Relationship between ferroptosis and mitophagy in renal fibrosis: a systematic review.

Renal fibrosis, characterised by glomerulosclerosis and tubulointerstitial fibrosis, is a typical pathological alteration in the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD). However, the limited and expensive options for treating renal fibrosis place a heavy financial burden on patients and healthcare systems. Therefore, it is significant to find an effective treatment for renal fibrosis. Ferroptosis, a non-traditional form of cell death, has been found to play an important role in acute kidney injury (AKI), tumours, neurodegenerative diseases, and so on. Moreover, a growing body of research suggests that ferroptosis might be a potential target of renal fibrosis. Meanwhile, mitophagy is a type of selective autophagy that can selectively degrade damaged or dysfunctional mitochondria as a form of mitochondrial quality control, reducing the production of reactive oxygen species (ROS), the accumulation of which is the main cause of renal fibrosis. Additionally, as a receptor of mitophagy, NIX can release beclin1 to induce mitophagy, which can also bind to solute carrier family 7 member 11 (SLC7A11) to block the activity of cystine/glutamate antitransporter (system Xc-) and inhibit ferroptosis, thereby suggesting a link between mitophagy and ferroptosis. However, there have been only limited studies on the relationship among mitophagy, ferroptosis and renal fibrosis. In this paper, we review the mechanisms of mitophagy, and describe how ferroptosis and mitophagy are related to renal fibrosis in an effort to identify potential novel targets for the treatment of renal fibrosis.

Mechanism of voltage gating in the voltage-sensing phosphatase Ci-VSP.

Conformational changes in voltage-sensing domains (VSDs) are driven by the transmembrane electric field acting on the protein charges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes as well as the major conformations visited during a complete functional gating cycle in the isolated VSD of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1 to S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated "Up" and resting "Down" states, the simulations predict two Ci-VSD conformations: a deeper resting state ("down-minus") and an extended activated ("up-plus") state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. The present results show that these four states are visited sequentially in a stepwise manner during voltage activation, each step translocating one arginine or the equivalent of ∼1 e0 across the membrane electric field, yielding a transfer of ∼3 e0 charges in total for the complete process.

MIUH Inhibits the Hippocampal Neuron Growth in Fetal Rat by Affecting the PTEN Pathway.

Mild intrauterine hypoperfusion (MIUH) can induce placental dysfunction and lead to long-term changes during the process of brain development. A better understanding of the mechanism of MIUH will help in the development of new neuroprotective strategies for the placental chamber. To better understand the mechanism of the effect of MIUH on the neural development of offspring, we constructed a model of MIUH in pregnant rats. The proliferation, apoptosis, and autophagy of hippocampal neurons in fetal rats were studied via flow cytometry, immunofluorescence staining, JC-1 staining, western blotting, and real-time polymerase chain reaction at different time points (6, 24, 48, and 72 h). The results showed that MIUH significantly inhibited the proliferation of hippocampal neurons and promoted their apoptosis and autophagy. Simultaneously, MIUH could promote PTEN expression and affect the PTEN signaling pathway. bpV, an inhibitor of PTEN, could restore the inhibition of hippocampal nerve cell growth caused by MIUH. MIUH may inhibit neuronal proliferation and promote neuronal apoptosis and autophagy by regulating the PTEN signaling pathway.

Immune cell infiltration landscape and immune marker molecular typing in preeclampsia.

Preeclampsia (PE) is an important topic in obstetrics. In this study, we used weighted gene co-expression network analysis (WGCNA) to screen the key modules related to immune cell infiltration and to identify the hub genes for the molecular subtyping of PE. We first downloaded a set of PE transcriptional data (GSE75010; 157 samples: 80 PE and 77 non-PE) from the GEO database. We then analyzed the PE samples and non-PE samples for immune cell infiltration and screened cells with differences in such infiltration. Next, we downloaded the immune-related genes from an immune-related database to screen the expression profile of the immune-related genes. Then, we obtained a candidate gene set by screening the immune-related genes differentially expressed between the two groups. We used WGCNA to construct a weighted co-expression network for these candidate genes, mined co-expression modules, and then calculated the correlation between each module and immune cells with differential infiltration. We screened the modules related to infiltrating immune cells, identified the key modules' hub genes, and determined the key module genes that interacted with each other. Finally, we obtained the hub genes related to the infiltrating immune cells. We classified the preeclampsia patients by unsupervised cluster molecular typing, determined the difference of immune cell infiltration among the different PE subtypes, and calculated the expression of hub genes in these different subtypes. In conclusion, we found 41 hub genes that may be closely related to the molecular typing of PE.

[Diagnosis of a fetus with atelosteogenesis type 2 through combined prenatal ultrasonography and whole exome sequencing].

