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MFPLI: A Computational Framework for Assessing Biological Authenticity of Protein-Ligand Interactions Using Molecular Fingerprints and Structural Features

Traditional computational drug discovery approaches struggle to accurately evaluate the biological authenticity of protein-ligand binding conformations due to inherent limitations in empirical scoring functions and force field approximations. This study proposes MFPLI - a deep learning framework integrating multimodal physicochemical features to systematically assess the biological authenticity alignment between molecular docking poses and true co-crystal structures. By establishing a continuous surface characterization system for protein-ligand interfaces, we concurrently incorporate geometric curvature features (radius, shape index) and chemical interaction fields (electrostatic potential, hydrogen-bond networks, hydrophobicity gradients). A contrastive learning architecture based on Siamese equivariant graph neural networks was developed to enable discriminative analysis between co-crystal conformations and parameter-perturbed pseudo-conformations generated through inverse docking. The five-channel fusion model demonstrates robust performance on the time-split PoseBuster validation set (AUC=0.91), with predicted Euclidean distance deviation ({Delta}E) effectively distinguishing native co-crystal conformations from aberrant docking poses in 80% of samples. Notably, 71% of {Delta}E-negative samples concentrate within the [-0.3, 0] interval, reflecting physical consistency between model predictions and conformational transition processes. This framework establishes a novel paradigm for biological authenticity assessment in virtual screening for computer-aided drug discovery through synergistic modeling of surface topology and interaction chemistry.

molecular biology↗

SimMapNet: A Bayesian Framework for Gene Regulatory Network Inference Using Gene Ontology Similarities as External Hint

MotivationGene regulatory network (GRN) reconstruction is a fundamental challenge in computational biology, and is crucial for understanding gene interactions. In this study, we aim to incorporate Gene Ontology (GO) similarities into the construction of GRNs. Our key assumption is that genes with higher similarity in Molecular Function, Biological Process, or Cellular Component categories are more likely to be functionally related and, therefore, more likely to be connected in the network. We introduce SimMapNet, a Bayesian framework that estimates the precision matrix, which serves as the adjacency matrix in a Gaussian graphical model (GGM) for GRN inference. SimMapNet enhances network inference by integrating GO similarities, which inform the hyperparameters of the prior distribution through a kernel function, incorporating biological prior knowledge in a principled manner. ResultsWe evaluate SimMapNet on three datasets: two datasets from the SOS DNA-repair response pathway in Escherichia coli and one dataset from Drosophila melanogaster. The results demonstrate the algorithms superior performance compared to state-of-the-art methods such as GLASSO, GENIE3, and KBOOST in terms of F1-score. SimMapNet has low time complexity, making it suitable for constructing large networks. Our simulation results confirm that SimMapNet is particularly well-suited for scenarios with limited sample sizes, where traditional methods often struggle. Availability and implementationThe datasets and R package of SimMapNet are available in the github repository, https://github.com/maryam-shahdoust/SimMapNet.

bioinformatics↗

Manumycin Polyketides Act as Molecular Glues Between UBR7 and P53 to Impair Breast Cancer Pathogenicity

Molecular glues are an intriguing therapeutic modality that harness small-molecules to induce interactions between proteins that typically do not interact, thus enabling the creation of novel protein functions not naturally encoded in biology. While molecular glues such as thalidomide and rapamycin have catalyzed drug discovery efforts, such molecules are rare and have often been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention of disease. Historically, natural products have proven to be important sources of molecular glues and we postulated that natural products bearing multiple electrophilic sites may be an unexplored source of such molecules, potentially through multi-covalent attachment. Using activity-based protein profiling (ABPP)-based chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells to impair breast cancer pathogenicity through engaging in molecular glue interactions with the neo-substrate tumor-suppressor TP53, leading to the activation of p53 transcriptional activity and cell death. Our results reveal a previously undiscovered anti-cancer mechanism of this natural product family and highlight the potential for combining chemoproteomics and multi-covalent natural products for the discovery and characterization of new molecular glues.

biochemistry↗

C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2

Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERSTs utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology.

molecular biology↗

Systematic in vivo quantification of microRNA affinities

The majority of mammalian genes are under regulation by microRNAs, yet predicting the extent of miRNA-mediated repression has remained elusive. Here we systematically quantified the biological impact of miRNAs conserved in vertebrates using stable mouse embryonic stem cell lines expressing sensitive fluorescent reporters. Differentiation of these 163 lines to the three germ layers revealed that the majority of conserved miRNAs have detectable changes in activity. We determined in vivo target affinity KD of 115 miRNAs by integrating activity measurements, CRISPR/Cas miRNA knockouts and miRNA sequencing. Target affinities of individual miRNAs spanned several orders of magnitude, with highly expressed miRNAs having overall higher KD. Scaling miRNA expression levels by their respective KD recapitulated the relative number of Argonaute-bound targets for individual miRNA families. Our results provide a rationale to determine the set of miRNAs with a biological activity in a given cell type, KD values setting expression thresholds for target repression.

molecular biology↗

Protein Charge Neutralization is the Proximate Driver Dynamically Tuning a Nanoscale Bragg Reflector

Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing its refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that reduction in repulsive net charge of the purified, recombinant reflectin - either (for the first time) by generalized anionic screening with salt, or by pH titration - drives a finely tuned, precisely calibrated increase in size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. X-ray scattering analyses confirm the sphericity, size and low polydispersity of the assemblies. Precise proportionality between assembly size and charge-neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth. The resulting stability of reflectin assemblies with time ensures reciprocally precise control of the particle number concentration, thereby encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The results presented here strongly suggest that it is charge neutralization, rather than any change in aromatic content, that is the proximate driver of assembly, fine-tuning a colligative property-based nanostructured biological machine. A physical mechanism is proposed.

molecular biology↗

The "replacing surgery" of cpDNA: de novo chemical synthesis and in vivo functional testing of Chlamydomonas chloroplast genome

We have successfully designed and synthesized the 221,372-bp cpDNA SynCpV1.0 with the native cpDNA of Chlamydomonas reinhardtii as the template. Homoplasmic SynCpv1.0-harboring algal strains were obtained by biolistic transformation and selected with an ascending gradient of antibiotic pressure. Meanwhile, we were pleasantly surprised to find that SynCpV1.0 was able to re-introduce and replicate normally after the total DNA of transplastomic algal strains were transformed to Escherichia coli, it indicated that SynCpV1.0 was able to shuttle between C. reinhardtii and E. coli. Finally, we analyzed the photosynthetic properties of SynCpV1.0-harboring transplastomic strains, the results showed that they exhibited the same photosynthetic efficiency as the wild strain of C. reinhardtii CC125, and could rescue the photosynthetic defect in mutant strain of C. reinhardtii CC5168. Herein, we have performed the "replacing surgery" of cpDNA and established an ideal platform to complete multiple cycles of "Design-Build-Test" for optimizing the cpDNA of photosynthetic organisms. HighlightO_LIAn artificial cpDNA SynCpV1.0 is constructed by de novo chemical synthesis. C_LIO_LIThe "replacing surgery" of cpDNA was performed in the chloroplast of C. reinhardtii C_LIO_LIIt is found that artificial cpDNA was able to shuttle between Chlamydomonas chloroplast and E. coli. C_LIO_LIEstablish an ideal platform to complete multiple cycles of "Design-Build-Test" for optimizing the cpDNA. C_LI One-Sentence SummaryThe chloroplast genome can be replaced by a complete synthesized genome and performs the designed biological function in C. reinhardtii.

molecular biology↗

Simultaneous protein and RNA analysis in single extracellular vesicles, including viruses: SPIRFISH

