The Soluble Guanylate Cyclase Stimulator BAY 41-2272 Attenuates Transforming Growth Factor β1-Induced Myofibroblast Differentiation of Human Corneal Keratocytes

Corneal transparency, necessary for vision and depending on the high organization of stromal extracellular matrix, is maintained by keratocytes. Severe or continuous corneal injuries determine exaggerated healing responses resulting in the formation of irreversible fibrotic scars and vision impairment. Soluble guanylate cyclase (sGC) stimulation demonstrated antifibrotic effects in both experimental fibrosis and human lung and skin fibroblasts. Here, we assessed whether sGC stimulation with BAY 41-2272 could attenuate transforming growth factor β1 (TGFβ1)-induced myofibroblast differentiation of human corneal keratocytes. Cells were challenged with TGFβ1, with/without BAY 41-2272 preincubation, and subsequently assessed for viability, proliferation, migration, chemoinvasion, as well for the expression of myofibroblast/fibroblast activation markers and contractile abilities. Treatment with BAY 41-2272 did not affect keratocyte viability, while preincubation of cells with the sGC stimulator was able to inhibit TGFβ1-induced proliferation, wound healing capacity, and invasiveness. BAY 41-2272 was also able to attenuate TGFβ1-induced myofibroblast-like profibrotic phenotype of keratocytes, as demonstrated by the significant decrease in ACTA2, COL1A1, COL1A2, FN1 and PDPN gene expression, as well as in α-smooth muscle actin, α-1 chain of type I collagen, podoplanin, vimentin and N-cadherin protein expression. Finally, BAY 41-2272 significantly counteracted the TGFβ1-induced myofibroblast-like ability of keratocytes to contract collagen gels, reduced phosphorylated Smad3 protein levels, and attenuated gene expression of proinflammatory cytokines. Collectively, our data show for the first time that BAY 41-2272 is effective in counteracting keratocyte-to-myofibroblast transition, thus providing the rationale for the development of sGC stimulators as novel promising modulators of corneal scarring and fibrosis.     

The Transition of Photoreceptor Guanylate Cyclase Type 1 to the Active State

Membrane-bound guanylate cyclases (GCs), which synthesize the second messenger guanosine-3′, 5′-cyclic monophosphate, differ in their activation modes to reach the active state. Hormone peptides bind to the extracellular domain in hormone-receptor-type GCs and trigger a conformational change in the intracellular, cytoplasmic part of the enzyme. Sensory GCs that are present in rod and cone photoreceptor cells have intracellular binding sites for regulatory Ca²⁺-sensor proteins, named guanylate-cyclase-activating proteins. A rotation model of activation involving an α-helix rotation was described as a common activation motif among hormone-receptor GCs. We tested whether the photoreceptor GC-E underwent an α-helix rotation when reaching the active state. We experimentally simulated such a transitory switch by integrating alanine residues close to the transmembrane region, and compared the effects of alanine integration with the point mutation V902L in GC-E. The V902L mutation is found in patients suffering from retinal cone–rod dystrophies, and leads to a constitutively active state of GC-E. We analyzed the enzymatic catalytic parameters of wild-type and mutant GC-E. Our data showed no involvement of an α-helix rotation when reaching the active state, indicating a difference in hormone receptor GCs. To characterize the protein conformations that represent the transition to the active state, we investigated the protein dynamics by using a computational approach based on all-atom molecular dynamics simulations. We detected a swinging movement of the dimerization domain in the V902L mutant as the critical conformational switch in the cyclase going from the low to high activity state.     

Impairment of Anti-Aggregatory Responses to Nitric Oxide and Prostacyclin: Mechanisms and Clinical Implications in Cardiovascular Disease

The propensity towards platelet-rich thrombus formation increases substantially during normal ageing, and this trend is mediated by decreases in platelet responsiveness to the anti-aggregatory nitric oxide (NO) and prostacyclin (PGI₂) pathways. The impairment of soluble guanylate cyclase and adenylate cyclase-based signalling that is associated with oxidative stress represents the major mechanism of this loss of anti-aggregatory reactivity. Platelet desensitization to these autacoids represents an adverse prognostic marker in patients with ischemic heart disease and may contribute to increased thrombo-embolic risk in patients with heart failure. Patients with platelet resistance to PGI₂ also are unresponsive to ADP receptor antagonist therapy. Apart from ischemia, diabetes and aortic valve disease are also associated with impaired anti-aggregatory homeostasis. This review examines the association of impaired platelet cyclic nucleotide (i.e., cGMP and cAMP) signalling with the emerging evidence of thromboembolic risk in cardiovascular diseases, and discusses the potential therapeutic strategies targeting this abnormality.     

Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins

Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid–protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid–protein interactions in the mechanism of membrane proteins.     

Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg²⁺ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe²⁺ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.     

Alphafold Predictions Provide Insights into the Structural Features of the Functional Oligomers of All Members of the KCTD Family

Oligomerization endows proteins with some key properties such as extra-stabilization, long-range allosteric regulation(s), and partnerships not accessible to their monomeric counterparts. How oligomerization is achieved and preserved during evolution is a subject of remarkable scientific relevance. By exploiting the abilities of the machine-learning algorithms implemented in AlphaFold (AF) in predicting protein structures, herein, we report a comprehensive analysis of the structural states of functional oligomers of all members of the KCTD protein family. Interestingly, our approach led to the identification of reliable three-dimensional models for the pentameric states of KCNRG, KCTD6, KCTD4, KCTD7, KCTD9, and KCTD14 and possibly for KCTD11 and KCTD21 that are involved in key biological processes and that were previously uncharacterized from a structural point of view. Although for most of these proteins, the CTD domains lack any sequence similarity, they share some important structural features, such as a propeller-like structure with a central cavity delimited by five exposed and regular β-strands. Moreover, the structure of the related proteins KCTD7 and KCTD14, although pentameric, appears to be characterized by a different organization of the CTD region, with the five chains forming a circle-like structure with a large cavity. Our predictions also suggest that other members of the family, such as KCTD10, KCTD13, and TNFAIP1, present a strong propensity to assume dimeric states. Although the structures of the functional oligomers reported herein represent models that require additional validations, they provide a consistent and global view of KCTD protein oligomerization.     

Inhibition of Soluble Epoxide Hydrolase Ameliorates Phenotype and Cognitive Abilities in a Murine Model of Niemann Pick Type C Disease

Niemann–Pick type C (NPC) disease is a rare autosomal recessive inherited childhood neurodegenerative disease characterized by the accumulation of cholesterol and glycosphingolipids, involving the autophagy-lysosome system. Inhibition of soluble epoxide hydrolase (sEH), an enzyme that metabolizes epoxy fatty acids (EpFAs) to 12-diols, exerts beneficial effects in modulating inflammation and autophagy, critical features of the NPC disease. This study aims to evaluate the effects of UB-EV-52, an sEH inhibitor (sEHi), in an NPC mouse model (Npc) by administering it for 4 weeks (5 mg/kg/day). Behavioral and cognitive tests (open-field test (OF)), elevated plus maze (EPM), novel object recognition test (NORT) and object location test (OLT) demonstrated that the treatment produced an improvement in short- and long-term memory as well as in spatial memory. Furthermore, UB-EV-52 treatment increased body weight and lifespan by 25% and reduced gene expression of the inflammatory markers (i.e., Il-1β and Mcp1) and enhanced oxidative stress (OS) markers (iNOS and Hmox1) in the treated Npc mice group. As for autophagic markers, surprisingly, we found significantly reduced levels of LC3B-II/LC3B-I ratio and significantly reduced brain protein levels of lysosomal-associated membrane protein-1 (LAMP-1) in treated Npc mice group compared to untreated ones in hippocampal tissue. Lipid profile analysis showed a significant reduction of lipid storage in the liver and some slight changes in homogenated brain tissue in the treated NPC mice compared to the untreated groups. Therefore, our results suggest that pharmacological inhibition of sEH ameliorates most of the characteristic features of NPC mice, demonstrating that sEH can be considered a potential therapeutic target for this disease.     

