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Research Interests: Biochemistry, Chemistry, Kinetics, Calcium, Biological Chemistry, and 15 moreMedicine, Biological Sciences, ATPase, Animals, Calcium channels, Endoplasmic Reticulum, CHEMICAL SCIENCES, Lipid bilayers, Binding Site, Biochemistry and cell biology, Adenosine Triphosphate, Fluorescein, binding sites, Medical and Health Sciences, and Medical biochemistry and metabolomics
Research Interests: Biochemistry, Microbiology, Chemistry, Medical Microbiology, Membrane Biology, and 15 moreBiology, Medicine, Biological Sciences, Brain, Animals, Endoplasmic Reticulum, Enzyme, Clinical Sciences, CHEMICAL SCIENCES, Calpain, Lipid bilayers, Enzymatic Activity, Kinetic Parameter, Biochemistry and cell biology, and Decarboxylation
Research Interests: Chemistry, Electrophysiology, Biology, Enzyme Inhibitors, Cell Biology, and 15 moreBiological Chemistry, Apoptosis, Biological Sciences, Cell line, Humans, Animals, Cytochrome C, CHEMICAL SCIENCES, Ion, Isoenzymes, Cell Survival, Electric Conductivity, Antineoplastic Agents, hexokinase, and Medical and Health Sciences
Research Interests: Biochemistry, Chemistry, Biology, Calcium, Medicine, and 15 moreDopamine, Humans, ATPase, Female, Animals, Male, Endoplasmic Reticulum, Middle Aged, Adult, Hydrolysis, Acetic Anhydrides, Adenosine Triphosphate, Pharmacology and pharmaceutical sciences, In Vitro Techniques, and Muscle fibers skeletal
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We have reexamined the "uncoupling" of Ca2+ transport from ATP hydrolysis, which has been reported to be caused by trypsin cleavage of the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles at the second (slower) of two... more
We have reexamined the "uncoupling" of Ca2+ transport from ATP hydrolysis, which has been reported to be caused by trypsin cleavage of the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles at the second (slower) of two characteristic tryptic sites (Scott, T. L., and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137-144). We find that the loss of Ca2+ accumulation capacity in SR vesicles is poorly correlated with this cleavage under several conditions. The loss is accompanied by increased Ca2+ permeability but not by changes in the properties of the ATPase or ATP-Pi exchange activities of the vesicles. Proteoliposomes containing purified Ca2+-ATPase which has been cleaved in part at the two tryptic sites are as well coupled and impermeable to Ca2+ as proteoliposomes containing intact Ca2+-ATPase. We conclude that the loss of Ca2+ accumulation capacity in SR vesicles on tryptic treatment is due to cleavage of a SR membrane component other than the Ca2+-ATPase, possibly a component of the gated channels which function in Ca2+ release from SR, which leads to a Ca2+ leak. The hydrolytic and coupled transport functions of the Ca2+-ATPase itself may well be unaffected by the two tryptic cleavages.
Research Interests: Chemistry, Calcium, Biological Chemistry, Medicine, Biological Sciences, and 11 moreATPase, Animals, Endoplasmic Reticulum, Trypsin, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Membrane, CHEMICAL SCIENCES, Molecular weight, Rabbits, Sarcoplasmic reticulum, and Medical and Health Sciences
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SMAC/Diablo, a pro‐apoptotic protein, yet it is overexpressed in several cancer types. We have described a noncanonical function for SMAC/Diablo as a regulator of lipid synthesis during cancer cell proliferation and development. Here, we... more
SMAC/Diablo, a pro‐apoptotic protein, yet it is overexpressed in several cancer types. We have described a noncanonical function for SMAC/Diablo as a regulator of lipid synthesis during cancer cell proliferation and development. Here, we explore the molecular mechanism through which SMAC/Diablo regulates phospholipid synthesis. We showed that SMAC/Diablo directly interacts with mitochondrial phosphatidylserine decarboxylase (PSD) and inhibits its catalytic activity during synthesis of phosphatidylethanolamine (PE) from phosphatidylserine (PS). Unlike other phospholipids (PLs), PE is synthesized not only in the endoplasmic reticulum but also in mitochondria. As a result, PSD activity and mitochondrial PE levels were increased in the mitochondria of SMAC/Diablo‐deficient cancer cells, with the total amount of cellular PLs and phosphatidylcholine (PC) being lower as compared to SMAC‐expressing cancer cells. Moreover, in the absence of SMAC/Diablo, PSD inhibited cancer cell proliferatio...
