and a C-type natriuretic peptide (CNP). Interestingly, the phylogenetic analysis of the CNP precursor showed it as a linker between two related precursors found in Viperidae and Elapidae snakes. We suggest that these precursors constitute a monophyletic group derived from the vertebrate CNPs.

Estudo etnofarmacológico em entorno de floresta urbana como subsídio para a implantação da Fitoterapia no Sistema Único de Saúde / Ethnopharmacological study surrounding urban forest as a resource for the implementation of herbal medicine in the National Health System

RESUMO O estudo etnofarmacológico pode ser definido como exploração científica interdisciplinar dos agentes biologicamente ativos, tradicionalmente utilizados por populações humanas e que fazem parte de um acervo de conhecimento compartilhado. Desta forma o presente estudo teve como objetivo o estudo etnofarmacológico de plantas medicinais, no entorno de floresta urbana na Reserva Biológica Poço D’Anta em Juiz de Fora/MG visando a implantação da fitoterapia no Sistema Único de Saúde. Para este, realizou-se levantamento com três diferentes amostras: profissionais de saúde, domicílios em geral e especialistas locais. Quanto aos profissionais de saúde, pôde-se constatar que nenhum entrevistado soube conceituar o termo “Fitoterápico” e que não conheciam as políticas vigentes. Constatou-se que há aceitabilidade da implantação de Fitoterapia na saúde pública, porém, o conhecimento do tema é limitado. A partir das entrevistas nos domicílios em geral e com os especialistas locais, selecionou-se um total de 20 espécies botânicas para análise estatística e confirmação farmacológica. Esses resultados possibilitaram confrontar o conhecimento cultural com científico, com base em 14 espécies que poderiam ser cultivadas em horto na Reserva Biologica Poço D´Anta, com base em suas relevâncias locais. Os resultados obtidos podem subsidiar a aproximação do saber popular em relação ao científico, servindo de base para manutenção e fomento da implantação da Fitoterapia no sistema único de saúde.

ABSTRACT The ethnopharmacological study can be defined as an interdisciplinary scientific exploration of biologically active agents, traditionally used by human populations and part of a shared body of knowledge. Thus, the current study focused on the ethnopharmacological research of medicinal plants, in the surroundings of the urban forest in the Biological Reserve PoçoD’Anta in Juiz de Fora / MG, aiming on the implementation of the herbal medicine in the Public Health System. For this purpose, a survey was held with three different groups: health professionals, members of the community and local experts.Concerning the health professionals, it could be verified that none of the participants were able to explain the term Phytotherapic and neither they had knowledge about the relevant and applicable policies.The acceptability for the implantation of Phytotherapy for public health use was observed, but the knowledge about this subject is limited. From the interviews with members of the community and local experts, a total of 20 plant species were selected for a statistical analysis and pharmacological confirmation. These results made possible to compare the cultural knowledge with the scientific one, defining 14 species that could be grown in the garden of the Biological Reserve Poço D’Anta, based on their local relevance. The results can support the approximation of the popular knowledge with the scientific one, providing a basis for the maintenance and promotion of the Phytotherapy in the Public Health System.

Extrato de plantas medicinais como carrapaticida de Rhipicephalus (Boophilus) microplus / Herbal extract against Rhipicephalus (Boophilus) microplus ticks

O uso de plantas medicinais tem sido uma prática antiga da humanidade, contribuindo para a divulgação das virtudes terapêuticas de extratos de diferentes vegetais. Na busca de alternativas naturais eficazes para males que prejudicam indiretamente o homem, as plantas medicinais têm sido utilizadas na veterinária na tentativa de eliminar ou reduzir a ação dos carrapatos B. microplus, que podem trazer prejuízos a bovinos. Durante anos têm sido utilizado produtos químicos nesse controle, no entanto, os carrapatos adquirem resistência aos fármacos em decorrência do uso contínuo. Assim, objetivou-se avaliar o efeito de hidrolato e extratos aquosos de carqueja [Baccharis trimera (Less). D.C.], alfavaca (Ocimum gratissimum L.), necroton [Vernonia condensata (Backer) H. Rob.] camomila [Chamomilla recutita (L.) Rauschert], além do óleo essencial de alfavaca (Ocimum gratissimum L.). Os ensaios para larvas de B. microplus foram realizados com impregnação das substâncias em papel filtro. Os resultados obtidos demonstraram que todos os extratos aquosos foram ineficazes; o hidrolato de carqueja e de necroton apresentaram eficiência em torno de 30%, necessitando novos testes para comprovação. Dentre os resultados obtidos, destaca-se o hidrolato de alfavaca que apresentou eficiência de 76,7% na concentração de 100% e o óleo essencial puro de alfavaca que apresentou ação larvicidade 100% indicando o potencial carrapaticida dessa planta, especificamente no combate de B.microplus.