Effect of Soluble Adenylyl Cyclase (ADCY10) Inhibitors on the LH-Stimulated cAMP Synthesis in Mltc-1 Leydig Cell Line

In contrast to all transmembrane adenylyl cyclases except ADCY9, the cytosolic soluble adenylyl cyclase (ADCY10) is insensitive to forskolin stimulation and is uniquely modulated by calcium and bicarbonate ions. In the present paper, we focus on ADCY10 localization and a kinetic analysis of intracellular cAMP accumulation in response to human LH in the absence or presence of four different ADCY10 inhibitors (KH7, LRE1, 2-CE and 4-CE) in MTLC-1 cells. ADCY10 was immuno-detected in the cytoplasm of MLTC-1 cells and all four inhibitors were found to inhibit LH-stimulated cAMP accumulation and progesterone level in MLTC-1 and testosterone level primary Leydig cells. Interestingly, similar inhibitions were also evidenced in mouse testicular Leydig cells. In contrast, the tmAC-specific inhibitors ddAdo3′ and ddAdo5′, even at high concentration, exerted weak or no inhibition on cAMP accumulation, suggesting an important role of ADCY10 relative to tmACs in the MLTC-1 response to LH. The strong synergistic effect of HCO₃⁻ under LH stimulation further supports the involvement of ADCY10 in the response to LH.     

A Comparative Study to Decipher the Structural and Dynamics Determinants Underlying the Activity and Thermal Stability of GH-11 Xylanases

With the growing need for renewable sources of energy, the interest for enzymes capable of biomass degradation has been increasing. In this paper, we consider two different xylanases from the GH-11 family: the particularly active GH-11 xylanase from Neocallimastix patriciarum, NpXyn11A, and the hyper-thermostable mutant of the environmentally isolated GH-11 xylanase, EvXyn11^TS. Our aim is to identify the molecular determinants underlying the enhanced capacities of these two enzymes to ultimately graft the abilities of one on the other. Molecular dynamics simulations of the respective free-enzymes and enzyme–xylohexaose complexes were carried out at temperatures of 300, 340, and 500 K. An in-depth analysis of these MD simulations showed how differences in dynamics influence the activity and stability of these two enzymes and allowed us to study and understand in greater depth the molecular and structural basis of these two systems. In light of the results presented in this paper, the thumb region and the larger substrate binding cleft of NpXyn11A seem to play a major role on the activity of this enzyme. Its lower thermal stability may instead be caused by the higher flexibility of certain regions located further from the active site. Regions such as the N-ter, the loops located in the fingers region, the palm loop, and the helix loop seem to be less stable than in the hyper-thermostable EvXyn11^TS. By identifying molecular regions that are critical for the stability of these enzymes, this study allowed us to identify promising targets for engineering GH-11 xylanases. Eventually, we identify NpXyn11A as the ideal host for grafting the thermostabilizing traits of EvXyn11^TS.     

Advances in the Structural Strategies of the Self-Assembly of Photoresponsive Supramolecular Systems

Photosensitive supramolecular systems have garnered attention due to their potential to catalyze highly specific tasks through structural changes triggered by a light stimulus. The tunability of their chemical structure and charge transfer properties provides opportunities for designing and developing smart materials for multidisciplinary applications. This review focuses on the approaches reported in the literature for tailoring properties of the photosensitive supramolecular systems, including MOFs, MOPs, and HOFs. We discuss relevant aspects regarding their chemical structure, action mechanisms, design principles, applications, and future perspectives.     

Structural Perspective of Gliadin Peptides Active in Celiac Disease

Gluten fragments released in gut of celiac individuals activate the innate or adaptive immune systems. The molecular mechanisms associated with the adaptive response involve a series of immunodominant gluten peptides which are mainly recognized by human leucocyte antigen (HLA)-DQ2.5 and HLA-DQ8. Other peptides, such as A-gliadin P31–43, are not recognized by HLA and trigger innate responses by several routes not yet well detailed. Among the gluten fragments known to be active in Celiac disease, here we focus on the properties of all gluten peptides with known tri-dimensional structure either those locked into HLA-DQ complexes whose crystals were X-ray analyzed or characterized in solution as free forms. The aim of this work was to find the structural reasons why some gluten peptides prompt the adaptive immune systems while others do not, by apparently involving just the innate immune routes. We propose that P31–43 is a non-adaptive prompter because it is not a good ligand for HLA-DQ. Even sharing a similar ability to adopt polyproline II structure with the adaptive ones, the way in which the proline residues are located along the sequence disfavors a productive P31–43-HLA-DQ binding.     

