Membrane insertion of soluble CLIC1 into active chloride channels is triggered by specific divalent cations

The CLIC family of proteins display the unique feature of altering their structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, can be found in the cytoplasm or in nuclear, ER and plasma membranes, with membrane overexpression linked to tumour proliferation. The molecular switch promoting CLIC1 membrane insertion has been related to environmental factors, but still remains unclear. Here, we use solution NMR studies to confirm that both the soluble and membrane bound forms are in the same oxidation state. Our data from fluorescence assays and chloride efflux assays indicate that Ca2+ and Zn2+ trigger association to the membrane into active chloride channels. We use fluorescence microscopy to confirm that an increase of the intracellular Ca2+ leads to re-localisation of CLIC1 to both plasma and internal membranes. Finally, we show that soluble CLIC1 adopts an equilibrium of oligomeric species, and Ca2+/Zn2+ mediated membrane insertion promotes the formation of a tetrameric assembly. Thus, our results identify Ca2+ and Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion.SIGNIFICANCE STATEMENT CLIC1, a member of the CLIC family of proteins, is expressed as a soluble protein in cells but can insert in the membrane forming a chloride channel. This chloride channel form is upregulated in different types of cancers including glioblastoma and promote tumour invasiveness and metastasis. The factors promoting CLIC1 membrane insertion nor the mechanism of this process are yet understood. Here, we use a combination of solution NMR, biophysics and fluorescence microscopy to identify Ca2+ and Zn2+ binding as the switch to promote CLIC1 insertion into the membrane to form active chloride channels. We also provide a simple mechanism how such transition to the membrane occurs. Such understanding will enable subsequent studies on the structure of the chloride channel form and its inhibition

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic β loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies

A quantitative assay to study the lipid selectivity of membrane-associated systems using solution NMR

The activity of membrane proteins and compounds that interact with the membrane is modulated by the surrounding lipid composition. However, there are no simple methods that determine the composition of these annular phospholipids in eukaryotic systems. Herein, we describe a simple methodology that enables the identification and quantification of the lipid composition around membrane-associated compounds using SMA-nanodiscs and routine $^1H-^{31}P NMR$

An Updated View of the Trypanosoma cruzi Life Cycle: Intervention Points for an Effective Treatment

Chagas disease (CD) is a parasitic, systemic, chronic, and often fatal illness caused by infection with the protozoan Trypanosoma cruzi. The World Health Organization classifies CD as the most prevalent of poverty-promoting neglected tropical diseases, the most important parasitic one, and the third most infectious disease in Latin America. Currently, CD is a global public health issue that affects 6–8 million people. However, the current approved treatments are limited to two nitroheterocyclic drugs developed more than 50 years ago. Many efforts have been made in recent decades to find new therapies, but our limited understanding of the infection process, pathology development, and long-term nature of this disease has made it impossible to develop new drugs, effective treatment, or vaccines. This Review aims to provide a comprehensive update on our understanding of the current life cycle, new morphological forms, and genetic diversity of T. cruzi, as well as identify intervention points in the life cycle where new drugs and treatments could achieve a parasitic cure

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic β loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on E. coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted