Folding Energy Research Articles

Thermal Unfolding of the Human Serotonin Transporter: Differential Effect by Stabilizing and Destabilizing Mutations and Cholesterol on Thermodynamic and Kinetic Stability.

Folding-deficient mutants of solute carrier 6 (SLC6) family members have been linked to human diseases. The serotonin transporter [(SERT)/SLC6A4] is an important drug target in the treatment of depression, anxiety, and obsessive-compulsive disorders and-with structural information in several conformational states-one of the best understood transporters. Here, we surmised that thermal unfolding offered a glimpse on the folding energy landscape of SLC6 transporters. We carried out molecular dynamic (MD) simulations to understand the mechanistic basis for enhanced and reduced stability, respectively, of the thermostabilized variant SERT-Y110A/I291A/T439S, which had previously been used for crystallization of human SERT in the outward-facing state, and of the folding-deficient SERT-P601A/G602A. We also examined the hydrophobic mismatch caused by the absence of cholesterol to explore the contribution of cholesterol to protein stability. When compared with wild type SERT, the thermodynamic and kinetic stability of SERT-Y110A/I291A/T439S was enhanced. In the other instances, changes in these two components were not correlated: the mutations in SERT-P601A/G602A led to a drop in thermodynamic but an increase in kinetic stability. The divergence was even more pronounced after cholesterol depletion, which reduced thermodynamic stability but increased the kinetic stability of wild type SERT to a level comparable to that of SERT-Y110A/I291A/T439S. We conclude that the low cholesterol content of the endoplasmic reticulum facilitates progression of the folding trajectory by reducing the energy difference between folding intermediates and the native state. SIGNIFICANCE STATEMENT: Point mutations in solute carrier 6 (SLC6) family members cause folding diseases. The serotonin transporter [(SERT)/SLC6A4] is a target for antidepressants and the best understood SLC6. This study produced molecular dynamics simulations and examined thermal unfolding of wild type and mutant SERT variants to understand their folding energy landscape. In the folding-deficient SERT-P012A/G602A, changes in kinetic and thermodynamic stability were not correlated. Similarly, cholesterol depletion lowered thermodynamic but enhanced kinetic stability. These observations allow for rationalizing the action of pharmacochaperones.

Thermal adaptation of mRNA secondary structure: stability versus lability

Macromolecular function commonly involves rapidly reversible alterations in three-dimensional structure (conformation). To allow these essential conformational changes, macromolecules must possess higher order structures that are appropriately balanced between rigidity and flexibility. Because of the low stabilization free energies (marginal stabilities) of macromolecule conformations, temperature changes have strong effects on conformation and, thereby, on function. As is well known for proteins, during evolution, temperature-adaptive changes in sequence foster retention of optimal marginal stability at a species' normal physiological temperatures. Here, we extend this type of analysis to messenger RNAs (mRNAs), a class of macromolecules for which the stability-lability balance has not been elucidated. We employ in silico methods to determine secondary structures and estimate changes in free energy of folding (ΔGfold) for 25 orthologous mRNAs that encode the enzyme cytosolic malate dehydrogenase in marine mollusks with adaptation temperatures spanning an almost 60 °C range. The change in free energy that occurs during formation of the ensemble of mRNA secondary structures is significantly correlated with adaptation temperature: ΔGfold values are all negative and their absolute values increase with adaptation temperature. A principal mechanism underlying these adaptations is a significant increase in synonymous guanine + cytosine substitutions with increasing temperature. These findings open up an avenue of exploration in molecular evolution and raise interesting questions about the interaction between temperature-adaptive changes in mRNA sequence and in the proteins they encode.

Folding dynamics of polymorphic G-quadruplex structures.

G-quadruplexes (G4), found in numerous places within the human genome, are involved in essential processes of cell regulation. Chromosomal DNA G4s are involved for example, in replication and transcription as first steps of gene expression. Hence, they influence a plethora of downstream processes. G4s possess an intricate structure that differs from canonical B-form DNA. Identical DNA G4 sequences can adopt multiple long-lived conformations, a phenomenon known as G4 polymorphism. A detailed understanding of the molecular mechanisms that drive G4 folding is essential to understand their ambivalent regulatory roles. Disentangling the inherent dynamic and polymorphic nature of G4 structures thus is key to unravel their biological functions and make them amenable as molecular targets in novel therapeutic approaches. We here review recent experimental approaches to monitor G4 folding and discuss structural aspects for possible folding pathways. Substantial progress in the understanding of G4 folding within the recent years now allows drawing comprehensive models of the complex folding energy landscape of G4s that we herein evaluate based on computational and experimental evidence.

SARS-CoV-2 competes with host mRNAs for efficient translation by maintaining the mutations favorable for translation initiation.

During SARS-CoV-2 proliferation, the translation of viral RNAs is usually the rate-limiting step. Understanding the molecular details of this step is beneficial for uncovering the origin and evolution of SARS-CoV-2 and even for controlling the pandemic. To date, it is unclear how SARS-CoV-2 competes with host mRNAs for ribosome binding and efficient translation. We retrieved the coding sequences of all human genes and SARS-CoV-2 genes. We systematically profiled the GC content and folding energy of each CDS. Considering that some fixed or polymorphic mutations exist in SARS-CoV-2 and human genomes, all algorithms and analyses were applied to both pre-mutate and post-mutate versions. In SARS-CoV-2 but not human, the 5-prime end of CDS had lower GC content and less RNA structure than the 3-prime part, which was favorable for ribosome binding and efficient translation initiation. Globally, the fixed and polymorphic mutations in SARS-CoV-2 had created an even lower GC content at the 5-prime end of CDS. In contrast, no similar patterns were observed for the fixed and polymorphic mutations in human genome. Compared with human RNAs, the SARS-CoV-2 RNAs have less RNA structure in the 5-prime end and thus are more favorable of fast translation initiation. The fixed and polymorphic mutations in SARS-CoV-2 are further amplifying this advantage. This might serve as a strategy for SARS-CoV-2 to adapt to the human host.

Structure-based design of a photoswitchable affibody scaffold.

Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.

Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers

The functionality of an enzyme depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (Tm = 73.5 °C; ΔTm > 23 °C) affected the protein folding energy landscape. The introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded soluble domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations might (i) activate cryptic hinge-loop regions and (ii) establish secondary interfaces, where they make extensive noncovalent interactions between the intertwined protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from small-angle X-ray scattering (SAXS) and cross-linking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain swapping occurs exclusively in vivo. Crucially, the swapped-dimers exhibited advantageous catalytic properties such as an increased catalytic rate and elimination of substrate inhibition. These findings provide additional enzyme engineering avenues for next-generation biocatalysts.

Potential Achilles heels of SARS-CoV-2 are best displayed by the base order-dependent component of RNA folding energy

The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 "Achilles heel," the signal can be targeted by a new antiviral compound. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e. g. NSP3, ORF3a), we suggest that their nucleic acid level functions can be considered potential "Achilles heels" for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The ribosomal frameshifting element scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.

Delineating Characteristic Sequence and Structural Features of Precursor and Mature Piwi-interacting RNAs of Epithelial Ovarian Cancer

Background: Piwi-interacting RNAs (piRNAs) are an amazing class of small noncoding RNAs (sncRNAs) known for its promising role in germline and somatic cells. Myriad functional studies have been performed to unveil the true potential of this class of ncRNAs; however, global features encoded in their sequence and structure have not been explored. Objectives: We aim to identify the sequence and structural level characteristic features of piRNAs of normal ovary (NO), and two subtypes of epithelial ovarian cancer (EOCa)- endometrioid (ENOCa), and serous ovarian cancer (SOCa) that we had reported earlier and their precursors. Methods: We have performed sequence analysis of mature piRNAs and their upstream/downstream regions as well as structural analysis of precursor piRNAs (pre-piRNAs) by examining their minimal folding energy (MFE), adjusted minimal folding energy (AMFE) and minimal folding energy index (MFEI) etc using in silico approaches. Results: We observed enrichment of U at first position and G at several other positions of mature piRNAs, which might be associated with the processing of mature piRNAs similar to what is seen in the case of miRNAs and strong target binding, respectively. In addition, we found the richness of AU in and around 20 nts upstream and downstream of precursor piRNAs (pre-piRNAs). This characteristic feature of pre-piRNAs possibly contributes to lower MFE compared to random sequences and make its secondary structure less stable which decides biogenesis of piRNAs. We also noticed that MFE, AMFE and MFEI of pre-piRNAs are comparatively less than pre-miRNAs of metazoans, plants and viruses reported in other studies, which clearly discriminate pre-piRNAs from other RNA sequences including pre-miRNAs of other organisms. Conclusion: In summary, the present study reveals key characteristic features encoded within and around mature piRNAs as well as pre-piRNAs of NO and EOCa samples that distinguish piRNAs from miRNAs and other random RNA sequences. These findings might act as a cornerstone for better understanding of biogenesis and function of piRNAs as well as will aid in easier identification of new piRNAs from unknown stretch of sequences using the characteristic features.

Polyols, increasing global stability of cytochrome c, destabilize the thermal unfolding intermediate

Studies on the intermediate states of proteins provide essential information on folding pathway and energy landscape of proteins. Osmolytes, known to alter the stability of proteins, might also affect the structure and energy states of folding intermediates. This was examined using cytochrome c (Cyt) as a model protein which forms a spectroscopically detectable intermediate during thermal denaturation transition. Most of the secondary structure and the native heme-ligation were intact in the intermediate state of the protein. Denaturants, urea and guanidinium hydrochloride, and ionic salt destabilizes the intermediate and drive the protein to follow two-state transition. The effect of polyol class of osmolytes, glycol, glycerol, erythritol, xylitol and sorbitol (with OH-groups two to six), on the intermediate was studied using Soret absorbance and far-UV circular dichroism. With the increasing concentration of any of the polyols, the transition-midpoint temperature (Tm ) and the enthalpy change (ΔH) for native to intermediate transition were decreased. This indicated that the intermediate was destabilized by the polyols. However, the polyols increased the overall stability of the protein by increasing Tm and ΔH for intermediate to unfolded transition, except for glycol which destabilized the protein. These results show that the polyols could alter the energy state of the intermediate, and the effect of lower and higher polyols might be different on the stability and folding pathway of the protein. Communicated by Ramaswamy H. Sarma

Fundamental X-ray corona parameters of Swift/BAT AGN

ABSTRACT While X-ray emission from active galactic nuclei (AGN) is common, the detailed physics behind this emission is not well understood. This is in part because high quality broad-band spectra are required to precisely derive fundamental parameters of X-ray emission, such as the photon index, folding energy, and reflection coefficient. Here, we present values of such parameters for 33 AGN observed as part of the 105-month Swift/BAT campaign and with coordinated archival XMM-Newton and NuSTAR observations. We look for correlations between the various coronal parameters in addition to correlations between coronal parameters and physical properties, such as black hole mass and Eddington ratio. Using our empirical model, we find good fits to almost all of our objects. The folding energy was constrained for 30 of our 33 objects. When comparing Seyfert 1–1.9 to Seyfert 2 galaxies, a K–S test indicates that Seyfert 2 AGN have lower Eddington ratios and photon indices than Seyfert 1–1.9 objects with p-values of 5.6 × 10−5 and 7.5 × 10−3, respectively. We recover a known correlation between photon index and reflection coefficient as well as the X-ray Baldwin effect. Finally, we find that the inclusion of the high-energy Swift/BAT data significantly reduces the uncertainties of spectral parameters as compared to fits without the BAT data.

Potential treatment for chronic myeloid leukemia using microRNA: in silico comparison between plants and human microRNAs in targeting BCR-ABL1 gene

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the expression of the BCR-ABL1 fusion gene. Tyrosine kinase inhibitors (TKI) are used to treat CML, but mutations in the tyrosine kinase domain contribute to CML chemo-resistance. Therefore, finding alternative molecular-targeted therapy is important for the comprehensive treatment of CML. MicroRNAs (miRNA) are small non-coding regulatory RNAs which suppress the expression of their target genes by binding to the 3′ untranslated region (3′UTR) of the target mRNA. Hypothetically, the miRNA-mRNA interaction would suppress BCR-ABL1 expression and consequently reduce and inhibit CML cell proliferation. Thus, our objective was to determine the target interaction of human and plant miRNAs targeting the 3′UTR region of BCR-ABL1 in terms of miRNA binding conformity, protein interaction network, and pathways using in silico analysis. The 3′UTR sequence of BCR-ABL1 is obtained from Ensembl Genome Browser while the binding conformity was determined using the PsRNATarget Analysis Server, RNA22, Target Rank Server, and DIANA TOOLS. Protein-protein interaction network and pathway analysis are determined using STRING, Cytoscape, and KEGG pathway analysis.ResultsFive plants and five human miRNAs show strong binding conformity with 3′UTR of BCR-ABL1. The strongest binding conformity was shown by Oryza sativa’s Osa-miR1858a and osa-miR1858b with −24.4 kcal/mol folding energy and a p value of 0.0077. Meanwhile, in human miRNA, the hsa-miR-891a-3p shows the highest miTG score of 0.99 with −12 kcal/mol folding energy and a p value of 0.037. Apart from ABL1, osa-miR1858a/osa-miR1858b and hsa-miR891a-3p also target other 720 and 645 genes, respectively. The interaction network of Osa-miR1858a/osa-miR1858b and hsa-miR891a-3p identifies nineteen and twelve ABL1’s immediate neighboring proteins, respectively. The pathways analysis focuses on the RAS, MAPK, CML, and hematopoietic cell lineage pathway.ConclusionBoth plant and human miRNAs tested in this study could be a potential therapeutic prospect in CML treatment, but thermodynamically, osa-miR1858a/osa-miR1858b binding to ABL1 is more favorable. However, it is important to carry out more research in vitro and in vivo and clinical studies to assess its efficacy as a targeted therapy for CML.Graphical abstract

Folding and persistence times of intramolecular G-quadruplexes transiently embedded in a DNA duplex.

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence time increased. Our assay opens new perspectives for the measurement of G4 dynamics in double-stranded DNA mimicking a replication fork, which is important to understand their role in DNA replication and gene regulation at a mechanistic level.

Neutralism versus selectionism: Chargaff's second parity rule, revisited.

Of Chargaff's four "rules" on DNA base frequencies, the functional interpretation of his second parity rule (PR2) is the most contentious. Thermophile base compositions (GC%) were taken by Galtier and Lobry (1997) as favoring Sueoka's neutral PR2 hypothesis over Forsdyke's selective PR2 hypothesis, namely that mutations improving local within-species recombination efficiency had generated a genome-wide potential for the strands of duplex DNA to separate and initiate recombination through the "kissing" of the tips of stem-loops. However, following Chargaff's GC rule, base composition mainly reflects a species-specific, genome-wide, evolutionary pressure. GC% could not have consistently followed the dictates of temperature, since it plays fundamental roles in both sustaining species integrity and, through primarily neutral genome-wide mutation, fostering speciation. Evidence for a local within-species recombination-initiating role of base order was obtained with a novel technology that masked the contribution of base composition to nucleic acid folding energy. Forsdyke's results were consistent with his PR2 hypothesis, appeared to resolve some root problems in biology and provided a theoretical underpinning for alignment-free taxonomic analyses using relative oligonucleotide frequencies (k-mer analysis). Moreover, consistent with Chargaff's cluster rule, discovery of the thermoadaptive role of the "purine-loading" of open reading frames made less tenable the Galtier-Lobry anti-selectionist arguments.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10709-021-00119-5.

Energy landscape remodeling mechanism of Hsp70-chaperone-accelerated protein folding

Hsp70 chaperone is one of the key protein machines responsible for the quality control of protein production in cells. Facilitating in vivo protein folding by counteracting misfolding and aggregation is the essence of its biological function. Although the allosteric cycle during its functional actions has been well characterized both experimentally and computationally, the mechanism by which Hsp70 assists protein folding is still not fully understood. In this work, we studied the Hsp70-mediated folding of model proteins with rugged energy landscape by using molecular simulations. Different from the canonical scenario of Hsp70 functioning, which assumes that folding of substrate proteins occurs spontaneously after releasing from chaperones, our results showed that the substrate protein remains in contacts with the chaperone during its folding process. The direct chaperone-substrate interactions in the open conformation of Hsp70 tend to shield the substrate sites prone to form non-native contacts, which therefore avoids the frustrated folding pathway, leading to a higher folding rate and less probability of misfolding. Our results suggest that in addition to the unfoldase and holdase functions widely addressed in previous studies, Hsp70 can facilitate the folding of its substrate proteins by remodeling the folding energy landscape and directing the folding processes, demonstrating the foldase scenario. These findings add new, to our knowledge, insights into the general molecular mechanisms of chaperone-mediated protein folding.

A Mathematical Analysis of RNA Structural Motifs in Viruses

RNA stem-loop structures play an important role in almost every step of the viral replication cycle. In this contribution, a mathematical analysis is performed on a large dataset of RNA secondary structure elements in the coding regions of viruses by using topological indices that capture the Laplacian eigenvalues of the associated RNA graph representations and thereby enable structural classification, supplemented by folding energy and mutational robustness. The application of such an analysis for viral RNA structural motifs is described, being able to extract structural categories such as stem-loop structures of different sizes according to the tree-graph representation of the RNA structure, in our attempt to find novel functional motifs. While the analysis is carried on a large dataset of viral RNA structures, it can be applied more generally to other data that involve RNA secondary structures in biological agents.

In Silico and In Vitro Tailoring of a Chitosan Nanoformulation of a Human Metabolic Enzyme.

Enzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process.

Massively parallel gene expression variation measurement of a synonymous codon library

BackgroundCell-to-cell variation in gene expression strongly affects population behavior and is key to multiple biological processes. While codon usage is known to affect ensemble gene expression, how codon usage influences variation in gene expression between single cells is not well understood.ResultsHere, we used a Sort-seq based massively parallel strategy to quantify gene expression variation from a green fluorescent protein (GFP) library containing synonymous codons in Escherichia coli. We found that sequences containing codons with higher tRNA Adaptation Index (TAI) scores, and higher codon adaptation index (CAI) scores, have higher GFP variance. This trend is not observed for codons with high Normalized Translation Efficiency Index (nTE) scores nor from the free energy of folding of the mRNA secondary structure. GFP noise, or squared coefficient of variance (CV2), scales with mean protein abundance for low-abundant proteins but does not change at high mean protein abundance.ConclusionsOur results suggest that the main source of noise for high-abundance proteins is likely not originating at translation elongation. Additionally, the drastic change in mean protein abundance with small changes in protein noise seen from our library implies that codon optimization can be performed without concerning gene expression noise for biotechnology applications.

