An extracellular proteasome releases endostatin from human collagen XVIII.

Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

Maroteaux-Lamy syndrome: orofacial features after treatment by bone marrow transplant.

PURPOSE: The objective of the present study was to emphasise the oral and dental findings of a male patient with the Maroteaux-Lamy syndrome who successfully underwent bone marrow transplantation (BMT) at the age of 22 months. CASE REPORT: A 15-year-old boy was referred to the Dentistry Division of the Catholic University of Brasília, Brazil, for dental diagnosis. General characteristics of the Maroteaux-Lamy syndrome, such as a large head, a short neck, corneal opacity, an open mouth with macroglossia, enlargement of the skull and a long anteroposterior dimension, were observed. The patient had received the benefit of a BMT at an early stage. Therefore, characteristics were presented in a moderate form, except for the skeletal symptoms. DISCUSSION: Maroteaux-Lamy syndrome, also known as mucopolysaccharidosis type VI, is a lysosomal storage disorder that is caused by a deficiency of arylsulphatase B, which leads to an accumulation of dermatan sulphate in tissues and its increased excretion in urine. The deposition of mucopolysaccharides leads to a progressive disorder involving multiple organs. It is a rare condition that is inherited as an autosomal recessive trait. The characteristic features of this disease include retardation in growth, a large head, a short neck, corneal opacity, typical facies and spinal abnormalities. The main dental findings of this syndrome include gingival hyperplasia, hypertrophy of the maxillary alveolar ridge, macroglossia, unerupted dentition, malocclusions and dentigerous cyst-like follicles. BMT is a therapeutic treatment that is given to permanently replace any disorder caused due to the deficiency of enzymes in patients with storage diseases.

Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII.

Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.

Cysteine-protease activity elicited by Ca2+ stimulus in Plasmodium.

Bloodstage malaria parasites require proteolytic activity for key processes as invasion, hemoglobin degradation and merozoite escape from red blood cells (RBCs). We investigated by confocal microscopy the presence of cysteine-protease activity elicited by calcium stimulus in Plasmodium chabaudi and Plasmodium falciparum in free trophozoites or for the later parasite within RBC using fluorescence resonance energy transfer (FRET) peptides. Peptide probes access, to either free or intraerythrocytic parasites, was also tested by selecting a range of fluorescent peptides (653-3146 Da molecular mass) labeled with Abz or FITC. In the present work we show that Ca2+ stimulus elicited by treatment with either melatonin, thapsigargin, ionomicin or nigericin, promotes an increase of substrate hydrolysis, which was blocked by the specific cysteine-protease inhibitor E-64 and the intracellular Ca2+ chelator, BAPTA. When parasites were treated with cytoplasmic Ca2+ releasing compounds, a cysteine-protease was labeled in the parasite cytoplasm by the fluorescent specific irreversible inhibitor, Ethyl-Eps-Leu-Tyr-Cap-Lys(Abz)-NH2, where Ethyl-Eps is Ethyl-(2S,3S)-oxirane-2,3-dicarboxylate. In summary, we demonstrate that P. chabaudi and P. falciparum have a cytoplasmic dependent cysteine-protease activity elicited by Ca2+.

Atividade proteolítica relacionada à sinalização por melatonina e cálcio em Plasmodium / Proteolytic activity elicited by melatonin and calcium in Plasmodium

Durante o ciclo intraeritrocítico, o Plasmodium, parasita causador da malária, utiliza a proteólise em processos fundamentais do metabolismo, como invasão de novos eritrócitos, degradação da hemoglobina e escape eritrocitário dos merozoítos. Esta última fase ocorre de maneira característica em Plasmodium, quando todos os parasitas são liberados sincronicamente na corrente sangüínea, em um único evento. Tal fenômeno é desencadeado pela sinalização com a melatonina (Hotta et al., 2000), hormônio do organismo hospedeiro responsável pelo controle do ciclo circadiano em vertebrados. A sinalização por melatonina em Plasmodium ativa a fosfolipase C e libera Ca2+ para o citoplasma a partir de canais de Ca2+ sensíveis a IP3 presentes no retículo endoplasmático do parasita. Avaliamos a influência da sinalização por melatonina e compostos que aumentam o Ca2+ citoplasmático na atividade proteolítica de trofozoítos de P. chabaudi e P. falciparum, utilizando peptídeos com apagamento intramolecular da fluorescência. A permeação em parasitas isolados e intraeritrocíticos também foi avaliada a partir de microscopia confocal com peptídeos fluorescentes marcados com os fluoróforos Abz ou FITC, dentro de uma faixa de peso molecular (653-3146 Da). No presente trabalho observamos que o estímulo por Ca2+ desencadeado pelo tratamento dos parasitas com melatonina, tapsigargina, ionomicina e nigericina promovem o aumento da hidrólise de substratos peptídicos, bloqueada pelo inibidor de cisteinil-proteases E-64 e pelo quelante intracelular de Ca2+, BAPT A. A partir do tratamento dos trofozoítos com os compostos que aumentam o Ca2+ citoplasmático, pudemos marcar cisteinil-proteases neste mesmo compartimento, através da ligação irreversível com inibidor análogo ao E-64 ligado à sonda fluorescente. Em resumo, no presente trabalho demonstramos que P. falciparum e P. chabaudi apresentam atividade cisteinil-proteásica Ca2+ -dependente citoplasmática.