The use of medicinal plants has been a longstanding practice of mankind, helping to spread the therapeutic virtues of different plant extracts, due to their medicinal effects. In the search for effective natural alternatives for ailments that indirectly affect man, medicinal plants have been used in veterinary medicine, in an attempt to eliminate or reduce the action of B. microplus ticks, which can cause damage to cattle. For many years, chemical products have been used in this control. However, after some time ticks acquire drug resistance, as a result of continuous use. Thus, the objective of this paper was to evaluate the effect of hydrolates and aqueous extracts of carqueja [Baccharis trimera (Less). DC], alfavaca (Ocimum gratissimum L.), necroton [Vernonia condensata (Baker) H. Rob.], chamomile [Chamomilla recutita (L.) Rauschert] and also the essential oil of alfavaca (Ocimum gratissimum L.). The assays for larvae of B. microplus were carried out by impregnating the paper filter with the substances. The results showed that all the aqueous extracts were ineffective; the hydrolate of carqueja and necroton presented efficiencies around 30%, requiring further tests to prove. Among the results, we observed that the alfavaca hydrolate showed an efficiency of 76.7% at a concentration of 100% and the pure alfavaca oil showed a larvicidal action of 100%, indicating this plant's potential to reduce ticks, specifically in the control of B. microplus.

Identification of bradykinin-related peptides from Phyllomedusa nordestina skin secretion using electrospray ionization tandem mass spectrometry after a single-step liquid chromatography

Amphibian skin secretions are a source of potential new drugs with medical and biotechnological applications. Rich in peptides produced by holocrine-type serous glands in the integument, these secretions play different roles, either in the regulation of physiological skin functions or in the defense against predators or microorganisms. The aim of the present work was to identify novel peptides with bradykinin-like structure and/or activity present in the skin of Phyllomedusa nordestina. In order to achieve this goal, the crude skin secretion of this frog was pre-fractionated by solid phase extraction and separated by reversed-phase chromatography. The fractions were screened for low-molecular-mass peptides and sequenced by mass spectrometry. It was possible to identify three novel bradykinin-related peptides, namely: KPLWRL-NH2 (Pnor 3), RPLSWLPK (Pnor 5) and VPPKGVSM (Pnor 7) presenting vascular activities as assessed by intravital microscopy. Pnor 3 and Pnor 7 were able to induce vasodilation. On the other hand, Pnor 5 was a potent vasoconstrictor. These effects were reproduced by their synthetic analogues.(AU)

Identification of bradykinin: related peptides from Phyllomedusa nordestina skin secretion using electrospray ionization tandem mass spectrometry after a single-step liquid chromatography

Amphibian skin secretions are a source of potential new drugs with medical and biotechnological applications. Rich in peptides produced by holocrine-type serous glands in the integument, these secretions play different roles, either in the regulation of physiological skin functions or in the defense against predators or microorganisms. The aim of the present work was to identify novel peptides with bradykinin-like structure and/or activity present in the skin of Phyllomedusa nordestina. In order to achieve this goal, the crude skin secretion of this frog was pre-fractionated by solid phase extraction and separated by reversed-phase chromatography. The fractions were screened for low-molecular-mass peptides and sequenced by mass spectrometry. It was possible to identify three novel bradykinin-related peptides, namely: KPLWRL-NH2 (Pnor 3), RPLSWLPK (Pnor 5) and VPPKGVSM (Pnor 7) presenting vascular activities as assessed by intravital microscopy. Pnor 3 and Pnor 7 were able to induce vasodilation. On the other hand, Pnor 5 was a potent vasoconstrictor. These effects were reproduced by their synthetic analogues.

Phospholipases A2 isolated from Micrurus lemniscatus coral snake venom: behavioral, electroencephalographic, and neuropathological aspects.

The present study evaluated four phospholipase A2 (PLA2) (Mlx-8, Mlx-9, Mlx-11 and Mlx-12) isolated from Micrurus lemniscatus snake venom (Elapidae). The effects of intrahippocampal administration of these toxins have been determined on behavior, electroencephalography, and neuronal degeneration in rats. These four PLA2 toxins induced motor and EEG alterations in a dose-dependent manner. Behavioral convulsions were characterized by clonic movements and were often accompanied by EEG alterations. Mlx toxins were convulsive but weakly epileptogenic, since low rates of seizure discharges were observed in EEG records. Neuronal injury seemed to depend on the dose of the toxin used. The highest doses of toxins caused severe intoxication and death of some animals. The injury of hippocampal cells was characterized by massive neuronal loss and hippocampal gliosis. A high occurrence of compulsive scratching was observed. Moreover, the onset of seizures induced by Mlx toxins was markedly delayed. The similarities between the effects of Mlx PLA2s and those isolated from other Elapidae snakes venoms suggest that their toxicity are probably due to their specific binding to neuronal membranes and to the catalysis of phospholipid hydrolysis, producing lysophospholipids and fatty acids. These compounds likely disturb the membrane conformation, causing a marked increase in the release of neurotransmitters and concurrent inhibition of vesicle fission and recycling. These toxins can be a useful tool to investigate properties of endogenous secretory PLA2s and therefore may be important both to study mechanisms involved in neurotransmitter release at nerve terminals and to explain the convulsive properties of PLA2s toxins.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae.

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation.

A urbanização das leishmanioses e a baixa resolutividade diagnóstica em municípios da Região Metropolitana de Belo Horizonte / Leishmaniasis urbanization and low diagnosis capacity in the Metropolitan Region of Belo Horizonte

During the period from 1994 to 1999 cutaneous leishmaniasis was reported in 32 (89%) out of 36 municipalities in the Metropolitan Region of Belo Horizonte, Brazil, of which one (2,8%) municipality was classified as a very high risk area, 16 (44,5%) as high risk, seven (19,4%) as moderate risk areas and 12 (33,3%) as low risk. From 1994 to 1995, visceral leishmaniasis was reported in six (16%) municipalities whereas in 1998 - 1999 this number increased to 15 (42%). Annual numbers of cases during 1994 to 1999 were 30, 53, 64, 53 and 84, respectively. In 19 (61.3%) municipalities no reference center for the diagnosis of the infection was available, so that most of the patients (80%) were referred to Belo Horizonte. Twelve (39%) municipalities have a center for leishmaniasis evaluation, however in only eight (67%) of these basic specific diagnostic tests were available. Rapid and extensive increase of leishmaniasis associated with low diagnosis capacity has been observed in the metropolitan area of Belo Horizonte.

No período de 1994 a 1999, foram notificados casos de leishmaniose tegumentar em 32 (89%) dos 36 municípios da Região Metropolitana de Belo Horizonte. Em um (2,8%) município o risco de adquirir a doença foi considerado muito alto, em 16 (44.5%), médio em sete (19,4%) e baixo em 12 (33.3%). Leishmaniose visceral foi notificada em seis (17%) dos 36 municípios, nos anos 94 e 95, elevando-se para 15 (42%) no biênio 98/99. O total de casos de leishmaniose visceral notificados anualmente no período 94 a 99 foi 30, 53, 64, 60, 53, 84, respectivamente. Não há serviços referenciados para atendimento da doença em 19 (61,3%) de 31 municípios, sendo 80% dos pacientes encaminhados para Belo Horizonte. Em 12 (39%) municípios com serviços referenciados, somente oito (67%) dispõem de testes diagnósticos específicos para leishmaniose. Verificou-se rápida e extensa expansão das leishmanioses na região metropolitana de Belo Horizonte e baixa capacidade de resolução diagnóstica pelos seus municípios.