Small-Molecule Therapeutic Perspectives for the Treatment of Progeria

Hutchinson–Gilford progeria syndrome (HGPS), or progeria, is an extremely rare disorder that belongs to the class of laminopathies, diseases characterized by alterations in the genes that encode for the lamin proteins or for their associated interacting proteins. In particular, progeria is caused by a point mutation in the gene that codifies for the lamin A gene. This mutation ultimately leads to the biosynthesis of a mutated version of lamin A called progerin, which accumulates abnormally in the nuclear lamina. This accumulation elicits several alterations at the nuclear, cellular, and tissue levels that are phenotypically reflected in a systemic disorder with important alterations, mainly in the cardiovascular system, bones, skin, and overall growth, which results in premature death at an average age of 14.5 years. In 2020, lonafarnib became the first (and only) FDA approved drug for treating progeria. In this context, the present review focuses on the different therapeutic strategies currently under development, with special attention to the new small molecules described in recent years, which may represent the upcoming first-in-class drugs with new mechanisms of action endowed with effectiveness not only to treat but also to cure progeria.     

Molecular Mechanisms of Inhibition of Protein Amyloid Fibril Formation: Evidence and Perspectives Based on Kinetic Models

Inhibition of fibril formation is considered a possible treatment strategy for amyloid-related diseases. Understanding the molecular nature of inhibitor action is crucial for the design of drug candidates. In the present review, we describe the common kinetic models of fibril formation and classify known inhibitors by the mechanism of their interactions with the aggregating protein and its oligomers. This mechanism determines the step or steps of the aggregation process that become inhibited and the observed changes in kinetics and equilibrium of fibril formation. The results of numerous studies indicate that possible approaches to antiamyloid inhibitor discovery include the search for the strong binders of protein monomers, cappers blocking the ends of the growing fibril, or the species absorbing on the surface of oligomers preventing nucleation. Strongly binding inhibitors stabilizing the native state can be promising for the structured proteins while designing the drug candidates targeting disordered proteins is challenging.     

Inflammation in the Human Periodontium Induces Downregulation of the α1- and β1-Subunits of the sGC in Cementoclasts

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α₁- and β₁-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α₁- and β₁-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α₁- and β₁-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.     

Dynamical Mechanism Analysis of Three Neuroregulatory Strategies on the Modulation of Seizures

This paper attempts to explore and compare the regulatory mechanisms of optogenetic stimulation (OS), deep brain stimulation (DBS) and electromagnetic induction on epilepsy. Based on the Wilson–Cowan model, we first demonstrate that the external input received by excitatory and inhibitory neural populations can induce rich dynamic bifurcation behaviors such as Hopf bifurcation, and make the system exhibit epileptic and normal states. Then, both OS and DBS are shown to be effective in controlling the epileptic state to a normal low-level state, and the stimulus parameters have a broad effective range. However, electromagnetic induction cannot directly control epilepsy to this desired state, even if it can significantly reduce the oscillation frequency of neural populations. One main difference worth noting is that the high spatiotemporal specificity of OS allows it to target inhibitory neuronal populations, whereas DBS and electromagnetic induction can only stimulate excitatory as well as inhibitory neuronal populations together. Next, the propagation behavior of epilepsy is explored under a typical three-node feedback loop structure. An increase in coupling strength accelerates and exacerbates epileptic activity in other brain regions. Finally, OS and DBS applied to the epileptic focus play similar positive roles in controlling the behavior of the area of seizure propagation, while electromagnetic induction still only achieves unsatisfactory effects. It is hoped that these dynamical results can provide insights into the treatment of epilepsy as well as other neurological disorders.     

