RosettaDDGPrediction for high‐throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high‐throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease‐related variants that can benefit from analyses with structure‐based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high‐throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high‐throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication‐ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction.     

ArtiaX: An electron tomography toolbox for the interactive handling of sub‐tomograms in UCSF ChimeraX

Cryo‐electron tomography analysis involves the selection of macromolecular complexes to be used for subsequent sub‐tomogram averaging and structure determination. Here, we describe a plugin developed for UCSF ChimeraX that allows for the display, selection, and editing of particles within tomograms. Positions and orientations of selected particles can be manually set, modified and inspected in real time, both on screen and in virtual reality, and exported to various file formats. The plugin allows for the parallel visualization of particles stored in several meta data lists, in the context of any three‐dimensional image that can be opened with UCSF ChimeraX. The particles are rendered in user‐defined colors or using colormaps, such that individual classes or groups of particles, cross‐correlation coefficients, or other types of information can be highlighted to the user. The implemented functions are fast, reliable, and intuitive, exploring the broad range of features in UCSF ChimeraX. They allow for a fluent human–machine interaction, which enables an effective understanding of the sub‐tomogram processing pipeline, even for non‐specialist users.     

CellRegMap: a statistical framework for mapping context‐specific regulatory variants using scRNA‐seq

Single‐cell RNA sequencing (scRNA‐seq) enables characterizing the cellular heterogeneity in human tissues. Recent technological advances have enabled the first population‐scale scRNA‐seq studies in hundreds of individuals, allowing to assay genetic effects with single‐cell resolution. However, existing strategies to analyze these data remain based on principles established for the genetic analysis of bulk RNA‐seq. In particular, current methods depend on a priori definitions of discrete cell types, and hence cannot assess allelic effects across subtle cell types and cell states. To address this, we propose the Cell Regulatory Map (CellRegMap), a statistical framework to test for and quantify genetic effects on gene expression in individual cells. CellRegMap provides a principled approach to identify and characterize genotype–context interactions of known eQTL variants using scRNA‐seq data. This model‐based approach resolves allelic effects across cellular contexts of different granularity, including genetic effects specific to cell subtypes and continuous cell transitions. We validate CellRegMap using simulated data and apply it to previously identified eQTL from two recent studies of differentiating iPSCs, where we uncover hundreds of eQTL displaying heterogeneity of genetic effects across cellular contexts. Finally, we identify fine‐grained genetic regulation in neuronal subtypes for eQTL that are colocalized with human disease variants.     

7‐Epitaxol induces apoptosis in cisplatin‐resistant head and neck squamous cell carcinoma via suppression of AKT and MAPK signalling

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although cisplatin‐based chemotherapy is commonly used in HNSCC, frequent development of cisplatin resistance is a potential cause of poor HNSCC prognosis. In the present study, we investigated the anticancer efficacy of a major paclitaxel metabolite namely 7‐Epitaxol in cisplatin‐resistant HNSCC. The findings revealed that 7‐Epitaxol exerts cytotoxic effects in cisplatin‐resistant HNSCC cell lines by inducing cell cycle arrest and intrinsic and extrinsic apoptotic pathways. Specifically, 7‐Epitaxol increased Fas, TNF‐R1, DR5, DcR3 and DcR2 expressions, reduced Bcl‐2 and Bcl‐XL (anti‐apoptotic proteins) expressions, and increased Bid and Bim L/S (pre‐apoptotic proteins) expressions, leading to activation of caspase‐mediated cancer cell apoptosis. At the upstream cell signalling level, 7‐Epitaxol reduced the phosphorylation of AKT, ERK1/2 and p38 to trigger apoptosis. In vivo results showed that animals treated with 7‐Epitaxol show antitumor growth compared to control animals. Taken together, the study demonstrates the potential anticancer efficacy of 7‐Epitaxol in inducing apoptosis of cisplatin‐resistant HNSCC cells through the suppression of AKT and MAPK signalling pathways.     

Effects of Sirolimus treatment on patients with β‐Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4 + and CD8 + T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β‐Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β‐Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the {"type":"clinical-trial","attrs":{"text":"NCT03877809","term_id":"NCT03877809"}}NCT03877809 clinical trial. The approach was to verify IFN‐γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β‐Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN‐γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β‐Thalassemia patients are considered prone to immune deficiency.     

Shape shifting: The multiple conformational substates of the PTEN N‐terminal PIP2 ‐binding domain

The Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a chief regulator of a variety of cellular processes including cell proliferation, migration, growth, and death. It is also a major tumor suppressor gene that is frequently mutated or lost under cancerous conditions. PTEN encodes a dual‐specificity (lipid and protein) phosphatase that negatively regulates the PI3K/AKT/mTOR signaling pathway where the PIP₂‐binding domain (PBD) regulates the lipid phosphatase function. Unfortunately, despite two decades of research, a full‐length structure of PTEN remains elusive, leaving open questions regarding PTEN''s disordered regions that mediate protein stability, post‐translational modifications, protein–protein interactions, while also hindering the design of small molecules that can regulate PTEN''s function. Here, we utilized a combination of crosslinking mass spectrometry, in silico predicted structural modeling (including AlphaFold2), molecular docking, molecular dynamics simulations, and residue interaction network modeling to obtain structural details and molecular insight into the behavior of the PBD of PTEN. Our study shows that the PBD exists in multiple conformations which suggests its ability to regulate PTEN''s variety of functions. Studying how these specific conformational substates contribute to PTEN function is imperative to defining its function in disease pathogenesis, and to delineate ways to modulate its tumor suppressor activity.     

ER‐phagy: selective autophagy of the endoplasmic reticulum

Eukaryotic cells adequately control the mass and functions of organelles in various situations. Autophagy, an intracellular degradation system, largely contributes to this organelle control by degrading the excess or defective portions of organelles. The endoplasmic reticulum (ER) is an organelle with distinct structural domains associated with specific functions. The ER dynamically changes its mass, components, and shape in response to metabolic, developmental, or proteotoxic cues to maintain or regulate its functions. Therefore, elaborate mechanisms are required for proper degradation of the ER. Here, we review our current knowledge on diverse mechanisms underlying selective autophagy of the ER, which enable efficient degradation of specific ER subdomains according to different demands of cells.     

Three‐dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6‐bladed β‐propeller hydrolase family

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d‐xylonolactone. We have determined crystal structures of Cc XylC in complex with d‐xylonolactone isomer analogues d‐xylopyranose and (r)‐(+)‐4‐hydroxy‐2‐pyrrolidinone at high resolution. Cc XylC has a 6‐bladed β‐propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe²⁺. The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe²⁺. The structure of Cc XylC was compared with eight other ester hydrolases of the β‐propeller hydrolase family. The previously published crystal structures of other β‐propeller hydrolases contain either Ca²⁺, Mg²⁺, or Zn²⁺ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe²⁺ as a catalytic metal. This could further expand our understanding of utilization of Fe²⁺ not only in oxidative enzymes but also in hydrolases.     

TSGIT: An N‐ and C‐terminal tandem tag system for purification of native and intein‐mediated ligation‐ready proteins

A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.     

Role of vasorin,an anti‐apoptotic,anti‐TGF‐β and hypoxia‐induced glycoprotein in the trabecular meshwork cells and glaucoma

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open‐angle glaucoma patients compared to non‐glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti‐apoptotic and anti‐TGF‐β activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF‐β2‐induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride‐induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF‐α‐induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF‐β induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.     

Plasmodium chitinases: revisiting a target of transmission‐blockade against malaria

Malaria is a life‐threatening disease caused by one of the five species of Plasmodium, among which Plasmodium falciparum cause the deadliest form of the disease. Plasmodium species are dependent on a vertebrate host and a blood‐sucking insect vector to complete their life cycle. Plasmodium chitinases belonging to the GH18 family are secreted inside the mosquito midgut, during the ookinete stage of the parasite. Chitinases mediate the penetration of parasite through the peritrophic membrane, facilitating access to the gut epithelial layer. In this review, we describe Plasmodium chitinases with special emphasis on chitinases from P. falciparum and P. vivax, the representative examples of the short and long forms of this protein. In addition to the chitinase domain, chitinases belonging to the long form contain a pro‐domain and chitin‐binding domain. Amino acid sequence alignment of long and short form chitinase domains reveals multiple positions containing variant residues. A subset of these positions was found to be conserved or invariant within long or short forms, indicating the role of these positions in attributing form‐specific activity. The reported differences in affinities to allosamidin for P. vivax and P. falciparum were predicted to be due to different residues at two amino acid positions, resulting in altered interactions with the inhibitor. Understanding the role of these amino acids in Plasmodium chitinases will help us elucidate the mechanism of catalysis and the mode of inhibition, which will be the key for identification of potent inhibitors or antibodies demonstrating transmission‐blocking activity.     