A urbanização das leishmanioses e a baixa resolutividade diagnóstica em municípios da Região Metropolitana de Belo Horizonte / Leishmaniasis urbanization and low diagnosis capacity in the Metropolitan Region of Belo Horizonte

During the period from 1994 to 1999 cutaneous leishmaniasis was reported in 32 (89%) out of 36 municipalities in the Metropolitan Region of Belo Horizonte, Brazil, of which one (2,8%) municipality was classified as a very high risk area, 16 (44,5%) as high risk, seven (19,4%) as moderate risk areas and 12 (33,3%) as low risk. From 1994 to 1995, visceral leishmaniasis was reported in six (16%) municipalities whereas in 1998 - 1999 this number increased to 15 (42%). Annual numbers of cases during 1994 to 1999 were 30, 53, 64, 53 and 84, respectively. In 19 (61.3%) municipalities no reference center for the diagnosis of the infection was available, so that most of the patients (80%) were referred to Belo Horizonte. Twelve (39%) municipalities have a center for leishmaniasis evaluation, however in only eight (67%) of these basic specific diagnostic tests were available. Rapid and extensive increase of leishmaniasis associated with low diagnosis capacity has been observed in the metropolitan area of Belo Horizonte.(AU)

No período de 1994 a 1999, foram notificados casos de leishmaniose tegumentar em 32 (89%) dos 36 municípios da Região Metropolitana de Belo Horizonte. Em um (2,8%) município o risco de adquirir a doença foi considerado muito alto, em 16 (44.5%), médio em sete (19,4%) e baixo em 12 (33.3%). Leishmaniose visceral foi notificada em seis (17%) dos 36 municípios, nos anos 94 e 95, elevando-se para 15 (42%) no biênio 98/99. O total de casos de leishmaniose visceral notificados anualmente no período 94 a 99 foi 30, 53, 64, 60, 53, 84, respectivamente. Não há serviços referenciados para atendimento da doença em 19 (61,3%) de 31 municípios, sendo 80% dos pacientes encaminhados para Belo Horizonte. Em 12 (39%) municípios com serviços referenciados, somente oito (67%) dispõem de testes diagnósticos específicos para leishmaniose. Verificou-se rápida e extensa expansão das leishmanioses na região metropolitana de Belo Horizonte e baixa capacidade de resolução diagnóstica pelos seus municípios.(AU)

A mutation in human VAP-B--MSP domain, present in ALS patients, affects the interaction with other cellular proteins.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset Motor Neuron Disease (MND), characterized by motor neurons death in the cortex, brainstem and spinal cord. Ten loci linked to Familial ALS have been mapped. ALS8 is caused by a substitution of a proline by a serine in the Vesicle-Associated Membrane Protein-Associated protein-B/C (VAP-B/C). VAP-B belongs to a highly conserved family of proteins implicated in Endoplasmic Reticulum-Golgi and intra-Golgi transport and microtubules stabilization. Previous studies demonstrated that the P56S mutation disrupts the subcellular localization of VAP-B and that this position would be essential for Unfolded Protein Response (UPR) induced by VAP-B. In the present work we expressed and purified recombinant wild-type and P56S mutant VAP-B-MSP domain for the analysis of its interactions with other cellular proteins. Our findings suggest that the P56S mutation may lead to a less stable interaction of this endoplasmic reticulum protein with at least two other proteins: tubulin and GAPDH. These two proteins have been previously related to other forms of neurodegenerative diseases and are potential key points to understand ALS8 pathogenesis and other forms of MND. Understanding the role of these protein interactions may help the treatment of this devastating disease in the future.

Prolactin-induced changes in protein expression in human pancreatic islets.

Ex vivo islet cell culture prior to transplantation appears as an attractive alternative for treatment of type 1 diabetes. Previous results from our laboratory have demonstrated beneficial effects of human prolactin (rhPRL) treatment on human islet primary cultures. In order to probe into the molecular events involved in the intracellular action of rhPRL in these cells, we set out to identify proteins with altered expression levels upon rhPRL cell treatment, using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). An average of 300 different protein spots were detected, 14 of which were modified upon rhPRL treatment (p<0.01), of which 12 were successfully identified using MS and grouped according to their biological functions. In conclusion, our study provides, for the first time, information about proteins that could be critically involved in PRL's action on human pancreatic islets, and facilitate identification of new and specific targets involved in islet cell function and proliferation.

Proteinase activity regulation by glycosaminoglycans

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions

Proteinase activity regulation by glycosaminoglycans.

