RESUMO
AIMS: One promising approach for treatment of Alzheimer's disease (AD) is use of anti-amyloid therapies, based on the hypothesis that increases in amyloid-beta (Aß) deposits in brain are a major cause of AD. Several groups have focused on Aß immunotherapy with some success. Small molecules derivatives of Congo red have been shown to inhibit Aß aggregation and protect against Aß neurotoxicity in vitro. The agents described here are all small molecule Aß-binding agents (SMAßBA's) derivatives of Congo red. MAIN METHODS: Here, we have explored the anti-amyloid properties of these SMAßBA's in mice doubly transgenic for human prensenilin-1 (PS1) and APP gene mutations that cause early-onset AD. Mice were treated with either methoxy-X04, X:EE:B34 and X:034-3-OMe1. After treatment, brains were examined for Aß-deposition, using histochemistry, and soluble and insoluble Aß levels were determined using ELISA. KEY FINDINGS: A range of anti-amyloid activity was observed with these three compounds. PS1/APP mice treated with methoxy-X04 and X:EE:B34 showed decrease in total Aß load, a decrease in Aß fibril load, and a decrease in average plaque size. Treatment with methoxy-X04 also resulted in a decrease in insoluble Aß levels. The structurally similar compound, X:034:3-OMe1, showed no significant effect on any of these measures. The effectiveness of the SMAßBA's may be related to a combination of binding affinity for Aß and entry into brain, but other factors appear to apply as well. SIGNIFICANCE: These data suggest that SMAßBA's may significantly decrease amyloid burden in brain during the pathogenesis of AD and could be useful therapeutics alone, or in combination with immunotherapy.
RESUMO
The combined action of the antitumor antibiotic bleomycin and chloroethylnitrosourea (CCNU) was studied in human lymphocytes in vitro. All the experiments were carried out with 20 micrograms/ml bleomycin for a given treatment time. By adding 0.7 and 3.5 micrograms/ml CCNU at late G1-S phase we have demonstrated a considerable increase in both percent of aberrant cells and production of dicentrics and rings (5-fold, p less than 0.001). At late S-G2 the combined treatment led to a significant enhancement of breaks per cell (p less than 0.0001) and cells with more than 12 aberrations. A possible explanation could be the known repair-inhibitory potential of CCNU, but its pure clastogenic action still has to be considered. The results presented here point out the need for seeking chemotherapeutic regimens with reduced concentrations of the drugs in combination.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Bleomicina/toxicidade , Ciclo Celular/efeitos dos fármacos , Lomustina/toxicidade , Linfócitos/efeitos dos fármacos , Aberrações Cromossômicas , Sinergismo Farmacológico , Humanos , Troca de Cromátide IrmãRESUMO
Bleomycin (BLM) induced chromosomal damage in G2 phase and its repair kinetics in normal human lymphocytes were studied following different treatment schedules. As a first step, a dose-response curve was obtained (concentrations of 5-50 micrograms/ml). For repair kinetics studies, blood samples were treated with BLM at a concentration of 20 micrograms/ml. Continuous treatment produced equal numbers of breaks per cell (br/c) when the cells were treated 3, 4 or 5 h before fixation. If the treatment time was extended to 6 h, the level of br/c was increased 2-fold (p < 0.001) as a result of an increased number of cells with more than 3 br/c. The curves obtained after pulse treatment showed maximal chromosome damage at time 3 (45 min BLM treatment, followed by 2 h repair in drug free medium). When the time after treatment was extended to 4 h (treatment time 5), a 50% reduction in chromosome damage was measured. It was found out that at treatment points 3, 4 and 5 the differences in breaks per cell at the different schedules applied were statistically highly significant. If caffeine (CAF) was added, the continuous treatment, BLM+CAF, induced a statistically significant increase in the frequency of br/c at every treatment point, but the shape of the curve illustrating the kinetics of chromosomal damage remained unchanged. Moreover, the addition of CAF at continuous BLM treatment brings the level of br/c close to that measured at the pulse BLM treatment except for treatment time 3. When applied in a combination with BLM, CAF considerably modified the kinetics of chromosome damage for a pulse (BLM alone) treatment. The possible reasons for the changes in the level of br/c as well as a tentative scheme for assessment of chromosome damage repair capacity after BLM treatment are discussed.
Assuntos
Bleomicina/farmacologia , Aberrações Cromossômicas/genética , Reparo do DNA/fisiologia , Adulto , Cafeína/farmacologia , Células Cultivadas , DNA/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Interações Medicamentosas , Fase G2 , Humanos , Linfócitos/ultraestrutura , Fatores de TempoRESUMO
Mammalian bleomycin hydrolases (BH) are enzymes with proven exopeptidase activity responsible for deamidation of the beta-aminoalanine moiety in bleomycin and are thought to limit the therapeutic efficacy of the drug. We have recently determined that the highly conserved BH-like domain in the C-terminus of human bleomycin hydrolase (hBH) is critical both for in vitro aminopeptidase and bleomycin metabolizing activities. To determine if hBH protects mammalian cells against bleomycin clastogenic effect, we transfected CHO cells with plasmids encoding hBH or C-terminal truncated forms and evaluated the level of chromatid breaks after bleomycin exposure. CHO cells expressing hBH had 50% less chromatid breaks after bleomycin treatment compared with mock transfected cells. The eight amino acid bleomycin hydrolase-like domain in the C-terminus, which does not contain any of the putative active site amino acids, was essential for protection against bleomycin induced chromatid breaks. These results demonstrate that intracellular hBH levels can influence the clastogenic action of bleomycin and that the C-terminus has a functional role in the biological activity of hBH.
Assuntos
Bleomicina/metabolismo , Quebra Cromossômica , Cisteína Endopeptidases/química , Mutagênicos/metabolismo , Aminoácidos , Animais , Bleomicina/farmacologia , Células CHO , Cricetinae , Cisteína Endopeptidases/genética , Fase G2 , Humanos , Mutagênicos/farmacologia , Deleção de SequênciaRESUMO
Human bleomycin hydrolase (hBH) is a neutral cysteine protease genetically associated with increased risk for Alzheimer disease. We show here that ectopic expression of hBH in 293APPwt and CHOAPPsw cells altered the processing of amyloid precursor protein (APP) and increased significantly the release of its proteolytic fragment, beta amyloid (Abeta). We also found that hBH interacted and colocalized with APP as determined by subcellular fractionation, in vitro binding assay, and confocal immunolocalization. Metabolic labeling and pulse-chase experiments showed that ectopic hBH expression increased secretion of soluble APPalpha/beta products without changing the half-life of cellular APP. We also observed that this increased Abeta secretion was independent of hBH isoforms. Our findings suggest a regulatory role for hBH in APP processing pathways.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Cisteína Endopeptidases/metabolismo , Animais , Células CHO , Cricetinae , Cisteína Endopeptidases/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/metabolismo , Peptídeo Hidrolases/metabolismo , Frações Subcelulares/química , Radioisótopos de Enxofre , Transfecção , Ácidos Tri-Iodobenzoicos/químicaRESUMO
Amyloid precursor protein (APP) is the source of the neurotoxic amyloid beta (Abeta) peptide associated with Alzheimer's disease. Apolipoprotein A-I (apoA-I), a constituent of high-density lipoprotein complexes, was identified by a yeast two-hybrid system as a strong and specific binding partner of full-length APP (APPfl). This association between apoA-I and APPfl was localized to the extracellular domain of APP (APPextra). Furthermore, the interaction between apoA-I and APPfl was confirmed by coprecipitation using recombinant epitope-tagged APPextra and purified apoA-I. Several functional domains have been identified in APPextra, and we focused on a possible interaction between apoA-1 and the pathologically important Abeta peptide, because APPextra contains the nontransmembrane domain of Abeta. The binding between apoA-I and Abeta was saturable (K(d) = 6 nM), specific, and reversible. APPextra also competed with apoA-I for binding to Abeta. Direct evidence for this interaction was obtained by the formation of an SDS-resistant Abeta-apoA-I complex in polyacrylamide gels. Competitive experiments with apolipoprotein E (isoforms E2 and E4) showed that apoA-I had a higher binding affinity for Abeta. We also found that apoA-I inhibited the beta-sheet formation of Abeta with a mean inhibitory concentration close to that of alpha2-macroglobulin. Finally, we demonstrated that apoA-I attenuated Abeta-induced cytotoxicity. These results suggest apoA-I binds to at least one extracellular domain of APP and has a functional role in controlling Abeta aggregation and toxicity.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/farmacologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Ensaio de Imunoadsorção Enzimática , Espaço Extracelular/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Oxirredução , Células PC12/efeitos dos fármacos , Células PC12/metabolismo , Estrutura Terciária de Proteína/genética , Ratos , Dodecilsulfato de Sódio , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Bleomycin hydrolase (BH) is unusual among cysteine proteinases because it appears to form multihomomeric structures, inactivates the antitumor glycopeptide bleomycin, and contains a unique C-terminal amino acid sequence. We now demonstrate intrinsic endopeptidase activity associated with human BH (hBH) using artificial substrates and intracellular dimerization of hBH using a yeast two-hybrid assay. To determine domains important for homomeric interactions and catalysis, we constructed N- and C-terminal deletion mutants and identified an N-terminal region (hBH1-82) that interacted with two nonoverlaping hBH domains: one near the N-terminus (hBH14-103) and another neighboring the C-terminus (hBH358-455). In vitro hBH aggregated with a molecular mass of 235 kD corresponding to a homotetramer and the C-terminus was critical for this oligomerization since no tetramers were found when the last 40 amino acids were deleted. The penultimate 8 amino acids, which constitute a unique and highly conserved bleomycin hydrolase-like domain (BHYD), were essential for BH and aminopeptidase activity but not for endopeptidase activity or oligomer formation. Thus, the C-terminus of hBH has two independent roles controlling both the catalytic activity and oligomerization of hBH.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Bleomicina , Clonagem Molecular , Sequência Conservada , Cisteína Endopeptidases/genética , Primers do DNA/genética , Dimerização , Vetores Genéticos , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Bleomycin hydrolase (BH) is a highly conserved cysteine proteinase that deamidates and inactivates the anticancer drug bleomycin. Yeast BH self-assembles to form a homohexameric structure, which resembles a 20 S proteasome and may interact with other proteins. Therefore, we searched for potential human BH (hBH) partners using the yeast two-hybrid system with a HeLa cDNA library and identified the full-length human homologue of yeast ubiquitin-conjugating enzyme 9 (UBC9). Cotransformation assays using hBH deletion mutants revealed that the carboxyl terminus of hBH (amino acids 356-455), which contains two of the three essential catalytic amino acids, was not critical for protein binding in the yeast two-hybrid environment. In vitro translated human UBC9 was precipitated by glutathione S-transferase-hBH fusion protein but not by glutathione S-transferase. Efficient in vitro binding occurred in the absence of the first 24 amino acids of UBC9 and the catalytic Cys93 of UBC9. We confirmed that hBH and UBC9 interacted in vivo by affinity copurification of proteins overexpressed in mammalian cells. Using immunocytochemical analysis, hBH was colocalized with UBC9. Coexpression of hBH and UBC9 in mammalian cells did not markedly alter the bleomycin-hydrolyzing activity of hBH or apparent small ubiquitin-related modifier 1 addition. This is the first reported heteromeric interaction with hBH, and it suggests a role for hBH in intracellular protein processing and degradation.
Assuntos
Cisteína Endopeptidases/metabolismo , Ligases/metabolismo , Enzimas de Conjugação de Ubiquitina , Sequência de Aminoácidos , Animais , Antibióticos Antineoplásicos/metabolismo , Sequência de Bases , Bleomicina/metabolismo , Western Blotting , Células CHO , Linhagem Celular , Clonagem Molecular , Cricetinae , Cisteína Endopeptidases/biossíntese , Citoplasma/enzimologia , Evolução Molecular , Humanos , Membranas Intracelulares/enzimologia , Ligases/biossíntese , Dados de Sequência Molecular , Ligação Proteica , TransfecçãoRESUMO
Human bleomycin hydrolase (hBH) is a neutral cysteine protease that may regulate the secretion of soluble amyloid precursor protein (APP) and amyloid beta (A(beta)), which is a major constituent of the Alzheimer's disease-associated amyloid plaques. We have now determined that APP interacts with hBH by using yeast two hybrid methods and in vitro binding studies revealed that APP interacted with a 68 amino acid region that includes the catalytic domain of hBH. Ectopic expression of hBH increased the secretion of A(beta) but not of a second secreted protein, apolipoprotein A-I. Expression of hBH in which the catalytic cysteine 73 was mutated to serine failed to increase A(beta) secretion. These results indicate a critical role for cysteine 73 of hBH in mediating APP processing.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Cisteína Endopeptidases/metabolismo , Cisteína/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apolipoproteína A-I/metabolismo , Sequência de Bases , Células CHO , Cricetinae , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Primers do DNA , Camundongos , Mutagênese Sítio-DirigidaRESUMO
Bleomycin hydrolase (BH) is a cysteine proteinase that inactivates the anticancer drug bleomycin. Yeast BH forms a homohexameric structure that resembles a 20S proteasome and binds to single-stranded RNA and DNA. We now demonstrate that human BH (hBH) interacts and colocalizes with ribosomal proteins. Using a yeast two-hybrid system, we found hBH bound to human homologues of rat ribosomal proteins L11 and L29. The N-terminus of hBH (amino acids 14-175), which contains a catalytic Cys93, was critical for the binding to L11 in the two-hybrid environment. hBH precipitated 35S-labeled L11 and L29 in vitro, and hBH colocalized with L11 and L29 as determined by immunofluorescence. In addition to cytosolic bleomycin hydrolase, we found abundant bleomycin hydrolase activity associated with the ribosomal subcellular fraction by differential centrifugation. hBH was also detected by Western immunoblotting in a high-speed particulate fraction, where the majority of L11 and L29 were found. In vitro experiments showed recombinant hBH binds to Chinese hamster ovary cell microsomes. Thus, our data strongly suggest that hBH exists as both a free cytosolic and ribosome-associated protein.