OBJECTIVE: To explore the genetic basis for fetus with short limbs detected by prenatal ultrasonography. METHODS: Results of clinical imaging of the fetus was collected. Amniotic fluid sample was collected through amniocentesis for the extraction of fetal DNA. Whole exome sequencing was carried out to detect variants related to the clinical phenotypes. Candidate variant was verified by Sanger sequencing. RESULTS: Prenatal ultrasound showed that the fetus had short limbs but no other abnormality. Whole exome sequencing has identified that the fetus carried two heterozygous pathogenic variants c.484G>T and c.1436dupA of the SLC26A2 gene, for which its mother and father were heterozygous carriers, respectively. CONCLUSION: The fetus was diagnosed with atelosteogenesis type 2 by combined prenatal ultrasonography and whole exome sequencing, which may be attributed to the compound heterozygous variants of the SLC26A2 gene. Above findings provided evidence for the diagnosis of the fetus and genetic counseling.

Predicting the Conformational Variability of Abl Tyrosine Kinase using Molecular Dynamics Simulations and Markov State Models.

Understanding protein conformational variability remains a challenge in drug discovery. The issue arises in protein kinases, whose multiple conformational states can affect the binding of small-molecule inhibitors. To overcome this challenge, we propose a comprehensive computational framework based on Markov state models (MSMs). Our framework integrates the information from explicit-solvent molecular dynamics simulations to accurately rank-order the accessible conformational variants of a target protein. We tested the methodology using Abl kinase with a reference and blind-test set. Only half of the Abl conformational variants discovered by our approach are present in the disclosed X-ray structures. The approach successfully identified a protein conformational state not previously observed in public structures but evident in a retrospective analysis of Lilly in-house structures: the X-ray structure of Abl with WHI-P154. Using a MSM-derived model, the free energy landscape and kinetic profile of Abl was analyzed in detail highlighting opportunities for targeting the unique metastable states.

A Catalytically Disabled Double Mutant of Src Tyrosine Kinase Can Be Stabilized into an Active-Like Conformation.

Tyrosine kinases are enzymes playing a critical role in cellular signaling. Molecular dynamics umbrella sampling potential of mean force computations are used to quantify the impact of activating and inactivating mutations of c-Src kinase. The potential of mean force computations predict that a specific double mutant can stabilize c-Src kinase into an active-like conformation while disabling the binding of ATP in the catalytic active site. The active-like conformational equilibrium of this catalytically dead kinase is affected by a hydrophobic unit that connects to the hydrophobic spine network via the C-helix. The αC-helix plays a crucial role in integrating the hydrophobic residues, making it a hub for allosteric regulation of kinase activity and the active conformation. The computational free-energy landscapes reported here illustrate novel design principles focusing on the important role of the hydrophobic spines. The relative stability of the spines could be exploited in future efforts to artificially engineer active-like but catalytically dead forms of protein kinases.

Tyrosine Kinase Activation and Conformational Flexibility: Lessons from Src-Family Tyrosine Kinases.

Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.

The Activation of c-Src Tyrosine Kinase: Conformational Transition Pathway and Free Energy Landscape.

Tyrosine kinases are important cellular signaling allosteric enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Their activity must be tightly controlled, and malfunction can lead to a variety of diseases, particularly cancer. The nonreceptor tyrosine kinase c-Src, a prototypical model system and a representative member of the Src-family, functions as complex multidomain allosteric molecular switches comprising SH2 and SH3 domains modulating the activity of the catalytic domain. The broad picture of self-inhibition of c-Src via the SH2 and SH3 regulatory domains is well characterized from a structural point of view, but a detailed molecular mechanism understanding is nonetheless still lacking. Here, we use advanced computational methods based on all-atom molecular dynamics simulations with explicit solvent to advance our understanding of kinase activation. To elucidate the mechanism of regulation and self-inhibition, we have computed the pathway and the free energy landscapes for the "inactive-to-active" conformational transition of c-Src for different configurations of the SH2 and SH3 domains. Using the isolated c-Src catalytic domain as a baseline for comparison, it is observed that the SH2 and SH3 domains, depending upon their bound orientation, promote either the inactive or active state of the catalytic domain. The regulatory structural information from the SH2-SH3 tandem is allosterically transmitted via the N-terminal linker of the catalytic domain. Analysis of the conformational transition pathways also illustrates the importance of the conserved tryptophan 260 in activating c-Src, and reveals a series of concerted events during the activation process.

Computational study of the W260A activating mutant of Src tyrosine kinase.