Interest in using nanoparticles for delivery of therapeutic RNA has been steadily growing, provoking a need to precisely understand their structure and contents. Single-particle and single-molecule analysis techniques provide snapshots of single biological nanoparticles, including viruses, liposomes, and extracellular vesicles (EVs). While existing methods primarily focus on protein detection, RNA delivery is becoming increasingly prevalent. A method to simultaneously detect protein and internal RNA in the same particle would reveal variability in size, structure, and RNA packaging efficiency, enabling optimization of nanoparticle delivery. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor (SP-IRIS) with single-molecule fluorescence in-situ hybridization (smFISH). Using SPIRFISH, we detect HIV-1 envelope protein and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNA within EVs. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles. Teaser: A new single particle analysis technique simultaneously detects specific RNA and protein in biological nanoparticles.

molecular biology↗

Development of antisense tools to study Bodo saltans and its intracellular symbiont

Obligate symbioses are common in nature and present a particular challenge for functional genetic analysis. In many cases, the host is a non-model species with poor tools for genetic manipulation and the symbiont cannot be cultured or its gene expression manipulated to investigate function. Here we investigated the potential for using antisense inhibition to analyse host and symbiont gene function within an obligate aquatic symbiosis. We focused on the kinetoplastid host Bodo saltans and its bacterial symbiont, Candidatus Bodocaedibacter vickermanii, a member of Rickettsiales. We conclude that antisense inhibition is not feasible in the B. saltans and its symbiont, as the holobiont feeds on the antisense molecules - and increases in numbers - upon treatment with the antisense construct. Although our approach has proven unsuccessful, we have developed an array of protocols which can be used to study the biology of this microeukaryote and its microbial associates.

molecular biology↗

Glycosomal Aquaglyceroporin 1 Dual Role in Iron Homeostasis and Antimony Susceptibility in Leishmania amazonensis

Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major, localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9-mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (SbIII), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in SbIII EC50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance. Author SummaryLeishmaniases are neglected tropical diseases caused by parasites that survive and multiply inside vertebrates cells. These parasites rely on hosts nutrients like iron and must resist both host defenses and treatment with toxic drugs such as antimony. We studied a protein called Aquaglyceroporin 1 (AQP1) in Leishmania amazonensis, a species that causes skin lesions in South America. Unlike related species, where AQP1 is found on the parasites surface, we discovered that in L. amazonensis AQP1 is located in an internal organelle called glycosome. By deleting this protein from the parasite, we found that it plays a crucial role in iron balance, sensitivity to antimony drugs, and the parasites ability to cause disease. Unexpectedly, parasites without AQP1 were more resistant to antimony but still accumulated high levels of the drug, suggesting that Leishmania has other ways of taking up antimony. Our findings challenge the assumption that all Leishmania species use the same strategies to survive, and highlight the need to understand species-specific differences when designing treatments or analyzing parasite biology.

molecular biology↗

Comprehensive analysis of Arabidopsis thaliana DNA polymerase epsilon catalytic subunit A and B mutants - an insight into differentially expressed genes and protein-protein interactions

