Molecular Details Research Articles

Investigation of RNA-binding protein NOVA1 in silico: Comparison of the modern human V197 with the archaic I197 variant present in Neanderthals

By comparing the high-coverage archaic genome sequences to those of modern humans, specific genetic differences have been identified. For example, a human-specific substitution has been found in neuro-oncological ventral antigen 1 (NOVA1) – an RNA-binding protein that regulates the alternative splicing of neuronal pre-mRNA. The amino acid substitution results in an isoleucine-to-valine change at position 197 in NOVA1 (archaic: I197, modern human: V197). Previous studies have utilised gene editing technology to compare the archaic and modern human forms of NOVA1 in cortical organoids, however, the structural and molecular details require further investigation. Using an in silico approach, the modern human (WT) and archaic (V197I) structures of NOVA1 were generated. Moreover, the structure of NOVA1 containing a glycine-to-valine substitution at position 68 (G68V), which occurs at the RNA-binding interface, was examined for comparison. Protein-RNA docking was subsequently performed to model the interaction of NOVA1 variants with RNA and the complexes were evaluated further using classical molecular dynamics (MD) simulations. Based on the MM-PBSA analysis, the binding free energies were similar between the WT (−956.8 ± 32.6 kcal/mol), V197I (−975.4 ± 65.6 kcal/mol), and G68V (−946.7 ± 34.3 kcal/mol) complexes. The findings highlight the binding and stability of protein-RNA complexes with only modest structural changes observed in the archaic and G68V variants compared to the WT NOVA1 protein. Further clarification is required to enhance our understanding of the impact of NOVA1 mutations on alternative splicing and disease development. In particular, delineating the effect of multiple mutations in the NOVA1 gene is of importance.

The Salmonella enterica EnvE is an Outer Membrane Lipoprotein and Its Gene Expression Leads to Transcriptional Repression of the Virulence Gene msgA.

The envE gene of Salmonella enterica serovar Typhimurium is encoded within Salmonella Pathogenicity Island-11 (SPI-11) and is located immediately upstream of the virulence gene msgA (macrophage survival gene A) in the same transcriptional orientation. To date, the characteristics and roles of envE remain largely unexplored. In this study, we show that EnvE, a predicted lipoprotein, is localized on the outer membrane using sucrose gradient ultracentrifugation. Under oxidative stress conditions, envE transcription is suppressed, while msgA transcription is induced, indicating an inverse correlation between the mRNA levels of the two neighboring genes. Importantly, inactivation of envE leads to constitutive transcription of msgA regardless of the presence of oxidative stress. Moreover, trans-complementation of the envE mutant with a plasmid-borne envE fails to prevent the induction of msgA transcription, suggesting that envE functions as a cis-regulatory element rather than a trans-acting factor. We further show that both inactivation and complementation of envE confer wild-type levels of resistance to oxidative stress by ensuring the expression of msgA. Our data suggest that the S. enterica envE gene encodes an outer membrane lipoprotein, and its transcription represses msgA expression in a cis-acting manner, probably by transcriptional interference, although the exact molecular details are yet unclear.

CRISPR-AsCas12f1 couples out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage.

Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages. We find that following the protospacer DNA unwinding and non-target strand (NTS) DNA nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from the nick. Subsequently, DNA unwinding is extended to the out-of-protospacer region, and AsCas12f1 gradually digests the unwound DNA beyond the protospacer. Eventually, the single endonucleolytic target-strand DNA cleavage at 3 nt downstream of the protospacer readily dissociates the ternary AsCas12f1-sgRNA-DNA complex from the protospacer adjacent motif-distal end, leaving a staggered double-strand DNA break. The coupling between the unwinding and cleavage of both protospacer and out-of-protospacer DNA is promoted by Mg2+. Kinetic analysis on the engineered AsCas12f1-v5.1 variant identifies the only accelerated step of the protospacer NTS DNA trimming within the sequential DNA cleavage. Our findings provide a dynamic view of AsCas12f1 catalyzing DNA unwinding-coupled nucleolytic cleavage and help with practical improvements of Cas12f-based genome editing tools.

The Kinetics of Carbon-Carbon-Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity.

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b-ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon-carbon-bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS-mediated carbon-carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate-limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS-mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

The Kinetics of Carbon‐Carbon‐Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b‐ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon‐carbon‐bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS‐mediated carbon‐carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate‐limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS‐mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

Synaptic-like plasticity in 2D nanofluidic memristor from competitive bicationic transport.

Synaptic plasticity, the dynamic tuning of signal transmission strength between neurons, serves as a fundamental basis for memory and learning in biological organisms. This adaptive nature of synapses is considered one of the key features contributing to the superior energy efficiency of the brain. Here, we use molecular dynamics simulations to demonstrate synaptic-like plasticity in a subnanoporous two-dimensional membrane. We show that a train of voltage spikes dynamically modifies the membrane's ionic permeability in a process involving competitive bicationic transport. This process is shown to be repeatable after a given resting period. Because of a combination of subnanometer pore size and the atomic thinness of the membrane, this system exhibits energy dissipation of 0.1 to 100 aJ per voltage spike, which is several orders of magnitude lower than 0.1 to 10 fJ per spike in the human synapse. We reveal the underlying physical mechanisms at molecular detail and investigate the local energetics underlying this apparent synaptic-like behavior.

The homodimerization domain of the Stl repressor is crucial for efficient inhibition of mycobacterial dUTPase

The dUTPase is a key DNA repair enzyme in Mycobacterium tuberculosis, and it may serve as a novel promising anti-tuberculosis target. Stl repressor from Staphylococcus aureus was shown to bind to and inhibit dUTPases from various sources, and its expression in mycobacterial cells interfered with cell growth. To fine-tune and optimize Stl-induced inhibition of mycobacterial dUTPase, we aimed to decipher the molecular details of this interaction. Structural background of the complex between dUTPase and a truncated Stl lacking the repressor C-terminal homodimerization domain has been described, however, the effects of this truncation of Stl on enzyme binding and inhibition are still not known. Using several independent biophysical, structural and enzyme kinetic methods, here we show that lack of the repressor homodimerization domain strongly perturbs both enzyme binding and inhibition. We also investigated the role of a mycobacteria-specific loop in the Stl-interaction. Our results show that removal of this loop leads to a ten-fold increase in the apparent inhibition constant of Stl. We present a high-resolution three-dimensional structure of mycobacterial dUTPase lacking the genus-specific loop for structural insight. Our present data suggest that potent inhibition of mycobacterial dUTPase by Stl requires the wild-type full-length protein context.

Sequence and Phylogenetic Analysis of Influenza Virus (H1N1pdm2009) Circulating in Riyadh, Saudi Arabia

Influenza A virus (IAV) is the principal cause of seasonal flu and is often reported among pilgrims in Saudi Arabia (SA) due to their mass gatherings. The epidemiological, phylogenetic, and molecular details of A/H1N1pdm2009 in 200 clinical samples collected from hospitalized children in Riyadh during two epidemic seasons (2020/21 and 2021/22) are reported in this study. A total of 21 (10.50%) samples were positive for IAV, as determined using PCR. Fifteen isolates (71.42%) were identified as H1N1pdm2009: eight (53.33%) samples were from males, seven (46.67%) from females. The prevalence of H1N1pdm2009 isolates was significantly (p < 0.05) higher among the age group 15-64 years than the other age groups. A comparison of hemagglutinin (HA) and neuraminidase (NA) amino acid sequences between SA H1N1pdm and certain vaccine strains revealed 19 mutations relative to reference strain A/California/07/2009. Among them, eight (0.47%) were in HA, and eight (0.56%) were in NA sequences that differed from vaccine strains. All isolates of the 2020–2022 seasons exhibited N- and O-glycosylation sites comparable to vaccine strains. Phylogenetically their HA and NA genes are divided into different clades. Most of the studied isolates (five) belonged to clade 5a.1 of HA. These data identify the genetic makeup of circulating influenza virus subtypes.