“Cancer Reviews 2021” series: Dynamic EMT: a multi‐tool for tumor progression

The process of epithelial–mesenchymal transition (EMT) is fundamental for embryonic morphogenesis. Cells undergoing it lose epithelial characteristics and integrity, acquire mesenchymal features, and become motile. In cancer, this program is hijacked to confer essential changes in morphology and motility that fuel invasion. In addition, EMT is increasingly understood to orchestrate a large variety of complementary cancer features, such as tumor cell stemness, tumorigenicity, resistance to therapy and adaptation to changes in the microenvironment. In this review, we summarize recent findings related to these various classical and non‐classical functions, and introduce EMT as a true tumorigenic multi‐tool, involved in many aspects of cancer. We suggest that therapeutic targeting of the EMT process will—if acknowledging these complexities—be a possibility to concurrently interfere with tumor progression on many levels.     

Syn Wiki: Functional annotation of the first artificial organism Mycoplasma mycoides JCVI‐syn3A

The new field of synthetic biology aims at the creation of artificially designed organisms. A major breakthrough in the field was the generation of the artificial synthetic organism Mycoplasma mycoides JCVI‐syn3A. This bacterium possesses only 452 protein‐coding genes, the smallest number for any organism that is viable independent of a host cell. However, about one third of the proteins have no known function indicating major gaps in our understanding of simple living cells. To facilitate the investigation of the components of this minimal bacterium, we have generated the database SynWiki (http://synwiki.uni-goettingen.de/). SynWiki is based on a relational database and gives access to published information about the genes and proteins of M. mycoides JCVI‐syn3A. To gain a better understanding of the functions of the genes and proteins of the artificial bacteria, protein–protein interactions that may provide clues for the protein functions are included in an interactive manner. SynWiki is an important tool for the synthetic biology community that will support the comprehensive understanding of a minimal cell as well as the functional annotation of so far uncharacterized proteins.     

Cask methylation involved in the injury of insulin secretion function caused by interleukin1‐β

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.     

Integration of ‘omics’ data in aging research: from biomarkers to systems biology

Age is the strongest risk factor for many diseases including neurodegenerative disorders, coronary heart disease, type 2 diabetes and cancer. Due to increasing life expectancy and low birth rates, the incidence of age‐related diseases is increasing in industrialized countries. Therefore, understanding the relationship between diseases and aging and facilitating healthy aging are major goals in medical research. In the last decades, the dimension of biological data has drastically increased with high‐throughput technologies now measuring thousands of (epi) genetic, expression and metabolic variables. The most common and so far successful approach to the analysis of these data is the so‐called reductionist approach. It consists of separately testing each variable for association with the phenotype of interest such as age or age‐related disease. However, a large portion of the observed phenotypic variance remains unexplained and a comprehensive understanding of most complex phenotypes is lacking. Systems biology aims to integrate data from different experiments to gain an understanding of the system as a whole rather than focusing on individual factors. It thus allows deeper insights into the mechanisms of complex traits, which are caused by the joint influence of several, interacting changes in the biological system. In this review, we look at the current progress of applying omics technologies to identify biomarkers of aging. We then survey existing systems biology approaches that allow for an integration of different types of data and highlight the need for further developments in this area to improve epidemiologic investigations.     

Exosomal miR‐532‐5p induced by long‐term exercise rescues blood–brain barrier function in 5XFAD mice via downregulation of EPHA4

The breakdown of the blood–brain barrier, which develops early in Alzheimer''s disease (AD), contributes to cognitive impairment. Exercise not only reduces the risk factors for AD but also confers direct protection against cognitive decline. However, the exact molecular mechanisms remain elusive, particularly whether exercise can liberate the function of the blood–brain barrier. Here, we demonstrate that long‐term exercise promotes the clearance of brain amyloid‐β by improving the function of the blood–brain barrier in 5XFAD mice. Significantly, treating primary brain pericytes or endothelial cells with exosomes isolated from the brain of exercised 5XFAD mice improves cell proliferation and upregulates PDGFRβ, ZO‐1, and claudin‐5. Moreover, exosomes isolated from exercised mice exhibit significant changes in miR‐532‐5p. Administration or transfection of miR‐532‐5p to sedentary mice or primary brain pericytes and endothelial cells reproduces the improvement of blood–brain barrier function. Exosomal miR‐532‐5p targets EPHA4, and accordingly, expression of EphA4 is decreased in exercised mice and miR‐532‐5p overexpressed mice. A specific siRNA targeting EPHA4 recapitulates the effects on blood–brain barrier‐associated cells observed in exercised 5XFAD mice. Overall, our findings suggest that exosomes released by the brain contain a specific miRNA that is altered by exercise and has an impact on blood–brain barrier function in AD.