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Mitochondrial Ca uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca signaling, energy metabolism, and cell death. Ca transport across the inner and outer mitochondrial membranes (IMM, OMM,... more
Mitochondrial Ca uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca signaling, energy metabolism, and cell death. Ca transport across the inner and outer mitochondrial membranes (IMM, OMM, respectively), is mediated by several proteins, including the voltage-dependent anion channel 1 (VDAC1) in the OMM, and the mitochondrial Ca uniporter (MCU) and Na-dependent mitochondrial Ca efflux transporter, (the NCLX), both in the IMM. By transporting Ca across the OMM to the mitochondrial inner-membrane space (IMS), VDAC1 allows Ca access to the MCU, facilitating transport of Ca to the matrix, and also from the IMS to the cytosol. Intra-mitochondrial Ca controls energy production and metabolism by modulating critical enzymes in the tricarboxylic acid (TCA) cycle and fatty acid oxidation. Thus, by transporting Ca, VDAC1 plays a fundamental role in regulating mitochondrial Ca homeostasis, oxidative phosphorylation, and Ca crosstalk among mitochondria, cytoplasm, and the endoplasmic reticulum (ER). VDAC1 has also been recognized as a key protein in mitochondria-mediated apoptosis, and apoptosis stimuli induce overexpression of the protein in a Ca-dependent manner. The overexpressed VDAC1 undergoes oligomerization leading to the formation of a channel, through which apoptogenic agents can be released. Here, we review the roles of VDAC1 in mitochondrial Ca homeostasis, in apoptosis, and in diseases associated with mitochondria dysfunction.
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The photoaffinity analog of ATP, 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP... more
The photoaffinity analog of ATP, 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP stimulates up to fivefold the binding of ryanodine to its receptor. Photoactivation by ultraviolet light of the benzophenone group in the [alpha-32P]Bz2ATP results in covalent binding of [alpha-32P]Bz2ATP to the 450-kDa polypeptide, the ryanodine receptor's subunit. An apparent molar stiochiometry of Bz2ATP to the tetrameric ryanodine receptor complex of 1.146 +/- 0.087 (n = 2) was estimated. The covalent binding of [alpha-32P]Bz2ATP was inhibited by ATP and analogous compounds in the order: ATP = AdoPP[CH2]P = ADP = Ado = cAMP > AMP > ITP = GTP. Similar specificity was obtained for the stimulation of ryanodine binding by these nucleotides. ATP increased the ryanodine binding affinity by about sixfold. The polycationic dye ruthenium red, known as an inhibitor of Ca2+ release and ryanodine binding, inhibited the labeling of the ryanodine receptor by [alpha-32P]Bz2ATP. Tryptic digestion of the ryanodine receptor revealed a [alpha-32P]Bz2ATP-labeled 76-kDa tryptic fragment. Digestion of either the [alpha-32P]Bz2ATP-labeled 450-kDa or the 76-kDa polypeptides with S. aureus resulted in the appearance of four labeled fragments of 39, 33, 27 and 13 kDa, where the 39-kDa fragment is the precursor of the 27-kDa and 13-kDa fragments. The results suggest that the regulation of Ca2+ release by ATP involves an ATP binding site(s) located on the 27-kDa and 13-kDa fragments of the ryanodine receptor protein.
