Function of C1A barley peptidases and their inhibitors (cystatins) in barley seed germination : Funcion de las peptidasas C1A de cebada y sus inhibidores (cistatinas) en la germinacion de la semilla

Plant proteolysis is a metabolic process where specific enzymes called peptidases degrade proteins. In plants, this complex process involves broad metabolic networks and different sub-cellular compartments. Several types of peptidases take part in the proteolytic process, mainly cysteine-, serine-, aspartyl- and metallo- peptidases. Among the cysteine-peptidases, the papain-like or C1A peptidases (family C1, clan CA) are extensively present in land plants and are classified into catepsins L-, B-, H- and Flike. The catalytic mechanism of these C1A peptidases is highly conserved and involves the three amino acids Cys, His and Asn in the catalytic triad, and a Gln residue which seems essential for maintaining an active enzyme conformation. These proteins are synthesized as inactive precursors, which comprise an N-terminal signal peptide, a propeptide, and the mature protein. In barley, we have identified 33 cysteine-peptidases from the papain-like family, classifying them into 8 different groups. Five of them corresponded to cathepsins L-like (5 subgroups), 1 cathepsin B-like group, 1 cathepsin F-like group and 1 cathepsin H-like group. Besides, C1A peptidases are the specific targets of the plant proteinaceous inhibitors known as phytocystatins (PhyCys). The cystatin inhibitory mechanism is produced by a tight and reversible interaction with their target enzymes. In barley, the cystatin gene family is comprised by 13 members. In this work we have tried to elucidate the role of the C1A cysteine-peptidases and their specific inhibitors (cystatins) in the germination process of the barley grain. Therefore, we selected a representative member of each group/subgroup of C1A peptidases (1 cathepsin B-like, 1 cathepsin F-like, 1 cathepsin H-like and 5 cathepsins L-like). The molecular characterization of the cysteine-peptidases was done and the peptidase-inhibitor interaction was analyzed in vitro and in vivo. A study in the structural basis for specificity of pro-peptide/enzyme interaction in barley C1A cysteine-peptidases has been also carried out by inhibitory assays and the modeling of the three-dimensional structures. The barley grain maturation produces the accumulation of storage proteins (prolamins) in the endosperm which are mobilized during germination to supply the required nutrients until the photosynthesis is fully established. In this work, we have demonstrated the participation of the cysteine-peptidases and their inhibitors in the degradation of the different storage protein fractions (hordeins, albumins and globulins) present in the barley grain. Besides, transgenic barley plants overexpressing or silencing cysteine-peptidases or cystatins were obtained by Agrobacterium-mediated transformation of barley immature embryos to analyze their physiological function in vivo. Preliminary assays were carried out with the T1 grains of several transgenic lines. Comparing the knock-out and the overexpressing lines with the WT, alterations in the germination process were detected and were correlated with their grain hordein content. These data will be validated with the homozygous grains that are being produced through the double haploid technique by microspore culture. Resumen La proteólisis es un proceso metabólico por el cual se lleva a cabo la degradación de las proteínas de un organismo a través de enzimas específicas llamadas proteasas. En plantas, este complejo proceso comprende un entramado de rutas metabólicas que implican, además, diferentes compartimentos subcelulares. En la proteólisis participan numerosas proteasas, principalmente cisteín-, serín-, aspartil-, y metalo-proteasas. Dentro de las cisteín-proteasas, las proteasas tipo papaína o C1A (familia C1, clan CA) están extensamente representadas en plantas terrestres, y se clasifican en catepsinas tipo L, B, H y F. El mecanismo catalítico de estas proteasas está altamente conservado y la triada catalítica formada por los aminoácidos Cys, His y Asn, y a un aminoácido Gln, que parece esencial para el mantenimiento de la conformación activa de la proteína. Las proteasas C1A se sintetizan como precursores inactivos y comprenden un péptido señal en el extremo N-terminal, un pro-péptido y la proteína madura. En cebada hemos identificado 33 cisteín-proteasas de tipo papaína y las hemos clasificado filogenéticamente en 8 grupos diferentes. Cinco de ellos pertenecen a las catepsinas tipo L (5 subgrupos), un grupo a las catepsinas tipo-B, otro a las catepsinas tipo-F y un último a las catepsinas tipo-H. Las proteasas C1A son además las dianas específicas de los inhibidores protéicos de plantas denominados fitocistatinas. El mecanismo de inhibición de las cistatinas está basado en una fuerte interacción reversible. En cebada, se conoce la familia génica completa de las cistatinas, que está formada por 13 miembros. En el presente trabajo se ha investigado el papel de las cisteín-proteasas de cebada y sus inhibidores específicos en el proceso de la germinación de la semilla. Para ello, se seleccionó una proteasa representante de cada grupo/subgrupo (1 catepsina tipo- B, 1 tipo-F, 1 tipo-H, y 5 tipo-L, una por cada subgrupo). Se ha llevado a cabo su caracterización molecular y se ha analizado la interacción enzima-inhibidor tanto in vivo como in vitro. También se han realizado estudios sobre las bases estructurales que demuestran la especificidad en la interacción enzima/propéptido en las proteasas C1A de cebada, mediante ensayos de inhibición y la predicción de modelos estructurales de la interacción. Finalmente, y dado que durante la maduración de la semilla se almacenan proteínas de reserva (prolaminas) en el endospermo que son movilizadas durante la germinación para suministrar los nutrientes necesarios hasta que la nueva planta pueda realizar la fotosíntesis, en este trabajo se ha demostrado la participación de las cisteínproteasas y sus inhibidores en la degradación de las diferentes tipos de proteínas de reserva (hordeinas, albúmins y globulinas) presentes en el grano de cebada. Además, se han obtenido plantas transgénicas de cebada que sobre-expresan o silencian cistatinas y cisteín-proteasas con el fin de analizar la función fisiológica in vivo. Se han realizado análisis preliminares en las semillas T1 de varias líneas tránsgenicas de cebada y al comparar las líneas knock-out y las líneas de sobre-expresión con las silvestres, se han detectado alteraciones en la germinación que están además correlacionadas con el contenido de hordeinas de las semillas. Estos datos serán validados en las semillas homocigotas que se están generando mediante la técnica de dobles haploides a partir del cultivo de microesporas.

Factores transcripcionales de la clase DOF en Brachypodium distachyon: caracterización molecular de BdDOF24 durante la germinación de las semillas

La semilla es el órgano que garantiza la propagación y continuidad evolutiva de las plantas espermatofitas y constituye un elemento indispensable en la alimentación humana y animal. La semilla de cereales acumula en el endospermo durante la maduración, mayoritariamente, almidón y proteínas de reserva. Estas reservas son hidrolizadas en la germinación por hidrolasas sintetizadas en la aleurona en respuesta a giberelinas (GA), siendo la principal fuente de energía hasta que la plántula emergente es fotosintéticamente activa. Ambas fases del desarrollo de la semilla, están reguladas por una red de factores de transcripción (TF) que unen motivos conservados en cis- en los promotores de sus genes diana. Los TFs son proteínas que han desempeñado un papel central en la evolución y en el proceso de domesticación, siendo uno de los principales mecanismos de regulación génica; en torno al 7% de los genes de plantas codifican TFs. Atendiendo al motivo de unión a DNA, éstos, se han clasificado en familias. La familia DOF (DNA binding with One Finger) participa en procesos vitales exclusivos de plantas superiores y sus ancestros cercanos (algas, musgos y helechos). En las semillas de las Triticeae (subfamilia Pooideae), se han identificado varias proteínas DOF que desempeñan un papel fundamental en la regulación de la expresión génica. Brachypodium distachyon es la primera especie de la subfamilia Pooideae cuyo genoma (272 Mbp) ha sido secuenciado. Su pequeño tamaño, ciclo de vida corto, y la posibilidad de ser transformado por Agrobacterium tumefaciens (plásmido Ti), hacen que sea el sistema modelo para el estudio de cereales de la tribu Triticeae con gran importancia agronómica mundial, como son el trigo y la cebada. En este trabajo, se han identificado 27 genes Dof en el genoma de B. distachyon y se han establecido las relaciones evolutivas entre estos genes Dof y los de cebada (subfamilia Pooideae) y de arroz (subfamilia Oryzoideae), construyendo un árbol filogenético en base al alineamiento múltiple del dominio DOF. La cebada contiene 26 genes Dof y en arroz se han anotado 30. El análisis filogenético establece cuatro grupos de genes ortólogos (MCOGs: Major Clusters of Orthologous Genes), que están validados por motivos conservados adicionales, además del dominio DOF, entre las secuencias de las proteínas de un mismo MCOG. El estudio global de expresión en diferentes órganos establece un grupo de nueve genes BdDof expresados abundantemente y/o preferencialmente en semillas. El estudio detallado de expresión de estos genes durante la maduración y germinación muestra que BdDof24, ortólogo putativo a BPBF-HvDOF24 de cebada, es el gen más abundante en las semillas en germinación de B. distachyon. La regulación transcripcional de los genes que codifican hidrolasas en la aleurona de las semillas de cereales durante la post‐germinación ha puesto de manifiesto la existencia en sus promotores de un motivo tripartito en cis- conservado GARC (GA-Responsive Complex), que unen TFs de la clase MYB-R2R3, DOF y MYBR1-SHAQKYF. En esta tesis, se ha caracterizado el gen BdCathB de Brachypodium que codifica una proteasa tipo catepsina B y es ortólogo a los genes Al21 de trigo y HvCathB de cebada, así como los TFs responsables de su regulación transcripcional BdDOF24 y BdGAMYB (ortólogo a HvGAMYB). El análisis in silico del promotor BdCathB ha identificado un motivo GARC conservado, en posición y secuencia, con sus ortólogos en trigo y cebada. La expresión de BdCathB se induce durante la germinación, así como la de los genes BdDof24 y BdGamyb. Además, los TFs BdDOF24 y BdGAMYB interaccionan en el sistema de dos híbridos de levadura e in planta en experimentos de complementación bimolecular fluorescente. En capas de aleurona de cebada, BdGAMYB activa el promotor BdCathB, mientras que BdDOF24 lo reprime; este resultado es similar al obtenido con los TFs ortólogos de cebada BPBF-HvDOF24 y HvGAMYB. Sin embargo, cuando las células de aleurona se transforman simultáneamente con los dos TFs, BdDOF24 tiene un efecto aditivo sobre la trans-activación mediada por BdGAMYB, mientras que su ortólogo BPBF-HvDOF24 produce el efecto contrario, revirtiendo el efecto de HvGAMYB sobre el promotor BdCathB. Las diferencias entre las secuencias deducidas de las proteínas BdDOF24 y BPBF-HvDOF24 podrían explicar las funciones opuestas que desempeñan en su interacción con GAMYB. Resultados preliminares con líneas de inserción de T-DNA y de sobre-expresión estable de BdGamyb, apoyan los resultados obtenidos en expresión transitoria. Además las líneas homocigotas knock-out para el gen BdGamyb presentan alteraciones en anteras y polen y no producen semillas viables. ABSTRACT The seed is the plant organ of the spermatophytes responsible for the dispersion and survival in the course of evolution. In addition, it constitutes one of the most importan elements of human food and animal feed. The main reserves accumulated in the endosperm of cereal seeds through the maturation phase of development are starch and proteins. Its degradation by hydrolases synthetized in aleurone cells in response to GA upon germination provides energy, carbon and nitrogen to the emerging seedling before it acquires complete photosynthetic capacity. Both phases of seed development are controlled by a network of transcription factors (TFs) that interact with specific cis- elements in the promoters of their target genes. TFs are proteins that have played a central role during evolution and domestication, being one of the most important regulatory mechanisms of gene expression. Around 7% of genes in plant genomes encode TFs. Based on the DNA binding motif, TFs are classified into families. The DOF (DNA binding with One Finger) family is involved in specific processes of plants and its ancestors (algae, mosses and ferns). Several DOF proteins have been described to play important roles in the regulation of genes in seeds of the Triticeae tribe (Pooideae subfamily). Brachypodium distachyon is the first member of the Pooideae subfamily to be sequenced. Its small size and compact structured genome (272 Mbp), the short life cycle, small plant size and the possibility of being transformed with Agrobacterium tumefaciens (Ti-plasmid) make Brachypodium the model system for comparative studies within cereals of the Triticeae tribe that have big economic value such as wheat and barley. In this study, 27 Dof genes have been identified in the genome of B. distachyon and the evolutionary relationships among these Dof genes and those frome barley (Pooideae subfamily) and those from rice (Oryzoideae subfamily) have been established by building a phylogenetic tree based on the multiple alignment of the DOF DNA binding domains. The barley genome (Hordeum vulgare) contains 26 Dof genes and in rice (Oryza sativa) 30 genes have been annotated. The phylogenetic analysis establishes four Major Clusters of Orthologous Genes (MCOGs) that are supported by additional conserved motives out of the DOF domain, between proteins of the same MCOG. The global expression study of BdDof genes in different organs and tissues classifies BdDof genes into two groups; nine of the 27 BdDof genes are abundantly or preferentially expressed in seeds. A more detailed expression analysis of these genes during seed maturation and germination shows that BdDof24, orholog to barley BPBF-HvDof24, is the most abundantly expressed gene in germinating seeds. Transcriptional regulation studies of genes that encode hydrolases in aleurone cells during post-germination of cereal seeds, have identified in their promoters a tripartite conserved cis- motif GARC (GA-Responsive Complex) that binds TFs of the MYB-R2R3, DOF and MYBR1-SHAQKYF families. In this thesis, the characterization of the BdCathB gene, encoding a Cathepsin B-like protease and that is ortholog to the wheat Al21 and the barley HvCathB genes, has been done and its transcriptional regulation by the TFs BdDOF24 and BdGAMYB (ortholog to HvGAMYB) studied. The in silico analysis of the BdCathB promoter sequence has identified a GARC motif. BdCathB expression is induced upon germination, as well as, those of BdDof24 and BdGamyb genes. Moreover, BdDOF24 and BdGAMYB interact in yeast (Yeast 2 Hybrid System, Y2HS) and in planta (Bimolecular Fluorecence Complementation, BiFC). In transient assays in aleurone cells, BdGAMYB activates the BdCathB promoter, whereas BdDOF24 is a transcriptional repressor, this result is similar to that obtained with the barley orthologous genes BPBF-HvDOF24 and HvGAMYB. However, when aleurone cells are simultaneously transformed with both TFs, BdDOF24 has an additive effect to the trans-activation mediated by BdGAMYB, while its ortholog BPBF-HvDOF24 produces an opposite effect by reducing the HvGAMYB activation of the BdCathB promoter. The differences among the deduced protein sequences between BdDOF24 and BPBF-HvDOF24 could explain their opposite functions in the interaction with GAMYB protein. Preliminary results of T-DNA insertion (K.O.) and stable over-expression lines of BdGamyb support the data obtained in transient expression assays. In addition, the BdGamyb homozygous T-DNA insertion (K.O.) lines have anther and pollen alterations and they do not produce viable seeds.

Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes

Previously, we showed that retinoic acid (RA) binds to the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) with high affinity, suggesting that M6P/IGF2R may be a receptor for RA. Here, we show that RA, after 2–3 h of incubation with cultured neonatal-rat cardiac fibroblasts, dramatically alters the intracellular distribution of M6P/IGF2R as well as that of cathepsin B (a lysosomal protease bearing M6P). Immunofluorescence techniques indicate that this change in intracellular distribution is characterized by a shift of the proteins from the perinuclear area to cytoplasmic vesicles. The effect of RA was neither blocked by an RA nuclear receptor antagonist (AGN193109) nor mimicked by a selective RA nuclear-receptor agonist (TTNPB). Furthermore, the RA-induced translocation of cathepsin B was not observed in M6P/IGF2R-deficient P388D1 cells but occurred in stably transfected P388D1 cells expressing the receptor, suggesting that the effect of RA might be the result of direct interaction with M6P/IGF2R, rather than the result of binding to the nuclear receptors. These observations not only support the idea that M6P/IGF2R mediates an RA-response pathway but also indicate a role for RA in control of intracellular trafficking of lysosomal enzymes. Therefore, our observations may have important implications for the understanding of the diverse biological effects of retinoids.

The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

Ruolo di Clusterina nella progressione del tumore prostatico

Clusterina (CLU) è una proteina ubiquitaria, presente nella maggior parte dei fluidi corporei e implicata in svariati processi fisiologici. Dalla sua scoperta fino ad oggi, CLU è risultata essere una proteina enigmatica, la cui funzione non è ancora stata compresa appieno. Il gene codifica per 3 varianti trascrizionali identificate nel database NCBI con i codici: NM_001831 (CLU 1 in questo lavoro di tesi), NR_038335 (CLU 2 in questo lavoro di tesi) e NR_045494 (CLU 3 in questo lavoro di tesi). Tutte le varianti sono trascritte come pre-mRNA contenenti 9 esoni e 8 introni e si differenziano per l’esone 1, la cui sequenza è unica e caratteristica di ogni variante. Sebbene in NCBI sia annotato che le varianti CLU 2 e CLU 3 non sono codificanti, tramite analisi bioinformatica è stato predetto che da tutti e tre i trascritti possono generarsi proteine di differente lunghezza e localizzazione cellulare. Tra tutte le forme proteiche ipotizzate, l’unica a essere stata isolata e sequenziata è quella tradotta dall’AUG presente sull’esone 2 che dà origine a una proteina di 449 aminoacidi. Il processo di maturazione prevede la formazione di un precursore citoplasmatico (psCLU) che subisce modificazioni post-traduzionali tra cui formazione di ponti disolfuro, glicosilazioni, taglio in due catene denominate β e α prima di essere secreta come eterodimero βα (sCLU) nell’ambiente extracellulare, dove esercita la sua funzione di chaperone ATP-indipendente. Oltre alla forma extracellulare, è possibile osservare una forma intracellulare con localizzazione citosolica la cui funzione non è stata ancora completamente chiarita. Questo lavoro di tesi si è prefissato lo scopo di