AlloReverse: multiscale understanding among hierarchical allosteric regulations.

Increasing data in allostery are requiring analysis of coupling relationships among different allosteric sites on a single protein. Here, based on our previous efforts on reversed allosteric communication theory, we have developed AlloReverse, a web server for multiscale analysis of multiple allosteric regulations. AlloReverse integrates protein dynamics and machine learning to discover allosteric residues, allosteric sites and regulation pathways. Especially, AlloReverse could reveal hierarchical relationships between different pathways and couplings among allosteric sites, offering a whole map of allostery. The web server shows a good performance in re-emerging known allostery. Moreover, we applied AlloReverse to explore global allostery on CDC42 and SIRT3. AlloReverse predicted novel allosteric sites and allosteric residues in both systems, and the functionality of sites was validated experimentally. It also suggests a possible scheme for combined therapy or bivalent drugs on SIRT3. Taken together, AlloReverse is a novel workflow providing a complete regulation map and is believed to aid target identification, drug design and understanding of biological mechanisms. AlloReverse is freely available to all users at https://mdl.shsmu.edu.cn/AlloReverse/ or http://www.allostery.net/AlloReverse/.

A unique human cord blood CD8+CD45RA+CD27+CD161+ T-cell subset identified by flow cytometric data analysis using Seurat.

Advances in single-cell level analytical techniques, especially cytometric approaches, have led to profound innovation in biomedical research, particularly in the field of clinical immunology. This has resulted in an expansion of high-dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis is thus becoming untenable to handle these challenges. Furthermore, most newly developed computational methods lack flexibility and interoperability, hampering their accessibility and usability. Here, we adapted Seurat, an R package originally developed for single-cell RNA sequencing (scRNA-seq) analysis, for high-dimensional flow cytometric data analysis. Based on a 20-marker antibody panel and analyses of T-cell profiles in both adult blood and cord blood (CB), we showcased the robust capacity of Seurat in flow cytometric data analysis, which was further validated by Spectre, another high-dimensional cytometric data analysis package, and conventional manual analysis. Importantly, we identified a unique CD8+ T-cell population defined as CD8+CD45RA+CD27+CD161+ T cell that was predominantly present in CB. We characterised its IFN-γ-producing and potential cytotoxic properties using flow cytometry experiments and scRNA-seq analysis from a published dataset. Collectively, we identified a unique human CB CD8+CD45RA+CD27+CD161+ T-cell subset and demonstrated that Seurat, a widely used package for scRNA-seq analysis, possesses great potential to be repurposed for cytometric data analysis. This facilitates an unbiased and thorough interpretation of complicated high-dimensional data using a single analytical pipeline and opens a novel avenue for data-driven investigation in clinical immunology.

Diet, commensal microbiota-derived extracellular vesicles, and host immunity.

The gut microbiota has co-evolved with its host, and commensal bacteria can influence both the host's immune development and function. Recently, a role has emerged for bacterial extracellular vesicles (BEVs) as potent immune modulators. BEVs are nanosized membrane vesicles produced by all bacteria, possessing the membrane characteristics of the originating bacterium and carrying an internal cargo that may include nucleic acid, proteins, lipids, and metabolites. Thus, BEVs possess multiple avenues for regulating immune processes, and have been implicated in allergic, autoimmune, and metabolic diseases. BEVs are biodistributed locally in the gut, and also systemically, and thus have the potential to affect both the local and systemic immune responses. The production of gut microbiota-derived BEVs is regulated by host factors such as diet and antibiotic usage. Specifically, all aspects of nutrition, including macronutrients (protein, carbohydrates, and fat), micronutrients (vitamins and minerals), and food additives (the antimicrobial sodium benzoate), can regulate BEV production. This review summarizes current knowledge of the powerful links between nutrition, antibiotics, gut microbiota-derived BEV, and their effects on immunity and disease development. It highlights the potential of targeting or utilizing gut microbiota-derived BEV as a therapeutic intervention.