One of the main replicative enzymes in most eukaryotes, DNA polymerase {varepsilon} (POLE), is composed of four subunits, namely a single catalytic and three regulatory subunits. In Arabidopsis, the catalytic subunit of POLE is encoded by two genes: Arabidopsis thaliana DNA POLYMERASE EPSILON CATALYTIC SUBUNIT A (AtPOL2A) and B (AtPOL2B). Although studies have shown AtPOL2A to be involved in various biological processes, the role of AtPOL2B is unclear. Here, we investigated the transcriptomes of both atpol2a and atpol2b mutants, and the promoter sequences to provide a better insight into the targets of AtPOL2s at the molecular level. In the present study, leaf cDNA libraries of four AtPOL2 mutants (atpol2a-1 and atpol2b-1, -2 and - 3) were sequenced using the Illumina platform. Analysis of gene expression profiles identified a total of 198, 76, 141 and 67 differentially expressed genes in atpol2a-1, atpol2b-1, atpol2b-2 and atpol2b-3, respectively; the majority of pericentromeric transposable elements were transcriptionally active in atpol2a-1 as compared to atpol2b mutants and wild type. Protein-protein interaction network analysis and molecular docking identified three (CER1, RPA1E and AT5G60250) and two (PR1 and AT5G48490) proteins as potential interactors (cluster size > 60 and balanced score < -900) of AtPOL2A and AtPOL2B, respectively; Interestingly, these five proteins also showed a significant interaction between POLE catalytic subunit of Saccharomyces cerevisiae. Our in silico promoter analysis showed that the AtPOL2A promoter sequence is overrepresented with cis-acting regulatory elements (CREs) associate with cell cycle regulation, meristematic/reproductive tissue-specific pattern of expression and MYB protein recognition, whereas the AtPOL2B promoter sequence was mainly enriched with stress-responsive elements. The information provided here has led to the identification of targets of AtPOL2s at the molecular level and CREs putatively associated with the regulation of AtPOL2s. To our knowledge, this study provides the first comparative transcriptome profiling of single-gene mutants of AtPOL2s.

molecular biology↗

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells

The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, and its rapid national and international spread pose a global health emergency. Coronaviruses use their spike proteins to select and enter target cells and insights into nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. Finally, we show that the serum form a convalescent SARS patient neutralized 2019-nCoV-S-driven entry. Our results reveal important commonalities between 2019-nCoV and SARS-coronavirus infection, which might translate into similar transmissibility and disease pathogenesis. Moreover, they identify a target for antiviral intervention. One sentence summaryThe novel 2019 coronavirus and the SARS-coronavirus share central biological properties which can guide risk assessment and intervention.

molecular biology↗

HSATII RNA is induced via E2F3a and a non-canonical ATM-regulated DNA damage response pathway

Pericentromeric human satellite II (HSATII) repeats are normally silent, but can be actively transcribed in tumor cells, where increased HSATII copy number is associated with a poor prognosis in colon cancer, and in human cytomegalovirus (HCMV)-infected cells, where the RNA facilitates viral replication. Here, we report that HCMV infection or treatment of ARPE-19 diploid epithelial cells with the DNA-damaging agents, etoposide and zeocin, induced HSATII RNA expression, and a kinase-independent function of ATM was required for the induction. Additionally, various breast cancer cell lines growing in adherent, 2-dimensional cell culture expressed HSATII RNA at different levels, and levels were markedly increased when cells were either infected with HCMV or treated with zeocin. High levels of HSATII RNA expression correlated with enhanced migration of breast cancer cells, and knockdown of HSATII RNA reduced cell migration and the rate of cell proliferation. Our investigation links high expression of HSATII RNA to the DNA damage response, centered on a non-canonical function of ATM, and demonstrates a role for the satellite RNA in tumor cell proliferation and movement. SIGNIFICANCEHSATII RNA is associated with cancer progression, immunostimulation and, as we recently reported, it plays an important role in herpesvirus infections. However, the understanding of cellular processes responsible for the expression of HSATII RNA has been limited. Our current investigation identified a non-canonical, ATM kinase-independent DNA-damage response pathway as a common cellular mechanism regulating HSATII RNA induction in virus-infected cells or cells treated with DNA-damaging agents. Additionally, our study provides a link between expression of HSATII RNA and the cellular growth and migration phenotypes of cancer cells, establishing a new paradigm to study the biological consequences of HSATII RNA expression, i.e., treatment of normal diploid and tumor cells with DNA-damaging agents.

molecular biology↗

Human Tear Metabolomics Using Liquid Chromatography-Q Exactive-HF Mass Spectrometry

AimTo putatively identify and characterise human tear metabolites in a normal subject on an untargeted platform of liquid chromatography-Q exactive-HF mass spectrometry. MethodsFour samples of unstimulated tears were collected from both eyes on four consecutive days between 1 - 2 pm using a microcapillary tube and pooled from both eyes each day. Untargeted analysis of the tears was performed by chromatographic separation of constituent metabolites in both CSH-C18RP (Charged Surface Hybrid-C18 Reversed Phase) and SeQuant ZIC-pHILIC (Zwitterionic-polymeric Hydrophilic Interaction Liquid Chromatography) columns, followed by heated electrospray ionization (HESI) and the acquisition of mass spectra using QExactive-HF mass spectrometer. Compound Discoverer software (v2.0) was used for data analysis. ResultEighty-two metabolites were tentatively identified. Seventy compounds (85.4 %) were observed in all four samples with a coefficient of variation (CV) less than 25 %. Fifty-nine metabolites (71.9 %) were novel in the healthy tears. Amino acids were the most frequently detected metabolites in the tears (28 %), followed by carbohydrates (12.2 %), carboxylic acids (8.5 %), carnitines (6.1 %) and glycerophospholipids (4.9 %), respectively. ConclusionThe current untargeted platform is capable of detecting a range of tear metabolites across several biological categories. This study provides a baseline for further ocular surface studies.

molecular biology↗

Programmable Cleavage of Double-stranded DNA by Combined Action of Argonaute CbAgo from Clostridium butyricum and Nuclease Deficient RecBC Helicase from E.coli

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Discovery of mesophilic Argonautes active at physiological temperature places pAgos closer to their possible application for genome editing as a simpler alternative to CRISPR/Cas nucleases. Currently, the use of mesophilic pAgos as programmable DNA endonucleases is hampered by their poor action on double-stranded DNA (dsDNA), mainly due to their inability to invade the DNA duplex. The present study demonstrates that efficient in vitro cleavage of double-stranded DNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo-C). Properties of CbAgo and characteristics of simultaneous cleavage of complementary DNA strands in concurrence with DNA strand unwinding by RecBexo-C were thoroughly explored using 0.3-25 kb DNA substrates. When combined with RecBexo-C helicase, CbAgo was capable of cleaving target sequences located 11-12.5 kb from the ends of linear dsDNA at 37{o}C. Our study demonstrates that CbAgo with RecBexo-C can be programmed to generate dsDNA fragments flanked with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. At present, the combination of CbAgo and RecBexo-C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of dsDNA at any desired location.

molecular biology↗

Claspin-dependent and -independent Chk1 activation by a panel of biological stresses

Replication stress has been suggested to be an ultimate trigger of carcinogenesis. Oncogenic signal, such as overexpression of CyclinE, has been shown to induce replication stress. Here, we show that various biological stresses, including heat, oxidative stress, osmotic stress, LPS, hypoxia, and arsenate induce activation of Chk1, a key effector kinase for replication checkpoint. Some of these stresses indeed reduce the fork rate, inhibiting DNA replication. Analyses of Chk1 activation in the cell population with western analyses showed that Chk1 activation by these stresses is largely dependent on Claspin. On the other hand, single cell analyses with Fucci cells indicated that while Chk1 activation during S phase is dependent on Claspin, that in G1 is mostly independent of Claspin. We propose that various biological stresses activate Chk1 either directly by stalling DNA replication fork or by some other mechanism that does not involve replication inhibition. The former pathway predominantly occurs in S phase and depends on Claspin, while the latter pathway, which may occur throughout the cell cycle, is largely independent of Claspin. Our findings provide evidence for novel links between replication stress checkpoint and other biological stresses and points to the presence of unknown mechanisms of Chk1 activation in mammalian cells.

molecular biology↗

Development of QSAR Models to Identify Mycobacterium tuberculosis enoyl-ACP-reductase Enzyme Inhibitors