Research Interests: Biochemistry, Chemistry, Photochemistry, Medicine, Animals, and 12 moreCalcium channels, Muscles, Ryanodine Receptor, European, Skeletal Muscle, Rabbits, Binding Site, Biochemistry and cell biology, Adenosine Triphosphate, Photoaffinity Labeling, binding sites, and Medical biochemistry and metabolomics
Research Interests: Chemistry, Biological Chemistry, Medicine, Biological Sciences, Humans, and 15 moreAnimals, Biological, CHEMICAL SCIENCES, Amino Acid Sequence, NAD, Binding Site, Nucleotide, Nucleotides, Adenosine Triphosphate, Adenosine monophosphate, Adenosine Diphosphate, Molecular Sequence Data, binding sites, fluorescent dyes, and Medical and Health Sciences
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Research Interests: Biology, Cell Biology, Apoptosis, Medicine, Cytochrome C, and 2 moreMitochondrion and VDAC(Mitochondrion and VDAC)
(Mitochondrion and VDAC)
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Ca(2+) is a ubiquitous cellular signal, with changes in intracellular Ca(2+) concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca(2+) uptake and... more
Ca(2+) is a ubiquitous cellular signal, with changes in intracellular Ca(2+) concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca(2+) uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca(2+) signaling, energy metabolism and cell death. Ca(2+) transport across the inner and outer mitochondrial membranes is mediated by several proteins, including channels, antiporters, and a uniporter. In this article, we present the background to several methods now established for assaying mitochondrial Ca(2+) transport activity across both mitochondrial membranes. The first of these is Ca(2+) transport mediated by the outer mitochondrial protein, the voltage-dependent anion-selective channel protein 1 (VDAC1, also known as porin 1), both as a purified protein reconstituted into a planar lipid bilayer (PLB) or into liposomes and as a mitochondrial membrane-embedded protein. The second method involves isolated mitochondria for assaying the activity of an inner mitochondrial membrane transport protein, the mitochondrial Ca(2+) uniporter (MCU) that transports Ca(2+) and is powered by the steep mitochondrial membrane potential. In the event of Ca(2+) overload, this leads to opening of the mitochondrial permeability transition pore (MPTP) and cell death. The third method describes how Na(+)-dependent mitochondrial Ca(2+) efflux mediated by mitochondrial NCLX, a member of the Na(+)/Ca(2+) exchanger superfamily, can be assayed in digitonin-permeabilized HEK-293 cells. The Ca(2+)-transport assays can be performed under various conditions and in combination with inhibitors, allowing detailed characterization of the transport activity of interest.
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In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time- and concentration-dependent fashion by prior treatment with the carboxyl reagent... more
In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time- and concentration-dependent fashion by prior treatment with the carboxyl reagent dicyclohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3-(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-pheny-lisoxazolium-3'-sulfonate (WRK) only slightly affects their ryanodine binding capacity. The amphipathic reagent N-ethoxy cabonyl-2-ethoxy-1,2-dihydroquinaline (EEDQ) inhibited ryanodine binding at relatively high concentrations. DCCD-modification of the SR decreased the binding affinities of the RyR for ryanodine and Ca2+ by about 3- and 18-fold, respectively. The single channel activity of SR membranes modified with DCCD and then incorporated into planar lipid bilayers is very low (5-8%) in comparison to control membranes. Application of DCCD to either the myoplasmic (cis) or luminal (trans) side of the reconstituted unmodified channels resulted in complete inhibition of their single channel activities. Similar results were obtained with the water soluble reagent WRK applied to the myoplasmic, but not to the luminal side. The DCCD-modified non-active channel is re-activated by addition of ryanodine in the presence of 250 microM Ca2+ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100 microM of Ca2+. The results suggest that a carboxyl residue(s) in the RyR is involved either in the binding of Ca2+, or in conformational changes that are produced by Ca2+ binding, and are required for the binding of ryanodine and the opening of the Ca2+ release channel.
Research Interests: Biophysics, Chemistry, Calcium, Medicine, Animals, and 14 moreCalcium channels, Endoplasmic Reticulum, Ryanodine Receptor, Bio-Nanotechnology and Membrane Molecular Biology, Time Factors, Carboxylic Acids, Protein Conformation, Lipid bilayers, Rabbits, Hydrogen-Ion Concentration, Biochemistry and cell biology, Sarcoplasmic reticulum, Cell Membrane, and binding sites(Calcium channels, Endoplasmic Reticulum, Ryanodine Receptor, Bio-Nanotechnology and Membrane Molecular Biology, Time Factors, Carboxylic Acids, Protein Conformation, Lipid bilayers, Rabbits, Hydrogen-Ion Concentration, Biochemistry and cell biology, Sarcoplasmic reticulum, Cell Membrane, and binding sites)
(Calcium channels, Endoplasmic Reticulum, Ryanodine Receptor, Bio-Nanotechnology and Membrane Molecular Biology, Time Factors, Carboxylic Acids, Protein Conformation, Lipid bilayers, Rabbits, Hydrogen-Ion Concentration, Biochemistry and cell biology, Sarcoplasmic reticulum, Cell Membrane, and binding sites)