GPR109A expressed on medullary thymic epithelial cells affects thymic Treg development.

Regulatory T cells (Treg) maintain immune homeostasis due to their anti-inflammatory functions. They can be generated either centrally in the thymus or in peripheral organs. Metabolites such as short-chain fatty acids produced by intestinal microbiota can induce peripheral Treg differentiation, by activating G-protein-coupled-receptors like GPR109A. In this study, we identified a novel role for GPR109A in thymic Treg development. We found that Gpr109a-/- mice had increased Treg under basal conditions in multiple organs compared with WT mice. GPR109A was not expressed on T cells but on medullary thymic epithelial cells (mTECs), as revealed by single-cell RNA sequencing in both mice and humans and confirmed by flow cytometry in mice. mTECs isolated from Gpr109a-/- mice had higher expression of autoimmune regulator (AIRE), the key regulator of Treg development, while the subset of mTECs that did not express Gpr109a in the WT displayed increased Aire expression and also enhanced signaling related to mTEC functionality. Increased thymic Treg in Gpr109a-/- mice was associated with protection from experimental autoimmune encephalomyelitis, with ameliorated clinical signs and reduced inflammation. This work identifies a novel role for GPR109A and possibly the gut microbiota, on thymic Treg development via its regulation of mTECs.

Breastfeeding is associated with enhanced intestinal gluconeogenesis in infants.

BACKGROUND: Breastfeeding (BF) confers metabolic benefits to infants, including reducing risks of metabolic syndrome such as obesity and diabetes later in life. However, the underlying mechanism is not yet fully understood. Hence, we aim to investigate the impacts of BF on the metabolic organs of infants. METHODS: Previous literatures directly studying the influences of BF on offspring's metabolic organs in both animal models and humans were comprehensively reviewed. A microarray dataset of intestinal gene expression comparing infants fed on breastmilk versus formula milk was analyzed. RESULTS: Reanalysis of microarray data showed that BF is associated with enhanced intestinal gluconeogenesis in infants. This resembles observations in other mammalian species showing that BF was also linked to increased gluconeogenesis. CONCLUSIONS: BF is associated with enhanced intestinal gluconeogenesis in infants, which may underpin its metabolic advantages through finetuning metabolic homeostasis. This observation seems to be conserved across species, hinting its biological significance.

Acetylation of calmodulin regulates synaptic plasticity and fear learning.

Synaptic plasticity is critical for brain function, including learning and memory. It is regulated by gene transcription and protein synthesis as well as posttranslational modifications at synapses. Although protein acetylation has been shown to be involved in the regulation of synaptic plasticity, this was mainly for histone protein acetylation. To investigate whether acetylation of nonhistone proteins is important for synaptic plasticity, we analyzed mouse brain acetylome and found that calmodulin (CaM), a ubiquitous Ca2+ sensor, was acetylated on three lysine residues, which were conserved across species. NMDA receptor-dependent long-term potentiation (LTP) is considered the most compelling form of synaptic plasticity. During LTP induction, activation of NMDA receptor triggers Ca2+ influx, and the Ca2+ binds with CaM and activates calcium/calmodulin-dependent protein kinase IIα (CaMKIIα), which is essential for LTP induction. By using home-generated and site-specific antibodies against acetylated CaM, we show that CaM acetylation is upregulated by neural activities in an NMDA receptor-dependent manner. Moreover, mutation of acetyllysines in CaM1 proteins disrupts synaptic plasticity and fear learning in a mouse model. We further demonstrate that acetylation of CaM reduces the binding free energy and increases the binding affinity toward CaMKIIα, a protein kinase pivotal to synaptic plasticity and learning. Taken together, our results demonstrate importance of CaM acetylation in regulating synaptic plasticity and learning.

Markov State Models and Molecular Dynamics Simulations Reveal the Conformational Transition of the Intrinsically Disordered Hypervariable Region of K-Ras4B to the Ordered Conformation.