Tuberculosis is a global concern due to its high prevalence in developing countries and the ability of mycobacteria to develop resistance to current treatment regimens. In this project, we propose the use of QSAR (Quantitative Structure-Activity Relationships) modeling as a means to identify and evaluate the inhibitory activity of candidate molecules for molecular improvement stages and/or in vitro assays. This approach allows for in silico estimation, reducing research time and costs. To achieve this, we utilized the SAR (Structure-Activity Relationships) study conducted by He, Alian, and Montellano (2007), which focused on a series of arylamides tested as inhibitors of the enzyme enoyl-ACP-reductase (InhA) in Mycobacterium tuberculosis. We developed both the Hansh-Fujita (classical) and CoMFA (Comparative Molecular Field Analysis) QSAR models. The classical QSAR model produced the most favorable statistical results using Multiple Linear Regression (MLR). It achieved an internal validation correlation factor R2 of 0.9012 and demonstrated predictive quality with a Stone-Geisser indicator Q2 of 0.8612. External validation resulted in a correlation factor R2 of 0.9298 and Q2 of 0.720, indicating a highly predictive mathematical model. The CoMFA Model obtained a Q2 of 0.6520 in internal validation, enabling the estimation of energy fields around the molecules. This information is crucial for molecular improvement efforts. We constructed a library of small molecules, analogous to those used in the SAR study, and subjected them to the classic QSAR function. As a result, we identified ten molecules with high estimated biological activity. Molecular docking analysis suggests that these ten analogs, identified by the classical QSAR model, exhibit favorable estimated free energy of binding. In conclusion, the QSAR methodology proves to be an efficient and effective tool for searching and identifying promising drug-like molecules.

bioinformatics↗

Transglutaminase 2 is an RNA-binding protein: Experimental verification and characterisation of a novel transglutaminase feature

Transglutaminase 2 (TG2) is a uniquely versatile protein with diverse catalytic activities, such as transglutaminase, protein disulfide isomerase, GTPase, protein kinase, and participates in several biological processes. According to information available in the RBP2GO database, TG2 can be an RNA-binding protein (RBP). RBPs participate in posttranscriptional gene expression regulation, influencing RNAs function, while RNA molecules can also modulate RBPs biological activity. Our goal was to confirm this novel character of TG2 in human umbilical cord vein endothelial cells (HUVEC), which physiologically express TG2. First, UV cross-linked RNA-protein complexes were isolated from immortalised HUVEC using orthogonal organic phase separation. Compared with the RBP2GO database, mass spectrometry identified 392 potential RBPs, including TG2 and 20 novel, endothelium-related RBPs. Total RNA from HUVEC pulled down recombinant human TG2. Complex formation between TG2 and a 43-mer RNA molecule with a secondary structure as well as a homo-oligomeric single-stranded poly(dG), but not poly(dA), could be observed in magnetic RNA-protein pull-down experiments. Experiments with TG2 inhibitors NC9 and GTP{gamma}S, which stabilise its open and closed conformation, respectively, revealed that the open conformation of the enzyme favoured RNA-binding. Biolayer interferometry revealed a high binding affinity between TG2 and RNA with a KD value of 88 nM. We propose that superficial residues on the catalytic core and C-terminal {beta}-barrel domains, being in a hidden position in the closed TG2, are involved in RNA binding. Our study demonstrates TG2s previously uncharacterised RNA-binding ability, opening new avenues for understanding its multi-functionality. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/591323v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@3c7acaorg.highwire.dtl.DTLVardef@a165f2org.highwire.dtl.DTLVardef@2c0293org.highwire.dtl.DTLVardef@15d3d81_HPS_FORMAT_FIGEXP M_FIG C_FIG Transglutaminase 2 (TG2) is a unique multifunctional protein demonstrating large conformational changes. It has various transglutaminase and other catalytic and non-catalytic activities which show conformation dependency. Our study has characterised a novel, open conformation-related biological activity of TG2 and its RNA-binding ability.

molecular biology↗