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Research Interests: Chemistry, Calcium, Medicine, Biological Sciences, Magnesium, and 15 moreATPase, Animals, Endoplasmic Reticulum, Liposomes, Biochemical, Membrane, CHEMICAL SCIENCES, Hydrolysis, Amidines, Chemical Modification, Acetic Anhydrides, Adenosine Triphosphate, Aldehydes, ATP hydrolysis, and Medical and Health Sciences
Research Interests: Biophysics, Chemistry, Medicine, Biological Sciences, Animals, and 13 moreCalcium channels, Ryanodine Receptor, Biochemical, CHEMICAL SCIENCES, Protein Conformation, Propranolol, Rabbits, Hydrogen-Ion Concentration, Sodium Chloride, Sarcoplasmic reticulum, binding sites, Medical and Health Sciences, and In Vitro Techniques
Research Interests: Biophysics, Chemistry, Fluorescence, Calcium, Biological Chemistry, and 15 moreBiological Sciences, Animals, Calcium channels, Biological, Endoplasmic Reticulum, Ryanodine Receptor, CHEMICAL SCIENCES, EGTA, Rabbits, Binding Site, Biochemistry and cell biology, Sarcoplasmic reticulum, binding sites, Medical and Health Sciences, and Medical biochemistry and metabolomics
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Mitochondrial SMAC/Diablo induces apoptosis by binding the inhibitor of apoptosis proteins (IAPs), thereby activating caspases and, subsequently, apoptosis. Previously, we found that despite its pro-apoptotic activity, SMAC/Diablo is... more
Mitochondrial SMAC/Diablo induces apoptosis by binding the inhibitor of apoptosis proteins (IAPs), thereby activating caspases and, subsequently, apoptosis. Previously, we found that despite its pro-apoptotic activity, SMAC/Diablo is overexpressed in cancer, and demonstrated that in cancer it possesses new essential and non-apoptotic functions that are associated with regulating phospholipid synthesis including modulating mitochondrial phosphatidylserine decarboxylase activity. Here, we demonstrate additional functions for SMAC/Diablo associated with inflammation and immunity. CRISPR/Cas9 SMAC/Diablo-depleted A549 lung cancer cells displayed inhibited cell proliferation and migration. Proteomics analysis of these cells revealed altered expression of proteins associated with lipids synthesis and signaling, vesicular transport and trafficking, metabolism, epigenetics, the extracellular matrix, cell signaling, and neutrophil-mediated immunity. SMAC-KO A549 cell-showed inhibited tumor g...
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Mesothelioma, an aggressive cancer with a poor prognosis, is linked to asbestos exposure. However, carbon nanotubes found in materials we are exposed to daily can cause mesothelioma cancer. Cancer cells reprogram their metabolism to... more
Mesothelioma, an aggressive cancer with a poor prognosis, is linked to asbestos exposure. However, carbon nanotubes found in materials we are exposed to daily can cause mesothelioma cancer. Cancer cells reprogram their metabolism to support increased biosynthetic and energy demands required for their growth and motility. Here, we examined the effects of silencing the expression of the voltage-dependent anion channel 1 (VDAC1), controlling the metabolic and energetic crosstalk between mitochondria and the rest of the cell. We demonstrate that VDAC1 is overexpressed in mesothelioma patients; its levels increase with disease stage and are associated with low survival rates. Silencing VDAC1 expression using a specific siRNA identifying both mouse and human VDAC1 (si-m/hVDAC1-B) inhibits cell proliferation of mesothelioma cancer cells. Treatment of xenografts of human-derived H226 cells or mouse-derived AB1 cells with si-m/hVDAC1-B inhibited tumor growth and caused metabolism reprogrammi...
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Research Interests: Genetics, Cell Biology, Programmed cell death, SOD, Molecular sciences, and 2 moreMitochondrion and VDAC(Mitochondrion and VDAC)
(Mitochondrion and VDAC)
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The mitochondrial voltage-dependent anion channel 1 (VDAC1) protein is involved in several essential cancer hallmarks, including energy and metabolism reprogramming and apoptotic cell death evasion. In this study, we demonstrated the... more
The mitochondrial voltage-dependent anion channel 1 (VDAC1) protein is involved in several essential cancer hallmarks, including energy and metabolism reprogramming and apoptotic cell death evasion. In this study, we demonstrated the ability of hydroethanolic extracts from three different plants, Vernonanthura nudiflora (Vern), Baccharis trimera (Bac), and Plantago major (Pla), to induce cell death. We focused on the most active Vern extract. We demonstrated that it activates multiple pathways that lead to impaired cell energy and metabolism homeostasis, elevated ROS production, increased intracellular Ca2+, and mitochondria-mediated apoptosis. The massive cell death generated by this plant extract’s active compounds involves the induction of VDAC1 overexpression and oligomerization and, thereby, apoptosis. Gas chromatography of the hydroethanolic plant extract identified dozens of compounds, including phytol and ethyl linoleate, with the former producing similar effects as the Vern...