incrementare le conoscenze in merito ai trascritti CLU 1 e CLU 2 e alla loro regolazione, oltre ad approfondire il ruolo della forma citosolica della proteina in relazione al signaling di NF-kB che svolge un ruolo importante nel processo di sviluppo e metastatizzazione del tumore. Nella prima parte, uno screening di differenti linee cellulari, quali cellule epiteliali di prostata e di mammella, sia normali sia tumorali, fibroblasti di origine polmonare e linfociti di tumore non-Hodgkin, ha permesso di caratterizzare i trascritti CLU 1 e CLU 2. Dall’analisi è emerso che la sequenza di CLU 1 è più corta al 5’ rispetto a quella depositata in NCBI con l’identificativo NM_001831 e il primo AUG disponibile per l’inizio della traduzione è localizzato sull’esone 2. È stato dimostrato che CLU 2, al contrario di quanto riportato in NCBI, è tradotto in proteina a partire dall’AUG presente sull’esone 2, allo stesso modo in cui viene tradotto CLU 1. Inoltre, è stato osservato che i livelli d’espressione dei trascritti variano notevolmente tra le diverse linee cellulari e nelle cellule epiteliali CLU 2 è espressa sempre a bassi livelli. In queste cellule, l’espressione di CLU 2 è silenziata per via epigenetica e la somministrazione di farmaci capaci di rendere la cromatina più accessibile, quali tricostatina A e 5-aza-2’-deossicitidina, è in grado di incrementarne l’espressione. Nella seconda parte, un’analisi bioinformatica seguita da saggi di attività in vitro in cellule epiteliali prostatiche trattate con farmaci epigenetici, hanno permesso di identificare, per la prima volta in uomo, una seconda regione regolatrice denominata P2, capace di controllare l’espressione di CLU 2. Rispetto a P1, il classico promotore di CLU già ampiamente studiato da altri gruppi di ricerca, P2 è un promotore debole, privo di TATA box, che nelle cellule epiteliali prostatiche è silente in condizioni basali e la cui attività incrementa in seguito alla somministrazione di farmaci epigenetici capaci di alterare le modificazioni post-traduzionali delle code istoniche nell’intorno di P2. Ne consegue un rilassamento della cromatina e un successivo aumento di trascrizione di CLU 2. La presenza di un’isola CpG differentemente metilata nell’intorno di P1 spiegherebbe, almeno in parte, i differenti livelli di espressione di CLU che si osservano tra le diverse linee cellulari. Nella terza parte, l’analisi del pathway di NF-kB in un modello sperimentale di tumore prostatico in cui CLU è stata silenziata o sovraespressa, ha permesso di capire come la forma citosolica di CLU abbia un ruolo inibitorio nei confronti dell’attività del fattore trascrizionale NF-kB. CLU inibisce la fosforilazione e l’attivazione di p65, il membro più rappresentativo della famiglia NF-kB, con conseguente riduzione della trascrizione di alcuni geni da esso regolati e coinvolti nel rimodellamento della matrice extracellulare, quali l’urochinasi attivatrice del plasminogeno, la catepsina B e la metallo proteinasi 9. È stato dimostrato che tale inibizione non è dovuta a un’interazione fisica diretta tra CLU e p65, per cui si suppone che CLU interagisca con uno dei componenti più a monte della via di segnalazione responsabile della fosforilazione ed attivazione di p65.