K-Ras4B, the most frequently mutated Ras isoform in human tumors, plays a vital part in cell growth, differentiation, and survival. Its tail, the C-terminal hypervariable region (HVR), is involved in anchoring K-Ras4B at the cellular plasma membrane and in isoform-specific protein-protein interactions and signaling. In the inactive guanosine diphosphate-bound state, the intrinsically disordered HVR interacts with the catalytic domain at the effector-binding region, rendering K-Ras4B in its autoinhibited state. Activation releases the HVR from the catalytic domain, with its ensemble favoring an ordered α-helical structure. The large-scale conformational transition of the HVR from the intrinsically disordered to the ordered conformation remains poorly understood. Here, we deploy a computational scheme that integrates a transition path-generation algorithm, extensive molecular dynamics simulation, and Markov state model analysis to investigate the conformational landscape of the HVR transition pathway. Our findings reveal a stepwise pathway for the HVR transition and uncover several key conformational substates along the transition pathway. Importantly, key interactions between the HVR and the catalytic domain are unraveled, highlighting the pathogenesis of K-Ras4B mild mutations in several congenital developmental anomaly syndromes. Together, these findings provide a deeper understanding of the HVR transition mechanism and the regulation of K-Ras4B activity at an atomic level.

Unraveling allosteric landscapes of allosterome with ASD.

Allosteric regulation is one of the most direct and efficient ways to fine-tune protein function; it is induced by the binding of a ligand at an allosteric site that is topographically distinct from an orthosteric site. The Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) was developed ten years ago to provide comprehensive information related to allosteric regulation. In recent years, allosteric regulation has received great attention in biological research, bioengineering, and drug discovery, leading to the emergence of entire allosteric landscapes as allosteromes. To facilitate research from the perspective of the allosterome, in ASD 2019, novel features were curated as follows: (i) >10 000 potential allosteric sites of human proteins were deposited for allosteric drug discovery; (ii) 7 human allosterome maps, including protease and ion channel maps, were built to reveal allosteric evolution within families; (iii) 1312 somatic missense mutations at allosteric sites were collected from patient samples from 33 cancer types and (iv) 1493 pharmacophores extracted from allosteric sites were provided for modulator screening. Over the past ten years, the ASD has become a central resource for studying allosteric regulation and will play more important roles in both target identification and allosteric drug discovery in the future.

Cross-talk of four types of RNA modification writers defines tumor microenvironment and pharmacogenomic landscape in colorectal cancer.

BACKGROUND: The four major RNA adenosine modifications, i.e., m6A, m1A, alternative polyadenylation, and adenosine-to-inosine RNA editing, are mediated mostly by the "writer" enzymes and constitute critical mechanisms of epigenetic regulation in immune response and tumorigenesis. However, the cross-talk and potential roles of these "writers" in the tumor microenvironment (TME), drug sensitivity, and immunotherapy remain unknown. METHODS: We systematically characterized mRNA expression and genetic alterations of 26 RNA modification "writers" in colorectal cancer (CRC), and evaluated their expression pattern in 1697 CRC samples from 8 datasets. We used an unsupervised clustering method to assign the samples into two patterns of expression of RNA modification "writers". Subsequently, we constructed the RNA modification "writer" Score (WM_Score) model based on differentially expressed genes (DEGs) responsible for the RNA modification patterns to quantify the RNA modification-related subtypes of individual tumors. Furthermore, we performed association analysis for WM_Score and characteristics of TME, consensus molecular subtypes (CMSs), clinical features, transcriptional and post-transcriptional regulation, drug response, and the efficacy of immunotherapy. RESULTS: We demonstrated that multi-layer alterations of RNA modification "writer" are associated with patient survival and TME cell-infiltrating characteristics. We identified two distinct RNA modification patterns, characterized by a high and a low WM_Score. The WM_Score-high group was associated with worse patient overall survival and with the infiltration of inhibitory immune cells, such as M2 macrophages, EMT activation, and metastasis, while the WM_Score-low group was associated with a survival advantage, apoptosis, and cell cycle signaling pathways. WM_Score correlated highly with the regulation of transcription and post-transcriptional events contributing to the development of CRC. In response to anti-cancer drugs, WM_Score highly negatively correlated (drug sensitive) with drugs which targeted oncogenic related pathways, such as MAPK, EGFR, and mTOR signaling pathways, positively correlated (drug resistance) with drugs which targeted in apoptosis and cell cycle. Importantly, the WM_Score was associated with the therapeutic efficacy of PD-L1 blockade, suggesting that the development of potential drugs targeting these "writers" to aid the clinical benefits of immunotherapy. CONCLUSIONS: Our study is the first to provide a comprehensive analysis of four RNA modifications in CRC. We revealed the potential function of these writers in TME, transcriptional and post-transcriptional events, and identified their therapeutic liability in targeted therapy and immunotherapy. This work highlights the cross-talk and potential clinical utility of RNA modification "writers" in cancer therapy.