ATP-Triggered Fe(CN)2CO Synthon Transfer from the Maturase HypCD to the Active Site of Apo-[NiFe]-Hydrogenase.

[NiFe]-hydrogenases catalyze the reversible activation of H2 using a unique NiFe(CN)2CO metal site, which is assembled by a sophisticated multiprotein machinery. The [4Fe-4S] cluster-containing HypCD complex, which possesses an ATPase activity with a hitherto unknown function, serves as the hub for the assembly of the Fe(CN)2CO subfragment. HypCD is also thought to be responsible for the subsequent transfer of the iron fragment to the apo-form of the catalytic hydrogenase subunit, but the underlying mechanism has remained unexplored. Here, we performed a thorough spectroscopic characterization of different HypCD preparations using infrared, Mössbauer, and NRVS spectroscopy, revealing molecular details of the coordination of the Fe(CN)2CO fragment. Moreover, biochemical assays in combination with spectroscopy, AlphaFold structure predictions, protein-ligand docking calculations, and crosslinking MS deciphered unexpected mechanistic aspects of the ATP requirement of HypCD, which we found to actually trigger the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase.

Molecular details and phosphoregulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells

Molecular details and phosphoregulation of the CENP-T-Mis12 complex interaction during mitosis in DT40 cells

Multi-species cryoEM calibration and workflow verification standard.

Cryogenic electron microscopy (cryoEM) is a rapidly growing structural biology modality that has been successful in revealing molecular details of biological systems. However, unlike established biophysical and analytical techniques with calibration standards, cryoEM has lacked comprehensive biological test samples. Here, a cryoEM calibration sample consisting of a mixture of compatible macromolecules is introduced that can not only be used for resolution optimization, but also provides multiple reference points for evaluating instrument performance, data quality and image-processing workflows in a single experiment. This combined test specimen provides researchers with a reference point for validating their cryoEM pipeline, benchmarking their methodologies and testing new algorithms.

A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.

The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.

Validation of metaxin-2 deficient C. elegans as a model for MandibuloAcral Dysplasia associated to mtx-2 (MADaM) syndrome

MandibuloAcral Dysplasia associated to MTX2 gene (MADaM) is a recently described progeroid syndrome (accelerated aging disease) whose clinical manifestations include skin abnormalities, growth retardation, and cardiovascular diseases. We previously proposed that mtx-2-deficient C. elegans could be used as a model for MADaM and to support this, we present here our comprehensive phenotypic characterization of these worms using atomic force microscopy (AFM), transcriptomic, and oxygen consumption rate analyses. AFM analysis showed that young mtx-2-less worms had a significantly rougher, less elastic cuticle which becomes significantly rougher and less elastic as they age, and abnormal mitochondrial morphology. mtx-2 C. elegans displayed slightly delayed development, decreased pharyngeal pumping, significantly reduced mitochondrial respiratory capacities, and transcriptomic analysis identified perturbations in the aging, TOR, and WNT-signaling pathways. The phenotypic characteristics of mtx-2 worms shown here are analogous to many of the human clinical presentations of MADaM and we believe this validates their use as a model which will allow us to uncover the molecular details of the disease and develop new therapeutics and treatments.

Structural Basis for Voltage Gating and Dalfampridine Binding in the Shaker Potassium Channel.

The generation and propagation of action potentials in neurons depend on the coordinated activation of voltage-dependent sodium and potassium channels. Potassium channels of the Shaker family regulate neuronal excitability through voltage-dependent opening and closing of their ion conduction pore. This family of channels is an important therapeutic target, particularly in multiple sclerosis where the inhibitor dalfampridine (4-aminopyridine) is used to improve nerve conduction. The molecular details of how the voltage sensor domain drives opening of the pore domain has been limited by the lack of closed-state structures, also impairing the search for novel drugs. Using AlphaFold2-based conformational sampling methods we identify a structural model for the closed Shaker potassium channel where movement of the voltage sensor drives the opening trough interactions between the S4-S5 linker and S6 helix. We show experimentally that breakage of a backbone hydrogen bond is a critical part of the activation pathway. Through docking we identify a hydrophobic cavity formed by the pore domain helices that binds dalfampridine in the closed state. Our results demonstrate how voltage sensor movement drives pore opening and provide a structural framework for developing new therapeutic agents targeting the closed state. We anticipate this work will enable structure-based drug design efforts focused on state-dependent modulation of voltage-gated ion channels for the treatment of neurological disorders.

