This Ph.D. research project focused on the study of channels composed of connexin 26 (Cx26) protein subunits. In particular, we analyzed conductance, permeability and/or structural differences of deafness-related mutations of Cx26 intercellular gap junction channels or unpaired hemichannels, using different molecular dynamics approaches and statistical mechanics.
The first part of this thesis introduces the structure of gap junction channels and the connexin proteins that form them. Next, it dwells on the function of gap junction channels and connexin hemichannels, particularly on their conductance and permeability, two fundamental properties explored in the rest of the work. This introductory section contains also a brief overview of electrophysiology experiments performed on connexin channels. It ends with a section on gating properties that modify conductance state and a discussion of disease-related connexin mutations. The second part presents the key results obtained during this Ph.D. work, beginning with the full atom model on which the work was based, and a discussion of the improvements achieved during this project. The rest of the results are subdivided into three topics: (a) structural analysis, (b) conductance and (c) permeability.
a) This section summarizes the results obtained for the mutant Cx26 C169Y, the analysis of which shows a large displacement of the extracellular loops and, consequently, of residues critically involved in hemichannel docking.
b) This section introduces two methods to compute the ionic conductance of a connexin hemichannel and applies them to three different models: the mutant Cx26 M34T, the WT Cx26 hemichannel in a Coarse-Grained representation and the full-atom WT hemichannel model bound to an antibody.
c) This section starts with the explanation of the method used to compute the potential of mean force (PMF) for the passage of a molecule through the channel pore and a possible interpretation of this potential. The PMF was then used to quantify permeation of different molecules (IP3, ATP) through Cx26 WT and V84L mutant hemichannels. The chapter ends with a structural analysis which accounts for the differences in the permeability of the two models.
This thesis also contains an overall discussion of the results highlighted above and a methodological appendix.
Abstract The co‐assembly of charged nanoparticles with oppositely charged molecular ions has emerged as a promising technique in the fabrication of nanoparticle superstructures. However, the underlying mechanism behind these molecular ions in mediating the repulsion between these charged nanoparticles remains elusive. Herein, coarse‐grained molecular dynamics simulations are used to elucidate the effects of valency, shape, and size of molecular anions on their co‐assembly with gold nanoparticles coated with positively charged ligands. The findings suggest that the valency, shape, and size of molecular anions significantly influence the repulsion and aggregating dynamics among these positively charged nanoparticles. Moreover, the free energy calculations reveal that ring‐shaped molecular anions with higher valences and larger sizes are more effective at reducing the repulsion between these gold nanoparticles and thus enhance the stability of the aggregate. This study contributes to a better understanding of the critical roles of valence, shape, and size of ions in mediating the electrostatic co‐assembly of nanoparticles with oppositely charged ions, and it also guides the future design of DNA templates and DNA origami in co‐assembly with oppositely charged nanoparticles.
Connexin hemichannels have been implicated in pathology-promoting conditions, including inflammation, numerous widespread human diseases, including cancer and diabetes, and several rare diseases linked to pathological point mutations.We analysed the literature focusing on antibodies capable of modulating hemichannel function, highlighting generation methods, applications to basic biomedical research and translational potential.Anti-hemichannel antibodies generated over the past 3 decades targeted mostly connexin 43, with a focus on cancer treatment. A slow transition from relatively unselective polyclonal antibodies to more selective monoclonal antibodies resulted in few products with interesting characteristics that are under evaluation for clinical trials. Selection of antibodies from combinatorial phage-display libraries, has permitted to engineer a monoclonal antibody that binds to and blocks pathological hemichannels formed by connexin 26, 30 and 32.All known antibodies that modulate connexin hemichannels target the two small extracellular loops of the connexin proteins. The extracellular region of different connexins is highly conserved, and few residues of each connexins are exposed. The search for new antibodies may develop an unprecedented potential for therapeutic applications, as it may benefit tremendously from novel whole-cell screening platforms that permit in situ selection of antibodies against membrane proteins in native state. The demonstrated efficacy of mAbs in reaching and modulating hemichannels in vivo, together with their relative specificity for connexins overlapping epitopes, should hopefully stimulate an interest for widening the scope of anti-hemichannel antibodies. There is no shortage of currently incurable diseases for which therapeutic intervention may benefit from anti-hemichannel antibodies capable of modulating hemichannel function selectively and specifically.
Abstract Cyclin-dependent kinase 7 (CDK7) plays a crucial role in cell cycle regulation and transcription, establishing it as a promising target for cancer therapy. Although the covalent inhibitor THZ1 effectively targets CDK7, it presents risks such as short half-life and potential off-target side effects. To address these limitations, we employed a computational workflow integrating virtual screening, molecular dynamics (MD) simulations, and free energy perturbation (FEP) method to design non-covalent CDK7 inhibitors with enhanced selectivity and safety profiles. MD simulations elucidated THZ1’s inhibitory mechanism and identified key molecular fragments within its structure. By incorporating fragments from known inhibitors, we introduced extensive non-covalent interactions within the binding pocket, leading to the identification of three novel non-covalent inhibitors with binding affinities comparable to or higher than that of THZ1. Our findings not only introduce promising CDK7 inhibitors but also present a robust computational framework that could accelerate the discovery of kinase-targeted therapeutics.
Abstract DNA encoded chemical libraries (DELs) link the powers of genetics and chemical synthesis via combinatorial optimization. Through combinatorial chemistry, DELs can grow to the unprecedented size of billions to trillions. To take full advantage of the DEL approach, linking the power of genetics directly to chemical structures would offer even greater diversity in a finite chemical world. Natural products have evolved an incredible structural diversity along with their biological evolution. Herein, we used traditional Chinese medicines (TCMs) as examples in a late‐stage modification toolbox approach to annotate these complex organic compounds with amplifiable DNA barcodes, which could be easily incorporated into a DEL. The method of end‐products labeling also generates a cluster of isomers with a single DNA tag at different sites. These isomers provide an additional spatial diversity for multiple accessible pockets of targeted proteins. Notably, a novel PARP1 inhibitor from TCM has been identified from the natural products enriched DEL ( n DEL).
Nuclear shape modulates cell behavior and function, while aberrant nuclear morphologies correlate with pathological phenotype severity. Nevertheless, functions of specific nuclear morphological features and underlying molecular mechanisms remain poorly understood. Here, we investigate a nucleus-intrinsic mechanism driving nuclear lobulation and segmentation concurrent with granulocyte specification, independently from extracellular forces and cytosolic cytoskeleton contributions. Transcriptomic regulation of cholesterol biosynthesis is equally concurrent with nuclear remodeling. Its putative role as a regulatory element is supported by morphological aberrations observed upon pharmacological impairment of several enzymatic steps of the pathway, most prominently the sterol ∆14-reductase activity of laminB-receptor and protein prenylation. Thus, we support the hypothesis of a nuclear-intrinsic mechanism for nuclear shape control with the putative involvement of the recently discovered GGTase III complex. Such process could be independent from or complementary to the better studied cytoskeleton-based nuclear remodeling essential for cell migration in both physiological and pathological contexts such as immune system function and cancer metastasis.
Introduction: The ζ subunit is a potent inhibitor of the F 1 F O -ATPase of Paracoccus denitrificans (PdF 1 F O -ATPase) and related α -proteobacteria different from the other two canonical inhibitors of bacterial ( ε ) and mitochondrial (IF 1 ) F 1 F O -ATPases. ζ mimics mitochondrial IF 1 in its inhibitory N-terminus, blocking the PdF 1 F O -ATPase activity as a unidirectional pawl-ratchet and allowing the PdF 1 F O -ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans , as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans , here, we assessed the evolution of ζ across the α -proteobacteria class. Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α -proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria. Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm- ζ ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm- ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF 1 F O -ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε , the data confirm that the primary inhibitory function of the α-proteobacterial F 1 F O -ATPase was transferred from ε to ζ and that ζ, ε, and IF 1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α -proteobacteria, are also discussed.
Abstract Connexins (Cxs) are fundamental in cell–cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca 2+ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability (“leaky”) or excessive activity (“hyperactive”). We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca 2+ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca 2+ uptake in response to elevated extracellular Ca 2+ concentrations ([Ca 2+ ] ex ) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca 2+ uptake, achieving a 50% inhibitory concentration (EC 50 ) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations. In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody’s applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs.
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