Mechanism of zinc ejection by disulfiram in nonstructural protein 5A.

Hepatitis C virus (HCV) is a notorious member of the Flaviviridae family of enveloped, positive-strand RNA viruses. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is a multi-domain protein which includes an N-terminal amphipathic membrane anchoring alpha helix, a highly structured domain-1, and two intrinsically disordered domains 2-3. The highly structured domain-1 contains a zinc finger (Zf)-site, and binding of zinc stabilizes the overall structure, while ejection of this zinc from the Zf-site destabilizes the overall structure. Therefore, NS5A is an attractive target for anti-HCV therapy by disulfiram, through ejection of zinc from the Zf-site. However, the zinc ejection mechanism is poorly understood. To disclose this mechanism based on three different states, A-state (NS5A protein), B-state (NS5A + Zn), and C-state (NS5A + Zn + disulfiram), we have performed molecular dynamics (MD) simulation in tandem with DFT calculations in the current study. The MD results indicate that disulfiram triggers Zn ejection from the Zf-site predominantly through altering the overall conformation ensemble. On the other hand, the DFT assessment demonstrates that the Zn adopts a tetrahedral configuration at the Zf-site with four Cys residues, which indicates a stable protein structure morphology. Disulfiram binding induces major conformational changes at the Zf-site, introduces new interactions of Cys39 with disulfiram, and further weakens the interaction of this residue with Zn, causing ejection of zinc from the Zf-site. The proposed mechanism elucidates the therapeutic potential of disulfiram and offers theoretical guidance for the advancement of drug candidates.

Markov State Models and Molecular Dynamics Simulations Provide Understanding of the Nucleotide-Dependent Dimerization-Based Activation of LRRK2 ROC Domain.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent cause of Parkinson's disease (PD). As a multidomain ROCO protein, LRRK2 is characterized by the presence of both a Ras-of-complex (ROC) GTPase domain and a kinase domain connected through the C-terminal of an ROC domain (COR). The bienzymatic ROC-COR-kinase catalytic triad indicated the potential role of GTPase domain in regulating kinase activity. However, as a functional GTPase, the detailed intrinsic regulation of the ROC activation cycle remains poorly understood. Here, combining extensive molecular dynamics simulations and Markov state models, we disclosed the dynamic structural rearrangement of ROC's homodimer during nucleotide turnover. Our study revealed the coupling between dimerization extent and nucleotide-binding state, indicating a nucleotide-dependent dimerization-based activation scheme adopted by ROC GTPase. Furthermore, inspired by the well-known R1441C/G/H PD-relevant mutations within the ROC domain, we illuminated the potential allosteric molecular mechanism for its pathogenetic effects through enabling faster interconversion between inactive and active states, thus trapping ROC in a prolonged activated state, while the implicated allostery could provide further guidance for identification of regulatory allosteric pockets on the ROC complex. Our investigations illuminated the thermodynamics and kinetics of ROC homodimer during nucleotide-dependent activation for the first time and provided guidance for further exploiting ROC as therapeutic targets for controlling LRRK2 functionality in PD treatment.