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

[Leishmaniasis urbanization and low diagnosis capacity in the Metropolitan Region of Belo Horizonte]. / A urbanização das leishmanioses e a baixa resolutividade diagnóstica em municípios da Região Metropolitana de Belo Horizonte.

During the period from 1994 to 1999 cutaneous leishmaniasis was reported in 32 (89%) out of 36 municipalities in the Metropolitan Region of Belo Horizonte, Brazil, of which one (2,8%) municipality was classified as a very high risk area, 16 (44,5%) as high risk, seven (19,4%) as moderate risk areas and 12 (33,3%) as low risk. From 1994 to 1995, visceral leishmaniasis was reported in six (16%) municipalities whereas in 1998 - 1999 this number increased to 15 (42%). Annual numbers of cases during 1994 to 1999 were 30, 53, 64, 53 and 84, respectively. In 19 (61.3%) municipalities no reference center for the diagnosis of the infection was available, so that most of the patients (80%) were referred to Belo Horizonte. Twelve (39%) municipalities have a center for leishmaniasis evaluation, however in only eight (67%) of these basic specific diagnostic tests were available. Rapid and extensive increase of leishmaniasis associated with low diagnosis capacity has been observed in the metropolitan area of Belo Horizonte.

Substrate specificity of human cathepsin D using internally quenched fluorescent peptides derived from reactive site loop of kallistatin.

Kallistatin, a serpin that specifically inhibits human tissue kallikrein, was demonstrated to be cleaved at the Phe-Phe bond in its reactive site loop (RSL) by cathepsin D. Internally quenched fluorescent peptides containing the amino acid sequence of kallistatin RSL were highly susceptible to hydrolysis by cathepsin D. Surprisingly, these peptides were efficiently hydrolyzed at Phe-Phe bond, despite having Lys and Ser at P2 and P2' positions, respectively, which was reported to be very unfavorable for substrates for cathepsin D. Due to the importance of cathepsin D in several physiological and pathological processes, we took the peptide containing kallistatin RSL sequence, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, as a reference substrate for a systematic specificity study of S3 to S3' protease subsites (EDDnp=N-[2,4-dinitrophenyl]-ethylenediamine and Abz=ortho-amino benzoic acid). We present in this paper some internally quenched fluorescent peptides that were efficient substrates for cathepsin D. They essentially differ from other previously described substrates by their higher kcat/Km values due, mainly, to low Km values, such as the substrate Abz-Ala-Ile-Ala-Phe-Phe-Ser-Arg-Gln-EDDnp (Km=0.27 microM, kcat=16.25 s(-1), kcat/Km=60185 microM(-1) x s(-1)).

Human tissue kallikrein S1 subsite recognition of non-natural basic amino acids.

We explored the unique substrate specificity of the primary S(1) subsite of human urinary kallikrein (hK1), which accepts both Phe and Arg, using internally quenched fluorescent peptides Abz-F-X-S-R-Q-EDDnp and Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp [Abz is o-aminobenzoic acid; EDDnp is N-(2,4-dinitrophenyl)ethylenediamine], which were based on the human kininogen sequence at the C-terminal region of bradykinin. Position X, which in natural sequence stands for Arg, received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(aminomethyl)-N-isopropylphenylalanine (Iaf), N(im)-(dimethyl)histidine [H(2Me)], 3-pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(aminomethyl)cyclohexylalanine (Ama), and 4-(aminocyclohexyl)alanine (Aca). Only Abz-F-Amf-S-R-Q-EDDnp and Abz-F-H(2Me)]-S-R-Q-EDDnp were efficiently hydrolyzed, and all others were resistant to hydrolysis. However, Abz-F-Ama-S-R-Q-EDDnp inhibited hK1 with a K(i) of 50 nM with high specificity compared to human plasma kallikrein, thrombin, plasmin, and trypsin. The Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp series were more susceptible to hK1, although the peptides with Gnf, Pya, and Ama were resistant to it. Unexpectedly, the peptides in which X is His, Lys, H(2Me), Amf, Iaf, Ppa, and Aca were cleaved at amino or at carboxyl sites of these amino acids, indicating that the S(1)' subsite has significant preference for basic residues. Human plasma kallikrein did not hydrolyze any peptide of this series except the natural sequence where X is Arg. In conclusion, the S(1) subsite of hK1 accepts amino acids with combined basic and aromatic side chain, although for the S(1)-P(1) interaction the preference is for aliphatic and basic side chains.