Polymerizable Peptide Monomers for the Targeted and Intracellular Delivery of Cancer Therapeutics

Thesis (Ph.D.)--University of Washington, 2016-06

Multifaceted roles of the lysosome in the establishment and expansion of tuberculous infection

Thesis (Ph.D.)--University of Washington, 2016-04

A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause dis- ease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consist- ing of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar com- pound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aerugi- nosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

O papel do inflamassoma na infecção induzida pelas formas saprofíticas do fungo Fonsecaea pedrosoi

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2016.

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function:implications for age-related macular degeneration (AMD)

Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D. Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test. Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure. Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

Immunofluorescence detection and localization of B[a]P and TCDD in earthworm tissues

An immunohistochemical method using antibodies against polycyclic aromatic hydrocarbons (PAHs) and dioxins was developed on frozen tissue sections of the earthworm Eisenia andrei exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50 ppm) and 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) (0.01, 0.1, 2 ppb) in spiked standard soils. The concentrations of B[a]P and TCDD in E. andrei exposed to the same conditions were also measured using analytical chemical procedures. The results demonstrated that tissues of worms exposed to even minimal amount of B[a]P and TCDD reacted positively and specifically to anti-PAHs and -dioxins antibody. Immunofluorescence revealed a much more intense staining for the gut compared to the body wall; moreover, positively immunoreactive amoeboid coelomocytes were also observed, i.e. cells in which we have previously demonstrated the occurrence of genotoxic damage. The double immunolabelling with antibodies against B[a]P/TCDD and the lysosomal enzyme cathepsin D demonstrated the lysosomal accumulation of the organic xenobiotic compounds, in particular in the cells of the chloragogenous tissue as well as in coelomocytes, involved into detoxification and protection of animals against toxic chemicals. The method described is timesaving, not expensive and easily applicable.

The clinical significance of cathepsin S expression in human astrocytomas

Early local invasion by astrocytoma. cells results in tumor recurrence even after apparent total surgical resection, leading to the poor prognosis associated with malignant astrocytomas. Proteolytic enzymes have been implicated in facilitating tumor cell invasion and the current study was designed to characterize the expression of the cysteine proteinase cathepsin S (CatS) in astrocytomas and examine its potential role in invasion. Immunohistochemical analysis of biopsies demonstrated that CatS was expressed in astrocytoma cells but absent from normal astrocytes, oligodendrocytes, neurones and endothelial cells. Microglial cells and macrophages were also positive. Assays of specific activity in 59 astrocytoma biopsies confirmed CatS expression and in addition demonstrated that the highest levels of activity were expressed in grade IV tumors. CatS activity was also present in astrocytoma cells in vitro and the extracellular levels of activity were highest in cultures derived from grade IV tumors. In vitro invasion assays were carried out using the U251MG cell line and the invasion rate was reduced by up to 61% in the presence of the selective CatS inhibitor 4-Morpholineurea-LeuHomoPhe-vinylsulphone. We conclude that CatS expression is up-regulated in astrocytoma. cells and provide evidence for a potential role for CatS in invasion.