Using human disease mutations to understand de novo DNA methyltransferase function.

DNA methylation is a repressive epigenetic mark that is pervasive in mammalian genomes. It is deposited by DNA methyltransferase enzymes (DNMTs) that are canonically classified as having de novo (DNMT3A and DNMT3B) or maintenance (DNMT1) function. Mutations in DNMT3A and DNMT3B cause rare Mendelian diseases in humans and are cancer drivers. Mammalian DNMT3 methyltransferase activity is regulated by the non-catalytic region of the proteins which contain multiple chromatin reading domains responsible for DNMT3A and DNMT3B recruitment to the genome. Characterising disease-causing missense mutations has been central in dissecting the function and regulation of DNMT3A and DNMT3B. These observations have also motivated biochemical studies that provide the molecular details as to how human DNMT3A and DNMT3B mutations drive disorders. Here, we review progress in this area highlighting recent work that has begun dissecting the function of the disordered N-terminal regions of DNMT3A and DNMT3B. These studies have elucidated that the N-terminal regions of both proteins mediate novel chromatin recruitment pathways that are central in our understanding of human disease mechanisms. We also discuss how disease mutations affect DNMT3A and DNMT3B oligomerisation, a process that is poorly understood in the context of whole proteins in cells. This dissection of de novo DNMT function using disease-causing mutations provides a paradigm of how genetics and biochemistry can synergise to drive our understanding of the mechanisms through which chromatin misregulation causes human disease.

Acetylation-Dependent Compaction of the Histone H4 Tail Ensemble.

Acetylation of the histone H4 tail (H4Kac) has been established as a significant regulator of chromatin architecture and accessibility; however, the molecular mechanisms that underlie these observations remain elusive. Here, we characterize the ensemble features of the histone H4 tail and determine how they change following acetylation on specific sets of lysine residues. Our comprehensive account is enabled by a robust combination of experimental and computational biophysical methods that converge on molecular details including conformer size, intramolecular contacts, and secondary structure propensity. We find that acetylation significantly alters the chemical environment of basic patch residues (16-20) and leads to tail compaction that is partially mediated by transient intramolecular contacts established between the basic patch and N-terminal amino acids. Beyond acetylation, we identify that the protonation state of H18, which is affected by the acetylation state, is a critical regulator of ensemble characteristics, highlighting the potential for interplay between the sequence context and post-translational modifications to define the ensemble features of intrinsically disordered regions. This study elucidates molecular details that could link H4Kac with the regulation of chromatin architecture, illuminating a small piece of the complex network of molecular mechanisms underlying the histone code hypothesis.

Microscopic view on the polarization-resolved S-SHG intensity of the vapor/liquid interface of pure water.

Second harmonic generation (SHG) is a nonlinear optical phenomenon where two photons at the frequency ω combine to form a single photon at the second-harmonic frequency 2ω. Since that second-order process is very weak in bulk isotropic media, optical SHG responses of interfaces provide a powerful and versatile technique to probe the molecular structure and dynamics of liquid interfaces. Both local dipole contributions and non-local quadrupole contributions can be interesting to investigate different properties of the interface, such as the molecular orientation or the charge density. However, a major difficulty is to comprehend the link between the S-SHG intensity and molecular details. This article reports a numerical approach to model the polarization-resolved SHG intensities of a model vapor/liquid interface of pure water. The influence of the interfacial local environment on the hyperpolarizability is taken into account using quantum mechanical/molecular mechanics calculations. The numerical predictions are in very good agreement with experiments. We detail the hypotheses made during the modeling steps and discuss the impact of various factors on the modeled SHG intensities, including the description of the exciting field in the interfacial layer, the effect of neighboring molecules on the second-harmonic polarization, and the presence of an additional static electric field at the interface.