Alpha1-antichymotrypsin and kallistatin hydrolysis by human cathepsin D.

In the present paper, we demonstrate that alpha1-antichymotrypsin, a serpin with high inhibitory specificity toward cathepsin G, and kallistatin, a human serpin with high specificity toward tissue kallikrein, are digested by cathepsin D. Alpha1-Antichymotrypsin was hydrolyzed essentially in the reactive center loop at L-S, A-L, or L-V bonds; kallistatin was split into small fragments, but we detected the cleavage at F-F and F-S bonds in its reactive center loop in the first 15 min of digestion. In contrast to alpha1-antichymotrypsin, kallistatin is irreversibly inactivated at pH 4.0. Synthetic internally quenched fluorescent peptides containing sequences similar to the reactive center loops of these serpins were hydrolyzed by cathepsin D. The peptides derived from kallistatin were hydrolyzed more efficiently, and particularly relevant was the high susceptibility of the substrates Abz-AIKFFSAQTNRHILRFNRQ-EDDnp (Km = 0.08 microM, kcat = 2.4 s(-1)) and Abz-AIKFFSAQTNRQ-EDDnp (Km = 0.8 microM, kcat = 17.8 s(-1)), which were hydrolyzed at the F-F bond. Therefore, besides the description of a new class of very efficient internally quenched substrates for cathepsin D, we give evidence for the downregulation role of this proteinase on alpha1-antichymotrypsin and kallistatin. The acidification of extracellular milieu by tumor cells can result in activation of cathepsin D; as a consequence, kinins can be released, improving blood supply and leaving more cathepsin G available for the degradation of extracellular matrix.

Identification of a major heparin-binding site in kallistatin.

Kallistatin is a heparin-binding serine proteinase inhibitor (serpin), which specifically inhibits human tissue kallikrein by forming a covalent complex. The inhibitory activity of kallistatin is blocked upon its binding to heparin. In this study we attempted to locate the heparin-binding site of kallistatin using synthetic peptides derived from its surface regions and by site-directed mutagenesis of basic residues in these surface regions. Two synthetic peptides, containing clusters of positive-charged residues, one derived from the F helix and the other from the region encompassing the H helix and C2 sheet of kallistatin, were used to assess their heparin binding activity. Competition assay analysis showed that the peptide derived from the H helix and C2 sheet displayed higher and specific heparin binding activity. The basic residues in both regions were substituted to generate three kallistatin double mutants K187A/K188A (mutations in the F helix) and K307A/R308A and K312A/K313A (mutations in the region between the H helix and C2 sheet), using a kallistatin P1Arg variant as a scaffold. Analysis of these mutants by heparin-affinity chromatography showed that the heparin binding capacity of the variant K187A/K188A was not altered, whereas the binding capacity of K307A/R308A and K312A/K313A mutants was markedly reduced. Titration analysis with heparin showed that the K312A/K313A mutant has the highest dissociation constant. Like kallistatin, the binding activity of K187A/K188A to tissue kallikrein was blocked by heparin, whereas K307A/R308A and K312A/K313A retained significant binding and inhibitory activities in the presence of heparin. These results indicate that the basic residues, particularly Lys(312)-Lys(313), in the region between the H helix and C2 sheet of kallistatin, comprise a major heparin-binding site responsible for its heparin-suppressed tissue kallikrein binding.

Specificity of human tissue kallikrein towards substrates containing Phe-Phe pair of amino acids.

We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK1) of substrates containing the Phe-Phe amino acid pair, after which hK1 cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1-14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2, 4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P9 to P'13, and the kinetic parameters of their hydrolysis by hK1 were determined. All these peptides were cleaved after the Phe-Phe pair. For comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe-Arg and Leu-Lys bonds, respectively. Hybrid peptides containing kallistatin-PCI sequences showed that the efficiency of hK1 activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P1 amino acid as well as on residues at the P- and P'-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe-Phe, and on Lys and Ile at the P3 and P4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp. All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe-Arg pair; however, hydrolysis after Phe-Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme-substrate interaction and/or particular substrate conformations.