Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from <i>Solanum tuberosum</i> subsp. <i>andigenum</i> cv. Churqueña

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.Facultad de Ciencias Exacta

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from <i>Solanum tuberosum</i> subsp. <i>andigenum</i> cv. Churqueña

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.Facultad de Ciencias Exacta

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs

Insights into the specificity and function of M14 metallocarboxypeptidases from structural and degradomic studies

Las proteasas son enzimas que escinden proteínas catalizando la hidrólisis del enlace peptídico. Todas las proteínas sufren proteólisis en algún momento u otro de su ciclo de vida y de este modo las proteasas regulan casi todos los procesos biológicos. Las carboxipeptidasas son proteasas que hidrolizan el enlace C-terminal de las proteínas y péptidos. En esta tesis se estudia a las carboxipeptidasas de la familia M14 en la clasificación de MEROPS (y que describiremos como metalocarboxipeptidasas). Las metalocarboxipeptidasas controlan importantes procesos biológicos, como son la regulación de la presión sanguínea, el equilibrio entre coagulación y fibrinólisis, o el procesamiento de neuropéptidos y hormonas peptídicas. Además, están implicadas en procesos patológicos como el cáncer o las enfermedades neurodegenerativas. La información disponible sobre la función biológica de estas enzimas es muy limitada, por lo que en la presente tesis se planteó como objetivo profundizar en el estudio de la función de varias metalocarboxipeptidasas. Con esta finalidad se realizaron ensayos cinéticos, estudios de biología celular y de determinación de la estructura tridimensional. Además, se desarrollaron y aplicaron distintas técnicas proteómicas para el estudio de los sustratos de carboxipeptidasas (y que se incluyen por lo tanto dentro del campo de la degradómica). Esta tesis se compone de cuatro trabajos de investigación independientes en los que se caracteriza funcional y estructuralmente a distintas metalocarboxipeptidasas. En el primer trabajo se determinó la estructura tridimensional de una isoforma corta del gen silver de Drosophila melanogaster, correspondiente al primer dominio de la carboxipeptidasa D de mamíferos. La mutación de este gen genera moscas adultas con cutículas pálidas o plateadas y con alas puntiagudas. Esta estructura tridimensional fue obtenida en presencia del inhibidor GEMSA a una resolución de 2,7 Å y se corresponde en general con la estructura canónica de otras carboxipeptidasas de su subfamilia. Presenta sin embargo como elemento distintivo un lazo flexible fácilmente accesible a las proteasas, por lo que la proteólisis podría constituir un mecanismo de regulación de esta enzima. En el segundo trabajo se incluye la caracterización bioquímica y funcional de la carboxipeptidasa A4 humana (CPA4), la cual ha sido asociada con la agresividad del cáncer de próstata. Se determinó que esta enzima es secretada al espacio extracelular y muestra un pH óptimo neutro. Mediante aproximaciones peptidómicas se identificaron distintos péptidos bioactivos como posibles sustratos de la CPA4: la neurotensina, diversas graninas y péptidos opioides tales como la Met- o Leu- encefalina. Estos péptidos están involucrados en la proliferación y diferenciación de las células de cáncer de próstata, y permiten explicar la asociación de esta enzima con la agresividad de este tipo de cáncer. En definitiva, la CPA4 funcionaría en el procesamiento de neuropéptidos a nivel extracelular. El tercer trabajo comprende el desarrollo de una nueva aproximación proteómica para el estudio de las preferencias de sustrato de las carboxipeptidasas. Esta técnica, basada en la tecnología COFRADIC, permite aislar miles de productos de la acción de carboxipeptidasas sobre una librería de péptidos (generada a partir de un proteoma celular). Esta técnica permitió describir las poco caracterizadas preferencias de sustrato de la carboxipeptidasa de mastocitos. En el cuarto trabajo se aplicó una técnica proteómica denominada "COFRADIC C-terminal" que permite la búsqueda de sustratos naturales de carboxipeptidasas. En particular, esta técnica se aplicó a la búsqueda de sustratos naturales de la carboxipeptidasa citosólica 1 (CCP1), la cual estaría asociada a mecanismos de degeneración y regeneración neuronal que aún se desconocen. Se ha propuesto que CCP1 tiene como sustratos a la tubulina y a otras proteínas que presentan residuos de glutámico en su extremo C-terminal. En este trabajo se pudieron identificar 7 nuevos posibles sustratos protéicos para CCP1.Proteases are enzymes that irreversibly cleave proteins by the catalysis of peptide-bond hydrolysis. With all proteins undergoing proteolysis at any point of their life cycle, proteases regulate virtually every biological process. Carboxypeptidases are proteolytic enzymes that catalyze the hydrolisis of peptidic bonds at the C-terminus of peptides and proteins. In this thesis, we studied carboxypeptidases from the M14 family (according to the MEROPS database classification), hereafter described as metallocarboxypeptidases. Metallocarboxypeptidases play key roles in controlling various biological processes, including blood coagulation/fibrinolysis, blood pressure regulation, pro-hormone and neuropeptide processing; and are also implicated in various pathological conditions such as cancer or neurodegenerative disorders. Despite the important functions performed by these enzymes, there is usually limited knowledge about their in vivo biological roles. The present thesis was aimed to gain insights into the understanding of the biological functions of different metallocarboxypeptidases. For this purpose, we applied different approaches that included kinetic studies, cell biological studies and structural characterization. In addition, we participated in the development of different proteomic tools for protease/carboxypeptidase substrate determination (degradomics). The present thesis consists of four independent research works that focus in the structural and functional characterization of different metallocarboxypeptidases. The first work presents the crystal structure of a short isoform of Drosophila melanogaster silver gene, which corresponds to the first repeat of a mammalian CPD. Silver gene is responsible for the silver mutation, characterized for adult flies that display cuticles that are pale and silvery in color, and pointed wings. This three-dimensional structure was solved in presence of an inhibitor (GEMSA) at 2.7 Å resolution and overall corresponds with the structure of other members of its subfamily of metallocarboxypeptidases. A unique structural element in the here presented structure is a surface hotspot targetable by peptidases, suggesting that this enzyme might be regulated (i.e., inactivated) by proteolysis. The second work comprises the biochemical and functional characterization of human carboxypeptidase A4 (CPA4), an enzyme that has been associated with prostate cancer aggressiveness. We found that this enzyme is secreted outside the cells and displays a neutral pH optimum that is compatible with a function in the extracellular environment. A peptidomic study identified several biologically relevant putative peptidic substrates of CPA4: neurotensin, granins, and opioid peptides such as Met- and Leu-enkephalin. These peptides are involved in the proliferation and differentiation of prostate cancer cells, potentially explaining the link between CPA4 and cancer aggressiveness. Altogether, CPA4 would function in the extracellular neuropeptide processing. The third work comprises the development of a novel proteomic approach to study the substrate preferences of carboxypeptidases. This technology can be described as a COFRADIC-based proteome-derived peptide library approach and allows for the enrichment of thousands of carboxypeptidase products from natural, proteome-derived peptide libraries. This approach served to delineate the previously little studied substrate specificity profile of mouse mast cell carboxypeptidase. In the fourth work we applied a proteomic tool (C-terminal COFRADIC) that allows searching for natural substrates of carboxypeptidases. Particularly, we searched for natural substrates of cytosolic carboxypeptidase 1 (CCP1) in a cellular system. This enzyme is considered to be a molecular link between neuronal degeneration and regeneration, although the molecular pathways in which CCP1 is implicated remain undefined. It has been proposed that CCP1 would posttranslationally modify tubulin and other proteins that present glutamate-stretches in their C-terminus. Here, we were able to identify seven new putative CCP1 protein substrates

Biochemical and MALDI-TOF mass spectrometric characterization of a novel native and recombinant cystine knot miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña

Altres ajuts: Bilateral Cooperation Program MinCyT-MICINN (project ES/09/24-AR2009/006)Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs

Substrate specificity and structural modeling of human Carboxypeptidase Z : a unique protease with a Frizzled-like domain

Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1' position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process

EUSO-SPB1 mission and science

International audienceThe Extreme Universe Space Observatory on a Super Pressure Balloon 1 (EUSO-SPB1) was launched in 2017 April from Wanaka, New Zealand. The plan of this mission of opportunity on a NASA super pressure balloon test flight was to circle the southern hemisphere. The primary scientific goal was to make the first observations of ultra-high-energy cosmic-ray extensive air showers (EASs) by looking down on the atmosphere with an ultraviolet (UV) fluorescence telescope from suborbital altitude (33 km). After 12 days and 4 h aloft, the flight was terminated prematurely in the Pacific Ocean. Before the flight, the instrument was tested extensively in the West Desert of Utah, USA, with UV point sources and lasers. The test results indicated that the instrument had sensitivity to EASs of ⪆3 EeV. Simulations of the telescope system, telescope on time, and realized flight trajectory predicted an observation of about 1 event assuming clear sky conditions. The effects of high clouds were estimated to reduce this value by approximately a factor of 2. A manual search and a machine-learning-based search did not find any EAS signals in these data. Here we review the EUSO-SPB1 instrument and flight and the EAS search

Design and implementation of the AMIGA embedded system for data acquisition

International audienceThe Auger Muon Infill Ground Array (AMIGA) is part of the AugerPrime upgrade of the Pierre Auger Observatory. It consists of particle counters buried 2.3 m underground next to the water-Cherenkov stations that form the 23.5 km2 large infilled array. The reduced distance between detectors in this denser area allows the lowering of the energy threshold for primary cosmic ray reconstruction down to about 1017 eV. At the depth of 2.3 m the electromagnetic component of cosmic ray showers is almost entirely absorbed so that the buried scintillators provide an independent and direct measurement of the air showers muon content. This work describes the design and implementation of the AMIGA embedded system, which provides centralized control, data acquisition and environment monitoring to its detectors. The presented system was firstly tested in the engineering array phase ended in 2017, and lately selected as the final design to be installed in all new detectors of the production phase. The system was proven to be robust and reliable and has worked in a stable manner since its first deployment