Transcriptome-wide analysis for glucocorticoid receptor-mediated mRNA decay reveals various classes of target transcripts

The glucocorticoid receptor (GR) can bind to DNA or RNA, eliciting transcriptional activation/repression or rapid mRNA degradation, respectively. Although GR-mediated transcriptional regulation has been well-characterized, the molecular details of rapid mRNA degradation induced by glucocorticoids (GCs) are not yet fully understood. Here, we demonstrate that GC-induced GR-mediated mRNA decay (GMD) takes place in the nucleus and the cytoplasm, acting on pre-mRNAs and mRNAs. We also performed cross-linking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) analysis for GMD factors (GR, YBX1, and HRSP12) and mRNA sequencing (mRNA-seq) analysis to identify endogenous GMD substrates. Our comprehensive CLIP-seq and mRNA sequencing analyses reveal that a range of cellular transcripts containing a common binding site for GR, YBX1, and HRSP12 are preferential targets for GMD, suggesting possible new functions of GMD in various biological events.

Devising a Breast Cancer Diagnosis Protocol through Machine Learning

AbstractBreast cancer is a life-threatening disease and has serious health implications. It is categorized based on receptors, including the estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), which are the focus of the present research We analyzed gene expression from data obtained from a functional genomics repository called Array Express. The accession numbers are E-GEOD-52194, E-GEOD-75367, and E-GEOD-58135, and the molecular details of these subsets of cancer receptors. Upon following a predefined computational pipeline, we identified 369 genes that had distinct patterns of gene expression profiles in cases of ER-positive (ER + ) and HER2-negative (HER2-) breast cancer. The support vector machine (SVM) and decision tree models of machine learning were used to evaluate the prognostic and diagnostic significance. Accuracy, sensitivity, and specificity were examined to gauge the effectiveness of these models. Then, a network analysis was performed to assess the significant biological process and signaling pathways of HER2- and ER+ breast cancer development. The present study facilitates an enhanced approach to these subcategories of breast cancer so that precise diagnoses can be made, and better and more focused treatment plans can be provided. The current research provides valuable information on the molecular and genetic basis of ER+ and HER2- breast cancer and has great potential for improving patients' treatment.

Long-read transcriptomics of Ostreid herpesvirus 1 uncovers a conserved expression strategy for the capsid maturation module and pinpoints a mechanism for evasion of the ADAR-based antiviral defence

Abstract Ostreid herpesvirus 1 (OsHV-1), a member of the family Malacoherpesviridae (order Herpesvirales), is a major pathogen of bivalves. However, the molecular details of the malacoherpesvirus infection cycle and its overall similarity to the replication of mammalian herpesviruses (family Orthoherpesviridae) remain obscure. Here, to gain insights into the OsHV-1 biology, we performed long read sequencing of infected blood clams, Anadara broughtonii, which yielded over one million OsHV-1 long reads. This data enabled the annotation of the viral genome with 78 gene units and 274 transcripts, of which 67 were polycistronic mRNAs, 35 ncRNAs and 20 natural antisense transcripts (NATs). Transcriptomics and proteomics data indicate preferential transcription and independent translation of the capsid scaffold protein as an OsHV-1 capsid maturation protease isoform. The conservation of this transcriptional architecture across Herpesvirales likely indicates its functional importance and ancient origin. Moreover, we traced RNA editing events using short read sequencing and supported the presence of inosine nucleotides in native OsHV-1 RNA, consistent with the activity of ADAR1. Our data suggests that, whereas RNA hyper-editing is concentrated in specific regions of the OsHV-1 genome, single nucleotide editing is more dispersed along the OsHV-1 transcripts. In conclusion, we reveal the existence of a conserved pan-Herpesvirales transcriptomic architecture of the capsid maturation module and uncover a transcription-based viral counter defence mechanism, which presumably facilitates the evasion of the host ADAR antiviral system.