AlloFinder: a strategy for allosteric modulator discovery and allosterome analyses.

Allostery tweaks innumerable biological processes and plays a fundamental role in human disease and drug discovery. Exploration of allostery has thus been regarded as a crucial requirement for research on biological mechanisms and the development of novel therapeutics. Here, based on our previously developed allosteric data and methods, we present an interactive platform called AlloFinder that identifies potential endogenous or exogenous allosteric modulators and their involvement in human allosterome. AlloFinder automatically amalgamates allosteric site identification, allosteric screening and allosteric scoring evaluation of modulator-protein complexes to identify allosteric modulators, followed by allosterome mapping analyses of predicted allosteric sites and modulators in human proteome. This web server exhibits prominent performance in the reemergence of allosteric metabolites and exogenous allosteric modulators in known allosteric proteins. Specifically, AlloFinder enables identification of allosteric metabolites for metabolic enzymes and screening of potential allosteric compounds for disease-related targets. Significantly, the feasibility of AlloFinder to discover allosteric modulators was tested in a real case of signal transduction and activation of transcription 3 (STAT3) and validated by mutagenesis and functional experiments. Collectively, AlloFinder is expected to contribute to exploration of the mechanisms of allosteric regulation between metabolites and metabolic enzymes, and to accelerate allosteric drug discovery. The AlloFinder web server is freely available to all users at http://mdl.shsmu.edu.cn/ALF/.

Emerging roles of allosteric modulators in the regulation of protein-protein interactions (PPIs): A new paradigm for PPI drug discovery.

Protein-protein interactions (PPIs) are closely implicated in various types of cellular activities and are thus pivotal to health and disease states. Given their fundamental roles in a wide range of biological processes, the modulation of PPIs has enormous potential in drug discovery. However, owing to the general properties of large, flat, and featureless interfaces of PPIs, previous attempts have demonstrated that the generation of therapeutic agents targeting PPI interfaces is challenging, rendering them almost "undruggable" for decades. To date, rapid progress in chemical and structural biology techniques has promoted the exploitation of allostery as a novel approach in drug discovery. By attaching to allosteric sites that are topologically and spatially distinct from PPI interfaces, allosteric modulators can achieve improved physiochemical properties. Thus, allosteric modulators may represent an alternative strategy to target intractable PPIs and have attracted intense pharmaceutical interest. In this review, we first briefly introduce the characteristics of PPIs and then present different approaches for investigating PPIs, as well as the latest methods for modulating PPIs. Importantly, we comprehensively review the recent progress in the development of allosteric modulators to inhibit or stabilize PPIs. Finally, we conclude with future perspectives on the discovery of allosteric PPI modulators, especially the application of computational methods to aid in allosteric PPI drug discovery.

The chemical diversity and structure-based discovery of allosteric modulators for the PIF-pocket of protein kinase PDK1.

Phosphoinositide-dependent protein kinase-1 (PDK1) is an important protein in mediating the PI3K-AKT pathway and is thus identified as a promising target. The catalytic activity of PDK1 is tightly regulated by allosteric modulators, which bind to the PDK1 Interacting Fragment (PIF) pocket of the kinase domain that is topographically distinct from the orthosteric, ATP binding site. Allosteric modulators by attaching to the less conserved PIF-pocket have remarkable advantages such as higher selectivity, less side effect, and lower toxicity. Targeting allosteric PIF-pocket of PDK1 has become the focus of recent attention. In this review, we summarise the current advances in the structure-based discovery of PDK1 allosteric modulators. We will first present the three-dimensional structure of PDK1 and illustrate the allosteric regulatory mechanism of PDK1 through the modulation of the PIF-pocket. Then, the recent advances of PDK1 allosteric modulators targeting the PIF-pocket will be recapitulated detailly according to the structural similarity of allosteric modulators.