Fancy-Shaped Gold–Platinum Nanocauliflowers for Improved Proton Irradiation Effect on Colon Cancer Cells

Enhancing the effectiveness of colorectal cancer treatment is highly desirable. Radiation-based anticancer therapy—such as proton therapy (PT)—can be used to shrink tumors before subsequent surgical intervention; therefore, improving the effectiveness of this treatment is crucial. The addition of noble metal nanoparticles (NPs), acting as radiosensitizers, increases the PT therapeutic effect. Thus, in this paper, the effect of novel, gold–platinum nanocauliflowers (AuPt NCs) on PT efficiency is determined. For this purpose, crystalline, 66-nm fancy shaped, bimetallic AuPt NCs were synthesized using green chemistry method. Then, physicochemical characterization of the obtained AuPt NCs by transmission electron microscopy (TEM), selected area electron diffraction (SAED), energy dispersive X-ray spectroscopy (EDS), and UV-Vis spectra measurements was carried out. Fully characterized AuPt NCs were placed into a cell culture of colon cancer cell lines (HCT116, SW480, and SW620) and a normal colon cell line (FHC) and subsequently subjected to proton irradiation with a total dose of 15 Gy. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test, performed after 18-h incubation of the irradiated cell culture with AuPt NCs, showed a significant reduction in cancer cell viability compared to normal cells. Thus, the radio-enhancing features of AuPt NCs indicate their potential application for the improvement in effectiveness of anticancer proton therapy.     

Effect of Ni Substitution on the Structural,Magnetic, and Electronic Structure Properties of Gd0.4Tb0.6(Co1−xNix)2 Compounds

The comprehensive research of magnetic and electronic structure properties of the new class of Gd_0.4Tb_0.6(Co_1−xNi_x)₂ compounds, crystallizing in the cubic Laves phase (C15), is reported. The magnetic study was completed with electrical resistivity and electronic structure investigations. The analysis of Arrott plots supplemented by a study of temperature dependency of Landau coefficients revealed that all compounds undergo a magnetic phase transition of the second type. Based on magnetic isotherms, magnetic entropy change (ΔS_M) was determined for many values of the magnetic field change (μ₀H), which varied from 0.1 to 7 T. For each compound, the ΔS_M had a maximum around the Curie temperature. Both values of the |ΔS_M^max| and relative cooling power RCP parameters increased with increasing nickel content. It is shown that structural disorder upon Co/Ni substitution influences some magnetic parameters. The magnetic moment values of Co atoms determined from different methods are quantitatively consistent. From the M(T) dependency, the exchange integrals J_RR, J_RT, and J_TT between rare-earths (R) and transition metal (T) moments were evaluated within the mean-field theory (MFT) approach. The experimental study of the electronic structure performed with the use of the X-ray photoelectron spectroscopy (XPS) was completed by calculations using the full-potential linearized augmented plane waves (FP-LAPW) method based on the density functional theory (DFT). The calculations explained experimentally observed changes in the XPS valence band spectra upon the Ni/Co substitution.     

The Influences of Sulphation,Salt Type,and Salt Concentration on the Structural Heterogeneity of Glycosaminoglycans

The increasing recognition of the biochemical importance of glycosaminoglycans (GAGs) has in recent times made them the center of attention of recent research investigations. It became evident that subtle conformational factors play an important role in determining the relationship between the chemical composition of GAGs and their activity. Therefore, a thorough understanding of their structural flexibility is needed, which is addressed in this work by means of all-atom molecular dynamics (MD) simulations. Four major GAGs with different substitution patterns, namely hyaluronic acid as unsulphated GAG, heparan-6-sulphate, chondroitin-4-sulphate, and chondroitin-6-sulphate, were investigated to elucidate the influence of sulphation on the dynamical features of GAGs. Moreover, the effects of increasing NaCl and KCl concentrations were studied as well. Different structural parameters were determined from the MD simulations, in combination with a presentation of the free energy landscape of the GAG conformations, which allowed us to unravel the conformational fingerprints unique to each GAG. The largest effects on the GAG structures were found for sulphation at position 6, as well as binding of the metal ions in the absence of chloride ions to the carboxylate and sulphate groups, which both increase the GAG conformational flexibility.     

Elucidating the Interaction between Pyridoxine 5′-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5′-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.