Tyrosine kinases are enzymes playing a critical role in cellular signaling. Mutations causing increased in kinase activity are often associated with cancer and various pathologies. One example in Src tyrosine kinases is offered by the substitution of the highly conserved tryptophan 260 by an alanine (W260A), which has been shown to cause an increase in activity. Here, molecular dynamics simulations based on atomic models are carried out to characterize the conformational changes in the linker region and the catalytic (kinase) domain of Src kinase to elucidate the impact of the W260A mutation. Umbrella sampling calculations show that the conformation of the linker observed in the assembled down-regulated state of the kinase is most favored when the kinase domain is in the inactive state, whereas the conformation of the linker observed in the re-assembled up-regulated state of the kinase is favored when the kinase domain is in the unphosphorylated active-like state. The calculations further indicate that there are only small differences between the WT and W260A mutant. In both cases, the intermediates states are very similar and the down-regulated inactive conformation is the most stable state. However, the calculations also show that the free energy cost to reach the unphosphorylated active-like conformation is slightly smaller for the W260A mutant compared with WT. A simple kinetic model is developed and submitted to a Bayesian Monte Carlo analysis to illustrate how such small differences can contribute to accelerate the trans-autophosphorylation reaction and yield a large increase in the activity of the mutant as observed experimentally.

Computational study of the "DFG-flip" conformational transition in c-Abl and c-Src tyrosine kinases.

Protein tyrosine kinases are crucial to cellular signaling pathways regulating cell growth, proliferation, metabolism, differentiation, and migration. To maintain normal regulation of cellular signal transductions, the activities of tyrosine kinases are also highly regulated. The conformation of a three-residue motif Asp-Phe-Gly (DFG) near the N-terminus of the long "activation" loop covering the catalytic site is known to have a critical impact on the activity of c-Abl and c-Src tyrosine kinases. A conformational transition of the DFG motif can switch the enzyme from an active (DFG-in) to an inactive (DFG-out) state. In the present study, the string method with swarms-of-trajectories was used to computationally determine the reaction pathway connecting the two end-states, and umbrella sampling calculations were carried out to characterize the thermodynamic factors affecting the conformations of the DFG motif in c-Abl and c-Src kinases. According to the calculated free energy landscapes, the DFG-out conformation is clearly more favorable in the case of c-Abl than that of c-Src. The calculations also show that the protonation state of the aspartate residue in the DFG motif strongly affects the in/out conformational transition in c-Abl, although it has a much smaller impact in the case of c-Src due to local structural differences.

Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity.

Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a "conformational selection" mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to the DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.

Activation pathway of Src kinase reveals intermediate states as targets for drug design.

Unregulated activation of Src kinases leads to aberrant signalling, uncontrolled growth and differentiation of cancerous cells. Reaching a complete mechanistic understanding of large-scale conformational transformations underlying the activation of kinases could greatly help in the development of therapeutic drugs for the treatment of these pathologies. In principle, the nature of conformational transition could be modelled in silico via atomistic molecular dynamics simulations, although this is very challenging because of the long activation timescales. Here we employ a computational paradigm that couples transition pathway techniques and Markov state model-based massively distributed simulations for mapping the conformational landscape of c-src tyrosine kinase. The computations provide the thermodynamics and kinetics of kinase activation for the first time, and help identify key structural intermediates. Furthermore, the presence of a novel allosteric site in an intermediate state of c-src that could be potentially used for drug design is predicted.

Explaining why Gleevec is a specific and potent inhibitor of Abl kinase.

Tyrosine kinases present attractive drug targets for specific types of cancers. Gleevec, a well-known therapeutic agent against chronic myelogenous leukemia, is an effective inhibitor of Abl tyrosine kinase. However, Gleevec fails to inhibit closely homologous tyrosine kinases, such as c-Src. Because many structural features of the binding site are conserved, the molecular determinants responsible for binding specificity are not immediately apparent. Some have attributed the difference in binding specificity of Gleevec to subtle variations in ligand-protein interactions (binding affinity control), whereas others have proposed that it is the conformation of the DFG motif, in which ligand binding is only accessible to Abl and not to c-Src (conformational selection control). To address this issue, the absolute binding free energy was computed using all-atom molecular dynamics simulations with explicit solvent. The results of the free energy simulations are in good agreement with experiments, thereby enabling a meaningful decomposition of the binding free energy to elucidate the factors controlling Gleevec's binding specificity. The latter is shown to be controlled by a conformational selection mechanism and also by differences in key van der Waals interactions responsible for the stabilization of Gleevec in the binding pocket of Abl.

Exchange frequency in replica exchange molecular dynamics.

The effect of the exchange-attempt frequency on sampling efficiency is studied in replica exchange molecular dynamics (REMD). We show that sampling efficiency increases with increasing exchange-attempt frequency. This conclusion is contrary to a commonly expressed view in REMD. Five peptides (1-21 residues long) are studied with a spectrum of exchange-attempt rates. Convergence rates are gauged by comparing ensemble properties between fixed length test REMD simulations and longer reference simulations. To show the fundamental correlation between exchange frequency and convergence time, a simple model is designed and studied, displaying the same basic behavior of much more complex systems.