Monoclonal antibodies targeting the disintegrin-like domain of ADAMDEC1 modulates the proteolytic activity and enables quantification of ADAMDEC1 protein in human plasma

Decysin-1 (ADAMDEC1) is an orphan ADAM-like metalloprotease with unknown biological function and a short domain structure. ADAMDEC1 mRNA has previously been demonstrated primarily in macrophages and mature dendritic cells. Here, we generated monoclonal antibodies (mAbs) against the mature ADAMDEC1 protein, as well as mAbs specific for the ADAMDEC1 pro-form, enabling further investigations of the metalloprotease. The generated mAbs bind ADAMDEC1 with varying affinity and represent at least six different epitope bins. Binding of mAbs to one epitope bin in the C-terminal disintegrin-like domain efficiently reduces the proteolytic activity of ADAMDEC1. A unique mAb, also recognizing the disintegrin-like domain, stimulates the caseinolytic activity of ADAMDEC1 while having no significant effect on the proteolysis of carboxymethylated transferrin. Using two different mAbs binding the disintegrin-like domain, we developed a robust, quantitative sandwich ELISA and demonstrate secretion of mature ADAMDEC1 protein by primary human macrophages. Surprisingly, we also found ADAMDEC1 present in human plasma with an approximate concentration of 0.5 nM. The presence of ADAMDEC1 both in human plasma and in macrophage cell culture supernatant were biochemically validated using immunoprecipitation and Western blot analysis demonstrating that ADAMDEC1 is secreted in a mature form

Modulation of cytochrome c release by mitochondrial redox status and caspase-2

The release of cytochrome c is an important event during apoptosis, induced by diverse stimuli. Our laboratory has previously proposed that cytochrome c release occurs via a two-step process, involving the detachment of the hemoprotein from its binding to the inner mitochondrial membrane, followed by its release into the cytosol through pores in the outer mitochondrial membrane - an event that is usually triggered by proapoptotic Bcl-2 family proteins, such as Bid, Bax and Bak. Cytochrome c specifically and stoichiometrically binds to cardiolipin, thus anchoring the hemoprotein to the inner mitochondrial membrane to participate in electron transport. Mitochondria are the main intracellular source of reactive oxygen species (ROS), and it has been shown that cardiolipin might become oxidized and lose its interaction with cytochrome c as a result of increased ROS production, or deficient ROS scavenging, within the mitochondria. This thesis investigates the mechanism of cytochrome c release from mitochondria, and how this may be modulated by caspase-2 or the mitochondrial redox status. Caspase-2 is one of the best conserved caspases among species, and it is unique in the sense that it shares features of both initiator and executioner caspases. We have demonstrated a new role for caspase-2 in apoptosis signaling, and propose a novel mechanism for cytochrome c release, mediated by caspase-2 and possibly involving pore formation in the mitochondrial membrane by this protease (papers I and II). Caspase-2 seemingly plays a role in apoptosis induction by exerting a direct effect on mitochondria, thereby releasing cytochrome c. Interestingly, this effect seems to also involve an interaction between caspase-2 and cardiolipin. On the contrary, we have shown that cardiolipin is not a pre-requisite for Bax-mediated cytochrome c release (Paper III). However, cardiolipin must be affected by protein binding or oxidation in order for solubilization of cytochrome c to occur, allowing release of the hemoprotein through the Bax-pores. One typical dissociation factor for cytochrome c is oxidation of cardiolipin. The glutathione (GSH; gamma-glu-cys-gly) system is one of the most important intracellular redox systems. This abundant tripeptide protects from ROS and has been linked to apoptosis by several observations. In paper IV, apart from describing a new method for GSH visualization in the cell, we also demonstrated the capability of mitochondria to scavenge GSH during oxidative stress. Moreover, papers V and VI indicated that mitochondrial Grx2 is a possible inhibitor of apoptosis, since knocking down the protein by siRNA (paper V) or overexpressing Grx2 (paper VI) influence cell death signaling, probably by preventing oxidation or degradation of cardiolipin (paper VI). It is clear that the mitochondrial redox environment is crucial for keeping cardiolipin reduced and preventing cytochrome c release. Lowering the level of Grx2, or other mitochondrial redox enzymes, may thus have a lethal effect on the cell. In conclusion, it is clear that the release of cytochrome c may occur by different mechanisms, depending on the apoptotic inducer and on the type of cell. While caspase-2 is able to form pores in the mitochondrial membrane, as well as promote dissociation of cytochrome c from cardiolipin, we cannot exclude that this protease also may work in concert with other pore forming agents, such as Bax. However, cardiolipin is not required for Bax pore-formation of the mitochondrial membrane. In addition, we have shown that mitochondria require a unctional redox system for protection from apoptosis

Mitochondrial cytochrome c release may occur by volume-dependent mechanisms not involving permeability transition.

The mechanisms regulating mitochondrial outer-membrane permeabilization and the release of cytochrome c during apoptosis remain controversial. In the present study, we show in an in vitro model system that the release of cytochrome c may occur via moderate modulation of mitochondrial volume, irrespective of the mechanism leading to the mitochondrial swelling. In contrast with mitochondrial permeability transition-dependent release of cytochrome c, in the present study mitochondria remain intact and functionally active

Glutathione and mitochondria

Glutathione (GSH) is the main non-protein thiol in cells whose functions are dependent on the redox-active thiol of its cysteine moiety that serves as a cofactor for a number of antioxidant and detoxifying enzymes. While synthesized exclusively in the cytosol from its constituent amino acids, GSH is distributed in different compartments, including mitochondria where its concentration in the matrix equals that of the cytosol. This feature and its negative charge at physiological pH imply the existence of specific carriers to import GSH from the cytosol to the mitochondrial matrix, where it plays a key role in defense against respiration-induced reactive oxygen species and in the detoxification of lipid hydroperoxides and electrophiles. Moreover, as mitochondria play a central strategic role in the activation and mode of cell death, mitochondrial GSH has been shown to critically regulate the level of sensitization to secondary hits that induce mitochondrial membrane permeabilization and release of proteins confined in the intermembrane space that once in the cytosol engage the molecular machinery of cell death. In this review, we summarize recent data on the regulation of mitochondrial GSH and its role in cell death and prevalent human diseases, such as cancer, fatty liver disease, and Alzheimer's disease. © 2014 Ribas, Garcia-Ruiz and Fernandez-Checa.Vicent Ribas is recipient of an Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) Post-doctoral Fellowship-BIOTRACK, supported by the European Community’s Seventh Framework Programme (EC FP7/2007-2013) under the grant agreement number 229673 and the Spanish Ministry of Economy and Competitiveness (MINECO) through the grant COFUND2013-40261. The work was supported by Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Fundació la Marató de TV3 and grants PI11/0325 (META) from the Instituto Salud Carlos III and grants, SAF2011-23031, and SAF2012-34831 from Plan Nacional de I+D, Spain; Fundación Mutua Madrileña and the center grant P50-AA-11999 (Research Center for Liver and Pancreatic Diseases, NIAAA/NIH)Peer Reviewe

Profiling constitutive proteolytic events in vivo

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)–MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments