Biology subjects

Ortega-Roldan, J. L.

Publications and source records attributed to Ortega-Roldan, J. L..

6 recordsLinked to original sources

bioRxiv2025

Mechanistic Elucidation of CLIC1 Membrane Insertion via Structural and Dynamic Modulation

Chloride Intracellular Channel 1 (CLIC1) is a metamorphic protein capable of transitioning from a soluble cytoplasmic state to a membrane-bound chloride channel. This conformational shift, crucial for physiological processes such as cell volume regulation, electrical excitability, and angiogenesis, is linked to pathological conditions including malignancies and cardiovascular diseases. Despite its significance, the molecular mechanism driving CLIC1s membrane insertion has remained elusive. Using an integrated structural biology approach combining NMR spectroscopy, SAXS, biophysical methods, and mutagenesis, we uncover the dynamic landscape underpinning CLIC1 function. Solution NMR and SAXS reveal that CLIC1 adopts a conformational ensemble in equilibrium, characterized by a compact ground state and a partially extended state exposing key membrane-interacting regions. Zn2+ binding acts as a critical trigger, inducing structural rearrangements, increasing protein flexibility, and promoting oligomerization essential for membrane insertion. Our findings demonstrate that structural flexibility, particularly within dynamic loop regions and interdomain linkers, is intrinsic to CLIC1s ability to adapt to membrane interactions. Zn2+-induced dimerization and tetramerization were identified as key steps preceding insertion, with mutations in the transmembrane (TM) region revealing pivotal roles for residues R29 and W35 in modulating protein dynamics, oligomerization and insertion eXiciency. This study provides a mechanistic framework for CLIC1s transition to its membrane-bound state, oXering insights into the interplay between conformational dynamics, oligomerization, and metal ion modulation. These findings pave the way for targeted strategies to regulate CLIC1 activity in pathological conditions, underscoring its potential as a therapeutic target.

biochemistry↗

bioRxiv2022

Elucidation of small molecule passive permeation across lipid membranes using conventional solution state NMR methods

Quantifying small molecule uptake across a biological membrane in any cell system is crucial for the development of efficacious and selective drugs. However, obtaining such data is not trivial, especially in bacterial systems. Herein, we present an assay which enables the determination of the degree of passive permeation and membrane interaction of mixtures of small molecules in vesicles of a desired lipid composition, including that of bacterial membranes. The assay employs highly accessible conventional solution NMR experiments, exploiting the paramagnetic relaxation enhancement effect, and allows the measurement of membrane permeation on mixtures of any number of small molecules which do not exhibit heterogeneous molecular signal overlap in under 20 minutes. As a proof-of -principle we apply this methodology to candidates from a class of supramolecular self-associating amphiphiles, members from which have been shown to interact with biological phospholipid membranes and elicit an antimicrobial effect, allowing the determination and comparison of their membrane permeability and membrane interaction properties.

biophysics↗

bioRxiv2022

Cell survival enabled by leakage of a labile metabolic intermediate

Many metabolic pathways are of ancient origin and have evolved over long periods of time (Noda-Garcia et al., 2018). Yet, new pathways can also emerge in short time scales in response, for instance, to the presence of anthropogenic chemicals in the environment (Copley, 2009). Models of metabolic pathway emergence and evolution often emphasize the acquisition of new reactions through horizontal gene transfer and promiscuous enzyme functionalities (Pal et al., 2005; Schulenburg & Miller, 2014; Copley, 2015; Noda-Garcia et al., 2018; Peracchi, 2018). A fundamentally different mechanism of metabolic innovation is revealed by the evolutionary repair experiments reported here. A block in the proline biosynthetic pathway that compromises cell survival is efficiently rescued by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a sequestered phosphorylated intermediate common to the biosynthetic pathways of proline and glutamine, thus generating a new route to proline. Metabolic intermediates may undergo a variety of chemical and enzymatic transformations, but are typically protected as shielded reaction intermediates or through channeling in multi-enzyme complexes and metabolons (Srere, 1987; Huang et al., 2001; Grunwald, 2018; Pareek et al., 2021). Our results show that intermediate leakage can readily occur and contribute to organismal adaptation. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways. We therefore anticipate applications of mutation-induced leakage in metabolic engineering.

molecular biology↗

bioRxiv2022

Structure-based discovery and in vitro validation of selective inhibitors of Chloride Intracellular Channel 4 protein.

Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumour and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approach 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 of the drugs. Both drugs 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 {beta} 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.

molecular biology↗

bioRxiv2021

Zn2+ triggered two-step mechanism of CLIC1 membrane insertion and activation into chloride channels

The CLIC protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and the plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains unclear. Here, cellular chloride efflux assays and immunofluorescence microscopy studies have identified Zn2+ intracellular release as the trigger for CLIC1 activation and membrane relocalisation. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing chloride efflux in a pH dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH activation of chloride efflux.

biochemistry↗

bioRxiv2019

The 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.\n\nSIGNIFICANCE STATEMENTCLIC1, 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.

biochemistry↗