RESUMEN

The role of proteases in cancer was originally thought to be limited to the breakdown of basement membranes and extracellular matrix (ECM), thereby promoting cancer cell invasion into surrounding normal tissues. It is now well understood that proteases play a much more complicated role in all stages of cancer progression and that not only tumor cells, but also stromal cells are an important source of proteases in the tumor microenvironment. Among all the proteolytic enzymes potentially associated with cancer, some proteases have taken on heightened importance due to their significant up-regulation and ability to participate at multiple stages of cancer progression and metastasis. In this review, we discuss some of the advances in understanding of the roles of several key proteases from different classes in the development and progression of cancer and the potential to leverage their upregulated activity for the development of novel targeted treatment strategies.

RESUMEN

The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.

Asunto(s)

Catepsina E/metabolismo , Precursores Enzimáticos/metabolismo , Neoplasias Pancreáticas/patología , Secuencia de Aminoácidos , Animales , Catepsina E/antagonistas & inhibidores , Catepsina E/química , Línea Celular Tumoral , Progresión de la Enfermedad , Precursores Enzimáticos/antagonistas & inhibidores , Precursores Enzimáticos/química , Regulación Neoplásica de la Expresión Génica , Concentración de Iones de Hidrógeno , Ratones , Inhibidores de Proteasas/farmacología , Neoplasias Pancreáticas

RESUMEN

The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU). In the full-length immunoglobulin form, U33 IgG labeled with near-infrared fluorophores or radionuclides allowed us to noninvasively detect active uPA in prostate cancer xenograft models using optical and single-photon emission computed tomography imaging modalities. U33 IgG labeled with (111)In had a remarkable tumor uptake of 43.2% injected dose per gram (%ID/g) 72 hours after tail vein injection of the radiolabeled probe in subcutaneous xenografts. In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissemination prostate cancer model that recapitulated metastatic human cancer. The favorable imaging properties were the direct result of U33 IgG internalization through an uPA receptor-mediated mechanism in which U33 mimicked the function of the endogenous inhibitor of uPA to gain entry into the cancer cell. Overall, our imaging probe targets a prostate cancer-associated protease, through a unique mechanism, allowing for the noninvasive preclinical imaging of prostate cancer lesions.

Asunto(s)

Neoplasias de la Próstata/enzimología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Radioisótopos de Indio , Masculino , Ratones Desnudos , Trasplante de Neoplasias , Especificidad de Órganos , Neoplasias de la Próstata/diagnóstico por imagen , Radiofármacos , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Óptica , Activador de Plasminógeno de Tipo Uroquinasa/genética

RESUMEN

Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.

Asunto(s)

Anticuerpos Antineoplásicos/uso terapéutico , Receptores ErbB/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Profármacos/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Cetuximab , Humanos , Inmunohistoquímica , Macaca fascicularis , Ratones , Ratones Desnudos , Profármacos/toxicidad , Piel/efectos de los fármacos , Piel/patología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

Asunto(s)

Péptido Hidrolasas/metabolismo , Especificidad por Sustrato , Proteasas Virales 3C , Animales , Carboxipeptidasas/metabolismo , Carcinoma Ductal Pancreático/enzimología , Catepsina E/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Cisteína Endopeptidasas/metabolismo , Exopeptidasas/metabolismo , Granzimas/metabolismo , Humanos , Ratones , Elastasa Pancreática/metabolismo , Biblioteca de Péptidos , Péptidos/metabolismo , Schistosoma mansoni , Espectrometría de Masas en Tándem , Proteínas Virales/metabolismo

RESUMEN

BACKGROUND: In order for tumors to grow and proliferate, they must avoid recognition by immune cells and subsequent death by apoptosis. Granzyme B (GrB), a protease located in natural killer cells, initiates apoptosis in target cells. Inhibition of GrB by PI-9, its natural inhibitor, can prevent apoptosis. Here we investigate whether PI-9 protects prostate cancer cells from apoptosis. METHODS: The expression of PI-9 was quantified by qPCR in several prostate cancer cell lines, and GrB activity was tested in each cell line. PI-9 was overexpressed in LNCaP cells, which lack endogenous PI-9. Apoptosis was induced by natural killer cells in LNCaP cells that either contained or lacked PI-9, and the percent cell death was quantified. Lastly, PI-9 levels were examined by qPCR and immunohistochemistry in prostate tumor tissue. RESULTS: Prostate cancer cell lines that expressed PI-9 could inhibit GrB. Overexpression of PI-9 protected LNCaP cells from natural killer cell-mediated apoptosis. Examination of the levels of PI-9 in tissue from prostate tumors showed that PI-9 could be upregulated in low grade tumors and stochastically dysregulated in high grade tumors. Additionally, PI-9 was found consistently in high grade prostatic intraepithelial neoplasia and atrophic lesions. CONCLUSIONS: These results indicate that overexpression of PI-9 can protect prostate cancer cells from apoptosis, and this effect may occur in human prostate tumors. These findings imply that early prostatic inflammation may trigger this increase in PI-9. This suggests that PI-9 upregulation is needed early in tumor progression, before additional protective mechanisms are in place.

Asunto(s)

Adenocarcinoma/metabolismo , Granzimas/antagonistas & inhibidores , Neoplasias de la Próstata/metabolismo , Serpinas/metabolismo , Adenocarcinoma/patología , Apoptosis/fisiología , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Masculino , Neoplasias de la Próstata/patología , Regulación hacia Arriba

RESUMEN

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1'-P2' residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

Asunto(s)

Variación Genética , Granzimas/metabolismo , Secuencia de Aminoácidos , Animales , Muerte Celular , Línea Celular , Regulación de la Expresión Génica , Granzimas/genética , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Nucleofosmina , Conformación Proteica , Especificidad de la Especie , Especificidad por Sustrato , Tubulina (Proteína)/metabolismo

RESUMEN

Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with beta1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/beta1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular/efectos de los fármacos , Fragmentos Fab de Inmunoglobulinas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Monoclonales , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Unión Proteica/inmunología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/inmunología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Transducción de Señal/inmunología , Spodoptera , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo

RESUMEN

MOTIVATION: Granzyme B (GrB) and caspases cleave specific protein substrates to induce apoptosis in virally infected and neoplastic cells. While substrates for both types of proteases have been determined experimentally, there are many more yet to be discovered in humans and other metazoans. Here, we present a bioinformatics method based on support vector machine (SVM) learning that identifies sequence and structural features important for protease recognition of substrate peptides and then uses these features to predict novel substrates. Our approach can act as a convenient hypothesis generator, guiding future experiments by high-confidence identification of peptide-protein partners. RESULTS: The method is benchmarked on the known substrates of both protease types, including our literature-curated GrB substrate set (GrBah). On these benchmark sets, the method outperforms a number of other methods that consider sequence only, predicting at a 0.87 true positive rate (TPR) and a 0.13 false positive rate (FPR) for caspase substrates, and a 0.79 TPR and a 0.21 FPR for GrB substrates. The method is then applied to approximately 25 000 proteins in the human proteome to generate a ranked list of predicted substrates of each protease type. Two of these predictions, AIF-1 and SMN1, were selected for further experimental analysis, and each was validated as a GrB substrate. AVAILABILITY: All predictions for both protease types are publically available at http://salilab.org/peptide. A web server is at the same site that allows a user to train new SVM models to make predictions for any protein that recognizes specific oligopeptide ligands.

Asunto(s)

Biología Computacional/métodos , Péptido Hidrolasas/química , Análisis de Secuencia de Proteína/métodos , Caspasas/química , Ligandos

RESUMEN

The use of plasmon coupling in metal nanoparticles has shown great potential for the optical characterization of many biological processes. Recently, we have demonstrated the use of "plasmon rulers" to observe conformational changes of single biomolecules in vitro. Plasmon rulers provide robust signals without photobleaching or blinking. Here, we show the first application of plasmon rulers to in vivo studies to observe very long trajectories of single biomolecules in live cells. We present a unique type of plasmon ruler comprised of peptide-linked gold nanoparticle satellites around a core particle, which was used as a probe to optically follow cell-signaling pathways in vivo at the single-molecule level. These "crown nanoparticle plasmon rulers" allowed us to continuously monitor trajectories of caspase-3 activity in live cells for over 2 h, providing sufficient time to observe early-stage caspase-3 activation, which was not possible by conventional ensemble analyses.

Asunto(s)

Caspasa 3/química , Caspasa 3/metabolismo , Nanopartículas del Metal/química , Técnicas de Sonda Molecular , Sondas Moleculares/química , Apoptosis/fisiología , Línea Celular , Activación Enzimática , Oro , Humanos , Cinética , Luz , Conformación Proteica , Dispersión de Radiación , Transducción de Señal , Resonancia por Plasmón de Superficie

RESUMEN

Dictyostelium myosin-II bipolar thick filament (BTF) assembly is heavily dependent on ionic strength and temperature and is reversible by the phosphorylation of just three threonines. Truncated tail fragments of Dictyostelium myosin-II are commonly used as models for BTF assembly, as they self-assemble into regular paracrystals that recapitulate the ionic strength and phosphorylation dependence of full-length Dictyostelium myosin-II BTF assembly. Here we show that Dictyostelium myosin-II tail fragment assembly is highly temperature dependent, similar to full-length Dictyostelium myosin-II. Assembly of paracrystals was far more robust at 4 degrees C than at higher temperatures. Pre-assembled paracrystals disassembled completely when shifted to 37 degrees C, indicating that assembly does not greatly improve the thermostability of these tail fragments. The melting temperatures of individual Dictyostelium myosin-II tail coiled-coils under both low and high ionic strength conditions that prohibit paracrystal assembly are extremely low, 21 degrees C and 28 degrees C, respectively. These data are consistent with reversible thermal denaturation of the coiled-coil as the most likely explanation for assembly incompetence under either very low ionic strength or high temperature conditions. Assembled paracrystals of a structurally similar fragment of nonmuscle myosin-IIA were far more thermodynamically stable than their Dictyostelium counterparts at the temperatures examined here.

Asunto(s)

Dictyostelium/metabolismo , Miosina Tipo II/metabolismo , Fragmentos de Péptidos/metabolismo , Temperatura , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Cristalografía , Dictyostelium/genética , Electricidad , Humanos , Microscopía Electrónica de Transmisión , Miosina Tipo II/química , Miosina Tipo II/genética , Miosina Tipo IIA no Muscular/química , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Concentración Osmolar , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Unión Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinámica , Temperatura de Transición

RESUMEN

The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates, and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein, Hip. Hsp90, Bag1L, and Hip were validated as GrB substrates in vitro, and mutational analysis confirmed the additional cleavage site in Hip. Because the role of Hip in apoptosis is unknown, its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization; therefore, it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer (NK) cell-mediated death. Hip is cleaved at both GrB cleavage sites during NK-mediated cell death in a caspase-independent manner, and its cleavage is due solely to GrB and not other granule components. Furthermore, Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T-cell line, suggesting that Hip is a substrate unique to GrB. RNA interference-mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis, indicating that proteolysis by GrB of Hip contributes to death induction. The small effect of RNA interference-mediated Hip deficiency on cytotoxicity is in agreement with the inherent redundancy of NK cell-mediated cell death. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contribute to understanding the interplay between stress response and apoptosis.

Asunto(s)

Factores de Coagulación Sanguínea/fisiología , Granzimas/química , Apoptosis , Factores de Coagulación Sanguínea/química , Fragmentación del ADN , Humanos , Células Jurkat , Células K562 , Células Asesinas Naturales/metabolismo , Chaperonas Moleculares/metabolismo , Estrés Oxidativo , Plásmidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Proteínas Ribosómicas , Factores de Tiempo

RESUMEN

Coiled-coils occur in a variety of proteins involved in mechanical and structural tasks in the cell. Their mechanical properties are important in various contexts ranging from hair elasticity to synaptic fusion. Beyond their importance in biology, coiled-coils have also attracted interest as programmable protein sequences for the design of novel hydrogels and materials. We have studied the elastic properties of the myosin coiled-coil at the single molecule level. The coiled-coil undergoes a massive structural transition at forces between 20 and 25 pN where the coil extends to about 2.5 times its original length. Unlike all other proteins investigated mechanically so far, this transition is reversible on a timescale of less than a second, making the coiled-coil a truly elastic protein.

Asunto(s)

Miosinas/química , Animales , Elasticidad , Músculo Esquelético/química , Conformación Proteica , Conejos , Termodinámica