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SummaryModern health care needs preventive vaccines and therapeutic treatments with stability against pathogen mutations to cope with current and future viral infections. At the beginning of the COVID-19 pandemic, our analytic and... more
SummaryModern health care needs preventive vaccines and therapeutic treatments with stability against pathogen mutations to cope with current and future viral infections. At the beginning of the COVID-19 pandemic, our analytic and predictive tool identified a set of eight short SARS-CoV-2 S-spike protein epitopes that had the potential to persistently avoid mutation. Here a combination of genetic, Systems Biology and protein structure analyses confirm the stability of our identified epitopes against viral mutations. Remarkably, this research spans the whole period of the pandemic, during which 93.9% of the eight peptides remained invariable in the globally predominant 43 circulating variants, including Omicron. Likewise, the selected epitopes are conserved in 97% of all 1,514 known SARS-CoV-2 lineages. Finally, experimental analyses performed with these short peptides showed their specific immunoreactivity. This work opens a new perspective on the design of next-generation vaccines ...
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The mitochondrial voltage-dependent anion channel-1 (VDAC1) protein functions in a variety of mitochondria-linked physiological and pathological processes, including metabolism and cell signaling, as well as in mitochondria-mediated... more
The mitochondrial voltage-dependent anion channel-1 (VDAC1) protein functions in a variety of mitochondria-linked physiological and pathological processes, including metabolism and cell signaling, as well as in mitochondria-mediated apoptosis. VDAC1 interacts with about 150 proteins to regulate the integration of mitochondrial functions with other cellular activities. Recently, we developed VDAC1-based peptides that have multiple effects on cancer cells and tumors including apoptosis induction. Here, we designed several cell-penetrating VDAC1 N-terminal-derived peptides with the goal of identifying the shortest peptide with improved cellular stability and activity. We identified the D-Δ(1-18)N-Ter-Antp comprising the VDAC1 N-terminal region (19–26 amino acids) fused to the Antp, a cell-penetrating peptide. We demonstrated that this peptide induced apoptosis, autophagy, senescence, cell volume enlargement, and the refusion of divided daughter cells into a single cell, it was responsi...
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Found at the outer mitochondrial membrane, the voltage-dependent anion channel, VDAC, assumes a crucial position in the cell, serving as the main interface between mitochondrial and cellular metabolisms by mediating transport of ions and... more
Found at the outer mitochondrial membrane, the voltage-dependent anion channel, VDAC, assumes a crucial position in the cell, serving as the main interface between mitochondrial and cellular metabolisms by mediating transport of ions and metabolites. VDAC thus functions as a gatekeeper, controlling cross-talk between mitochondria and the rest of the cell. Moreover, its location at the boundary between the mitochondria and the cytosol enables VDAC to interact with proteins that mediate and regulate the integration of
mitochondrial functions with other cellular activities. Here, we review current knowledge related to the roles played by VDAC in the regulation of cell life and cell death, with relation to cancer. The current concepts of altered metabolism in cancer cells are presented with specific emphasis on mitochondrial, more specifically VDAC1-bound hexokinase (HK), facilitating and promoting the high glycolytic tumor phenotype. In this respect, the up-regulation of HK expression in tumor cells and its binding to VDAC provide both a metabolic benefit and apoptosis-suppressive capacity that offers the cell a growth advantage and increases its resistance to chemotherapy. VDAC
has also been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and antiapoptotic Bcl-2-family of proteins, as well as due to its function in the release of apoptotic proteins located in the inter-membranal space. These and other functions point to VDAC1 as being a rational target for the development of a new generation of therapeutics.
mitochondrial functions with other cellular activities. Here, we review current knowledge related to the roles played by VDAC in the regulation of cell life and cell death, with relation to cancer. The current concepts of altered metabolism in cancer cells are presented with specific emphasis on mitochondrial, more specifically VDAC1-bound hexokinase (HK), facilitating and promoting the high glycolytic tumor phenotype. In this respect, the up-regulation of HK expression in tumor cells and its binding to VDAC provide both a metabolic benefit and apoptosis-suppressive capacity that offers the cell a growth advantage and increases its resistance to chemotherapy. VDAC
has also been recognized as a key protein in mitochondria-mediated apoptosis since it is the proposed target for the pro- and antiapoptotic Bcl-2-family of proteins, as well as due to its function in the release of apoptotic proteins located in the inter-membranal space. These and other functions point to VDAC1 as being a rational target for the development of a new generation of therapeutics.