Allosteric Modulators of Protein-Protein Interactions (PPIs).

Protein-protein interactions (PPIs) represent promising drug targets of broad-spectrum therapeutic interests due to their critical implications in both health and disease circumstances. Hence, they are widely accepted as the Holy Grail of drug development. Historically, PPIs were rendered "undruggable" for their large, flat, and pocket-less structures. Current attempts to drug these "intractable" targets include orthosteric and allosteric methodologies. Previous efforts employing orthosteric approaches like protein therapeutics and orthosteric small molecules frequently suffered from poor performance caused by the difficulties in directly targeting PPI interfaces. As structural biology progresses rapidly, allosteric modulators, which direct to the allosteric regulatory sites remote to the PPI surfaces, have gradually established as a potential solution. Allosteric pockets are topologically distal from the PPI orthosteric sites, and their ligands do not need to compete with the PPI partners, which helps to improve the physiochemical and pharmacological properties of allosteric PPI modulators. Thus, exploiting allostery to tailor PPIs is regarded as a tempting strategy in future PPI drug discovery. Here, we provide a comprehensive review of our representative achievements along the way we utilize allosteric effects to tame the difficult PPI systems into druggable targets. Importantly, we provide an in-depth mechanistic analysis of this success, which will be instructive to future related lead optimizations and drug design. Finally, we discuss the current challenges in allosteric PPI drug discovery. Their solutions as well as future perspectives are also presented.

Characteristics of Allosteric Proteins, Sites, and Modulators.

Allostery is considered one of the most direct and efficient ways to regulate biological macromolecule functions. Allostery is increasingly receiving attention in the field of drug discovery because of the unique advantages of allosteric modulators such as high selectivity and low toxicity. Because of technical breakthroughs in the allosteric studies, the understanding of the characteristics of allosteric entities such as allosteric proteins and their allosteric sites and modulators has made great strides. These features play a critical role in both the evolution of the allosteric concept and the prediction of allosteric interactions. In this chapter, we highlight the fundamental characteristics of allosteric proteins, allosteric sites, and allosteric modulators. Importantly, the applications of such principles in real cases are depicted in detail. Collectively, these characteristics are beneficial in aiding allosteric drug design and allosteric mechanism research.

Computational Insights into the Interactions between Calmodulin and the c/nSH2 Domains of p85α Regulatory Subunit of PI3Kα: Implication for PI3Kα Activation by Calmodulin.

Calmodulin (CaM) and phosphatidylinositide-3 kinase (PI3Kα) are well known for their multiple roles in a series of intracellular signaling pathways and in the progression of several human cancers. Crosstalk between CaM and PI3Kα has been an area of intensive research. Recent experiments have shown that in adenocarcinoma, K-Ras4B is involved in the CaM-PI3Kα crosstalk. Based on experimental results, we have recently put forward a hypothesis that the coordination of CaM and PI3Kα with K-Ras4B forms a CaM-PI3Kα-K-Ras4B ternary complex, which leads to the formation of pancreatic ductal adenocarcinoma. However, the mechanism for the CaM-PI3Kα crosstalk is unresolved. Based on molecular modeling and molecular dynamics simulations, here we explored the potential interactions between CaM and the c/nSH2 domains of p85α subunit of PI3Kα. We demonstrated that CaM can interact with the c/nSH2 domains and the interaction details were unraveled. Moreover, the possible modes for the CaM-cSH2 and CaM-nSH2 interactions were uncovered and we used them to construct a complete CaM-PI3Kα complex model. The structural model of CaM-PI3Kα interaction not only offers a support for our previous ternary complex hypothesis, but also is useful for drug design targeted at CaM-PI3Kα protein-protein interactions.

Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation.

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras-NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras.