Apical Loop Research Articles

Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into hepatocytes

Sporozoite forms of the Plasmodium parasite, the causative agent of malaria, are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytes. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection.

The Influence of Atrial Strain on Functional Capacity in Patients with the Fontan Circulation.

Speckle-tracking echocardiography has been used to assess atrial function. This analysis is feasible in univentricular hearts. The aim of this study was to characterize the relationship between atrial strain and functional capacity in the Fontan circulation. Apical four-chamber echocardiographic loops of 39 Fontan patients were reviewed. The dominant atrium was assessed for active (εact), conduit (εcon), and reservoir (εres) strain and εact/εres ratio. Cardiopulmonary exercise test was performed on the same day and oxygen uptake (VO2) at ventilatory threshold (VT) and peak VO2 were chosen as the dependent variables. Statistical analysis was performed using SPSS® version 23. Unpaired t test was used for binomial and continuous variable correlation; single and multivariable linear regression were used for continuous variable correlation. Statistical significance was defined as p value<0.05. VO2 at VT as a percentage of predicted VO2 was 36.8% (SD 10.7). Peak VO2 was 64.7% (SD 18.9) of the predicted value. In univariate analysis, both were associated with age, atrioventricular regurgitation, ejection fraction, εres, εcon, and εact/εres. In multivariate regression, higher VO2 at VT and peak VO2 were associated with younger age (p=0.003 and p=0.001, respectively) and higher εcon (p=0.026 and p=0.020). Evaluation of heart function is difficult in the Fontan circulation, hindered by complex ventricular morphology and lack of normative data. VO2 provides a good surrogate. Atrial strain parameters are compromised in these patients and associated with VO2. Therefore, whenever possible, atrial strain should be measured as it may provide a new method of risk stratification.

Aromatic 19 F-13 C TROSY-[19 F, 13 C]-Pyrimidine Labeling for NMR Spectroscopy of RNA.

We present the access to [5‐19F, 5‐13C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐19F, 5‐13C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic 19F–13C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13C–19F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐19F, 5‐13C]/[5‐19F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial 19F–13C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19F, 13C]‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.

Engineering Light-Up Aptamers for the Detection of RNA Hairpins through Kissing Interaction.

Aptasensors are biosensors that include aptamers for detecting a target of interest. We engineered signaling aptasensors for the detection of RNA hairpins from the previously described malachite green (MG) RNA aptamer. The top part of this imperfect hairpin aptamer was modified in such a way that it can engage loop-loop (so-called kissing) interactions with RNA hairpins displaying partly complementary apical loops. These newly derived oligonucleotides named malaswitches bind their cognate fluorogenic ligand (MG) exclusively when RNA-RNA kissing complexes are formed, whereas MG does not bind to malaswitches alone. Consequently, the formation of the ternary target RNA-malaswitch RNA-MG complex results in fluorescence emission, and malaswitches constitute sensors for detecting RNA hairpins. Malaswitches were designed that specifically detect precursors of microRNAs let7b and miR-206.

Systolic thickening fraction of inerventricular septum as a predictor of superresponse to cardiac resyncronisation therapy - concept of a helical ventriсular band

Aim: to assess morpho-functional properties of left ventricle (LV) in patients with superresponse (SR) to CRT using the helical ventriсular band concept (HVB).Materials and methods: 56 patients were examined (mean age 54.0±9.9 years) at baseline and during follow-up visit: 48.8±25.6 months. Patients were divided into groups: I group (n=34) with decrease of LV end-systolic volume (ESV) ≥30% (superresponders) and II group (n=22) - decrease of LV ESV ˂30% (nonsuperresponders).Results: apical loop descendens segment (DS) and ascendens segment (AS) of HVB were evaluated according to the concept of F. Torrent-Guasp et al. According to the logistic regression mechanical interventricular delay (MID) (OR 1.072, 95% CI 1.017-1.131; p=0.01) and systolic thickening fraction (STF) of interventricular septum (IVS) DS (OR 0.944, 95% CI 0.895 - 0.995; p = 0.033) had an independent relationship with CRT SR. According to the ROC analysis the sensitivity and specificity of this model were 72.7% and 66.7% (AUC=0.769; р=0.001). AS STF of IVS was higher in SR (22.5 [0.0;40.0]% и 0.0 [0.0;25.0]%; р=0.005). The survival rate in group I was 85.1%, in group II was 63.6% (Log-Rank test p=0.019).Conclusion: SR is associated with a higher AS STF of IVS, higher MID, also with a higher survival rate.

Structure-function analysis of the equine hepacivirus 5' untranslated region highlights the conservation of translational mechanisms across the hepaciviruses.

Equine hepacivirus (EHcV) (now also classified as hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5' untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5'UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5'UTR. The secondary structure was determined using selective 2' hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem-loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5'UTR, allowing us to investigate the roles of the stem-loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.

ADAM10 is Expressed by Ameloblasts, Cleaves the RELT TNF Receptor Extracellular Domain and Facilitates Enamel Development

MMP20 cleaves cadherins and may facilitate cell movement, however MMP20 is not known to cleave tight junction or desmosome proteins. Ameloblasts had not previously been screened for membrane anchored proteases that could contribute to cell movement. Here we performed a PCR screen for proteolyticlly active A Disintegrin And Metalloproteinase (ADAM) family members. These proteinases are termed sheddases because they have a transmembrane domain and their catalytic domain on the cell surface can function to release anchored proteins. Significantly, ADAMs can be targeted to specific substrates on the cell membrane through their interaction with tetraspanins. Six ADAMs (ADAM8, 9, 10, 15, 17, 19) were expressed in mouse enamel organs. We show that Adam10 expression begins in the apical loop, continues through the secretory stage and abruptly ends at the transition stage when ameloblast migration ceases. ADAM10 cleaves cadherins and tight junction plus desmosome proteins and is well characterized for its role in cell movement. ADAM10 facilitated LS8 cell migration/invasion through a Matrigel coated membrane and we demonstrate that ADAM10, but not ADAM17 cleaves the RELT extracellular domain. This striking result is significant because RELT mutations cause amelogenesis imperfecta (AI) and this directly links ADAM10 to an important role in enamel development.

P3445Subclinical myocardial dysfunction in pediatric kidney transplant recipients: a two-dimensional speckle-tracking echocardiography study

Abstract Chronic kidney disease is associated with increased risk of cardiovascular mortality and morbidity in pediatric patients as well. Renal transplantation results in improved survival, however, several factors contribute to markedly elevated cardiovascular complication rate compared to the healthy population. While major cardiac events occur rarely in pediatric population, detection of subclinical changes in cardiac morphology and function may be of high interest to effectively identify high-risk patients. Accordingly, our aim was to investigate left (LV)- and right ventricular (RV) morphology and function using conventional and two-dimensional (2D) speckle-tracking echocardiography (STE) in pediatric renal transplant recipients. Our study group consisted of 41 kidney transplanted children (RTX; mean age: 14±3 years, m/f: 25/16) and 39 age- and gender matched healthy controls. Using 2D echocardiography, LV and RV focused apical loops were obtained and LV end-diastolic volume index (EDVi), ejection fraction (EF), mass index (Mi), RV end-diastolic area index (EDAi) and fractional area change (FAC) were measured. Using STE, we have determined LV global longitudinal (GLS) and circumferential strain (GCS), RV GLS, and LV and RV early diastolic longitudinal strain rate (LSrE). LV EDVi did not differ between RTX and controls (51±13 vs. 52±10 mL/m2, p=NS), while LVMi was markedly higher in RTX patients (36±8 vs. 28±6 g/m2, p&lt;0.0001). LVEF was comparable between the two groups (62±5 vs. 62±3%; p=NS), while LV GLS was significantly lower in RTX (−20.6±2.1 vs. −21.8±2.1%, p=0.01) along with a tendential increase in LV GCS (−31.6±4.3 vs. −29.7±4.6%, p=0.06). LV LSrE was significantly lower in RTX patients (1.29±0.29 vs. 1.45±0.27 1/s, p&lt;0.05). RV EDAi did not differ between the two groups (11.2±2.3 vs. 11.6±2.0 cm2/m2, p=NS). Interestingly however, RTX patients had significantly higher RV FAC and RV GLS (FAC: 46±7 vs. 42±4%, GLS: −24.6±3.7 vs. −22.4±2.6%, both p&lt;0.01) along with lower RV LSrE (1.32±0.57 vs. 1.60±0.43, p&lt;0.05). Cardiac morphology and function shows distinct changes after RTX. Along with comparable ventricular dimensions, LV hypertrophy and subclinical systolic and diastolic dysfunction is present. RV systolic function is relatively increased accompanied by subclinical diastolic dysfunction of the chamber, which may refer to previous RV overload. STE may be a useful tool to reveal early myocardial dysfunction in pediatric kidney transplant recipients.

Crystal structure of an adenovirus virus-associated RNA

Adenovirus Virus-Associated (VA) RNAs are the first discovered viral noncoding RNAs. By mimicking double-stranded RNAs (dsRNAs), the exceptionally abundant, multifunctional VA RNAs sabotage host machineries that sense, transport, process, or edit dsRNAs. How VA-I suppresses PKR activation despite its strong dsRNA character, and inhibits the crucial antiviral kinase to promote viral translation, remains largely unknown. Here, we report a 2.7 Å crystal structure of VA-I RNA. The acutely bent VA-I features an unusually structured apical loop, a wobble-enriched, coaxially stacked apical and tetra-stems necessary and sufficient for PKR inhibition, and a central domain pseudoknot that resembles codon-anticodon interactions and prevents PKR activation by VA-I. These global and local structural features collectively define VA-I as an archetypal PKR inhibitor made of RNA. The study provides molecular insights into how viruses circumnavigate cellular rules of self vs non-self RNAs to not only escape, but further compromise host innate immunity.

A Small Cyclic β-Hairpin Peptide Mimics the Rbfox2 RNA Recognition Motif and Binds to the Precursor miRNA 20b.

The RNA recognition motif (RRM), which is the most abundant RNA-binding motif in eukaryotes, is a well-structured domain of about 90 amino acids, yet the β2β3 hairpin, corresponding to strands 2 and 3 of the β-sheet, and the intervening loop make essential interactions with RNA in many RRM complexes. A series of small cyclic peptide mimics of the β2β3 hairpin of Rbfox2 protein that recognize the terminal loop of precursor miR-20b have been designed to investigate whether the full RNA-binding protein can be mimicked with a minimal structurally preorganized peptide. Within a small library of seven cyclic peptides, a peptide with low-micromolar affinity for the miR-20b precursor was found. NMR spectroscopy titration data suggest that this peptide specifically targets the apical loop of pre-miR-20b. This work shows that it is possible to mimic RNA-binding proteins with designed stable peptides, which provide a starting point for designing or evolving small peptide mimetics of RRM proteins.

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides.

A synthetic riboswitch N1, inserted into the 5′-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6′ hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6′-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.

Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit Are Critical for Its Fusion Activity.

The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein.IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545-1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577-587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749-1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110-128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

ABSTRACTPackaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

Microprocessor depends on hemin to recognize the apical loop of primary microRNA.

Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction.

A Dual Interaction Between the 5'- and 3'-Ends of the Melon Necrotic Spot Virus (MNSV) RNA Genome Is Required for Efficient Cap-Independent Translation.

In eukaryotes, the formation of a 5′-cap and 3′-poly(A) dependent protein–protein bridge is required for translation of its mRNAs. In contrast, several plant virus RNA genomes lack both of these mRNA features, but instead have a 3′-CITE (for cap-independent translation enhancer), a RNA element present in their 3′-untranslated region that recruits translation initiation factors and is able to control its cap-independent translation. For several 3′-CITEs, direct RNA-RNA long-distance interactions based on sequence complementarity between the 5′- and 3′-ends are required for efficient translation, as they bring the translation initiation factors bound to the 3′-CITE to the 5′-end. For the carmovirus melon necrotic spot virus (MNSV), a 3′-CITE has been identified, and the presence of its 5′-end in cis has been shown to be required for its activity. Here, we analyze the secondary structure of the 5′-end of the MNSV RNA genome and identify two highly conserved nucleotide sequence stretches that are complementary to the apical loop of its 3′-CITE. In in vivo cap-independent translation assays with mutant constructs, by disrupting and restoring sequence complementarity, we show that the interaction between the 3′-CITE and at least one complementary sequence in the 5′-end is essential for virus RNA translation, although efficient virus translation and multiplication requires both connections. The complementary sequence stretches are invariant in all MNSV isolates, suggesting that the dual 5′–3′ RNA:RNA interactions are required for optimal MNSV cap-independent translation and multiplication.

RNA and RNP Structures that Contribute to Viral Pathogenesis

RNA viruses persist to pose serious threats to human health and global economies. Disease progression mediated by viral pathogenesis requires numerous intersections between host proteins and viral RNA (vRNA) elements. Host‐vRNA complexes drive essential processes in the replication cycles of viruses; as such, they represent novel targets for therapeutic intervention. Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family are cellular proteins often usurped by RNA viruses. My lab studies the molecular mechanisms by which HIV and Enterovirus 71 redirects hnRNPs to control viral gene expression. To that end, we have designed an integrated biophysical framework to elucidate the structures of vRNAs and protein‐vRNA complexes. Here, we describe 3D structures of conserved HIV and EV71 RNA elements that regulate splicing and translation. We further show that conformational adaptation and slow protein motions are fundamental properties by which hnRNP A1 and hnRNP H achieve cognate RNA recognition. To globally identify determinants of specificity, we used High Throughput Sequencing Equilibrium (HTS‐EQ) binding experiments to characterize the complete affinity distribution of human hnRNP A1 bound to the HIV ESS3 stem loop. HTS‐EQ involves competitive binding of hnRNP A1 to a completely randomized pool of ESS3 apical loop variants (n=16,384). Quantitative analysis of the resulting affinity distribution reveals RNA sequence; motif copy number; motif spacing; and secondary structure determine specificity by modulating the rates of productive hnRNP A1‐RNA encounters. Collectively, our work is offering mechanistic insights into the strategies by which viruses coopt cellular RNA binding proteins.Support or Funding InformationNIH R01 GM101979NIH U54 GM103297This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An unexpected RNA distal interaction mode found in an essential region of the hepatitis C virus genome.

The 3’X tail is a functionally essential 98-nt sequence located at the 3′-end of the hepatitis C virus (HCV) RNA genome. The domain contains two absolutely conserved dimer linkage sequence (DLS) and k nucleotide segments involved in viral RNA dimerization and in a distal base-pairing interaction with stem-loop 5BSL3.2, respectively. We have previously shown that domain 3’X forms an elongated structure comprising two coaxially stacked SL1’ and SL2’ stem-loops. This conformation favors RNA dimerization by exposing a palindromic DLS segment in an apical loop, but buries in the upper stem of hairpin SL2’ the k nucleotides involved in the distal contact with 5BSL3.2. Using nuclear magnetic resonance spectroscopy and gel electrophoresis experiments, here we show that the establishment of the complex between domain 3’X and stem-loop 5BSL3.2 only requires a rearrangement of the nucleotides forming the upper region of subdomain SL2’. The results indicate that the interaction does not occur through a canonical kissing loop mechanism involving the unpaired nucleotides of two terminal loops, but rather involves a base-paired stem and an apical loop and may result in a kissing three-way junction. On the basis of this information we suggest how the 3’X tail switches between monomer, homodimer and heterodimer states to regulate the HCV viral cycle.

Molecular Dynamics Simulations of a Riboswitch Binding Aminoglycoside Antibiotics

Riboswitches are regulatory elements in non-coding regions of mRNAs. By binding small ligands, they control the expression of the genes located downstream of the aptamer. The N1 synthetic riboswitch binds aminoglycoside antibiotics from the neomycin family. It consists of a 27-nucleotide-long hairpin with two flexible regions: a bulge and an apical loop. In N1 riboswitch, different aminoglycosides evoke a varying response: ribostamycin and neomycin inhibit the expression of genes in yeast and paromomycin is inactive, even though the two latter ligands differ by only one functional group in ring I. The NMR structures of the ribostamycin and paromomycin complexes with N1 are available. However, the structures of the N1 riboswitch with neomycin and without ligands are not known. To explain how such a small difference between neomycin and paromomycin affects the interactions and dynamics of the N1 riboswitch, we performed all-atom molecular dynamics simulations. Replica exchange method helped increase the sampling. The non-Watson-Crick hydrogen bonds differ between the riboswitch with and without ligands even though the secondary structure is similar. Aminoglycosides active in yeast (neomycin and ribostamycin) display significant stabilization of the apical loop nucleobase A17 upon binding with the riboswitch. In paromomycin complex, the A17 base is only partially stabilized and creates a frequent, but unstable hydrogen bonding with a terminal hydroxyl group in ring I of paromomycin. Surprisingly, this hydrogen bond was not captured in recent NMR experiments. The N1 riboswitch shares several similar patterns with another aminoglycoside binding site in ribosome called A-site. However, A-site does not discriminate between paromomycin and neomycin. Thus, we compare those two aminoglycoside binding sites from the point of view of their sequence, hydrogen bond network, and sodium ion concentration.

Conformational Dynamics of the HIV-1 Trans-Activation Response Element RNA Hairpin Bound to a Lab-Evolved Peptide

We report microsecond-length classical molecular dynamics simulations of a lab-evolved peptide bound to the HIV-1 trans-activation response element (TAR) RNA hairpin. An essential 5′-structure in all HIV-1 mRNAs, TAR interacts with HIV-1 Tat to promote HIV-1 genome transcription, and is considered a promising therapeutic target. Using yeast display, a U1A-derived TAR binding protein (TBP) was selected that binds TAR with subnanomolar affinity and extraordinarily high specificity over the native U1A U1hpII target, thereby preventing Tat-dependent transcription in vitro. A co-crystal structure of TBP in complex with TAR reveals the basis for its unprecedented affinity and specificity. The complex exhibits a Watson-Crick C30-G34 base pair in the six-nucleotide apical loop. From chemical modification experiments and sequence conservation, this base pair has been posited as a prerequisite for Tat binding. We investigated the role of a short TBP peptide - responsible for the preponderance of TAR readout - in stabilizing the observed TAR structure using molecular dynamics simulations of TAR in the apo state and in complex with the full TBP protein and the short TBP peptide. To define discrete conformational states, we performed principal component analysis and clustered trajectories based on the resulting principal components. We observed that the apical loop C30-G34 base pair and a major groove U23-A27-U38 base triple are stable features of peptide-bound TAR on the microsecond timescale. In agreement with NMR studies of free TAR, dissolution of the major groove base triple occurs in trajectories of the apo state. The occupancy of contacts between the protein and the TAR major groove is greater for the arginine residues whose mutation to alanine has a greater impact on the binding affinity, as measured by isothermal calorimetry.

The evaluation of nailfold videocapillaroscopy findings in patients with type 2 diabetes with and without diabetic retinopathy.

OBJECTIVE:Diabetes mellitus (DM) is a chronic hyperglycemic state and is associated with microvascular structural alterations. This study aimed to investigate the diameters of capillary loops and morphostructural changes using nailfold video capillaroscopy (NVC) in patients with type 2 DM with and without diabetic retinopathy (DR).METHODS:This cross-sectional, single-center study was conducted in patients with type 2 DM who were followed in outpatient clinics of ophthalmology and internal medicine. General demographic data were collected from patients. An ophthalmologist examined all patients in terms of DR. A rheumatologist blinded to the clinical data performed NVC. The diameters of apical, arterial, and venous loop of capillaries were measured, and the microvascular changes of capillaries were scored.RESULTS:In this study, 44 patients with type 2 DM with DR (47.7% males) and 20 patients with type 2 DM without DR (55% males) were included. In our study, patients with type 2 DM with DR had more frequent capillary hemorrhage, more frequent ectasia, more frequent giant capillary, and more frequent neo-angiogenesis than patients with type 2 DM without DR. However, these findings were not statistically significant.CONCLUSION:Further controlled studies with large sample size are needed to determine the characteristic NVC findings of DR in patients with type 2 DM.