Neutralism Versus Selectionism: Chargaff's Second Parity Rule, Revisited

Of Chargaff's four rules on DNA base frequencies, the functional interpretation of his second parity rule (PR2) is the most contentious. Thermophile base compositions (GC%) were taken by Galtier and Lobry (1997) as favoring Sueoka's neutral PR2 hypothesis over Forsdyke's selective PR2 hypothesis, namely that mutations improving local within-species recombination efficiency had generated a genome-wide potential for the strands of duplex DNA to separate and initiate recombination through the kissing of the tips of stem-loops. However, following Chargaff's GC rule, base composition mainly reflects a species-specific, genome-wide, evolutionary pressure. GC% could not have consistently followed the dictates of temperature, since it plays fundamental roles in both sustaining species integrity and, through primarily neutral genome-wide mutation, fostering speciation. Evidence for a local within-species recombination-initiating role of base order was obtained with a novel technology that masked the contribution of base composition to nucleic acid folding energy. Forsdyke's results were consistent with his PR2 hypothesis, appeared to resolve some root problems in biology and provided a theoretical underpinning for alignment-free taxonomic analyses using relative oligonucleotide frequencies (k-mer analysis). Moreover, consistent with Chargaff's cluster rule, discovery of the thermoadaptive role of the purine-loading of open reading frames made less tenable the Galtier-Lobry anti-selectionist arguments.

The Electrostatic Features of Dengue Virus Capsid Assembly

Dengue virus causes serious diseases and deaths in the world. Understanding the fundamental mechanisms of dengue virus is highly demanded to develop treatments for dengue virus caused diseases. Here, we present a computational work which focused on the stability of dengue viral capsid. The interactions among E proteins on the dengue viral capsid were studied using several computational approaches. It was found that the electrostatic distribution on a single E protein monomer is highly inhomogeneous, which makes an E protein strongly binding with another E protein. This is the reason why all the E proteins form homodimers as the basic units on the whole dengue viral capsids. The pKa calculations of E proteins demonstrated that the folding energy of an E protein is low and stable in the range of pH 6–10, which is different from many other proteins that are stable at certain pH. The pH dependence of binding energy of E protein homodimer shows that the binding energy is low and independent from pH when the pH is also in the range of 6–10. This finding implies that the dengue virus can survive in a wide range of pH, which can explain why the dengue virus is so widely distributed in the world and spreads fast.

TRFTars: predicting the targets of tRNA-derived fragments

BackgroundtRNA-derived fragments (tRFs) are 14–40-nucleotide-long, small non-coding RNAs derived from specific tRNA cleavage events with key regulatory functions in many biological processes. Many studies have shown that tRFs are associated with Argonaute (AGO) complexes and inhibit gene expression in the same manner as miRNAs. However, there are currently no tools for accurately predicting tRF target genes.MethodsWe used tRF-mRNA pairs identified by crosslinking, ligation, and sequencing of hybrids (CLASH) and covalent ligation of endogenous AGO-bound RNAs (CLEAR)-CLIP to assess features that may participate in tRF targeting, including the sequence context of each site and tRF-mRNA interactions. We applied genetic algorithm (GA) to select key features and support vector machine (SVM) to construct tRF prediction models.ResultsWe first identified features that globally influenced tRF targeting. Among these features, the most significant were the minimum free folding energy (MFE), position 8 match, number of bases paired in the tRF-mRNA duplex, and length of the tRF, which were consistent with previous findings. Our constructed model yielded an area under the receiver operating characteristic (ROC) curve (AUC) = 0.980 (0.977–0.983) in the training process and an AUC = 0.847 (0.83–0.861) in the test process. The model was applied to all the sites with perfect Watson–Crick complementarity to the seed in the 3′ untranslated region (3′-UTR) of the human genome. Seven of nine target/nontarget genes of tRFs confirmed by reporter assay were predicted. We also validated the predictions via quantitative real-time PCR (qRT-PCR). Thirteen potential target genes from the top of the predictions were significantly down-regulated at the mRNA levels by overexpression of the tRFs (tRF-3001a, tRF-3003a or tRF-3009a).ConclusionsPredictions can be obtained online, tRFTars, freely available at http://trftars.cmuzhenninglab.org:3838/tar/, which is the first tool to predict targets of tRFs in humans with a user-friendly interface.