Binding Domain Research Articles

8644 Dynamic Insights into Glucocorticoid Receptor Dysfunction: Novel patient-derived T437I Mutation and Its Impact on Glucocorticoid Receptor Function

Abstract Disclosure: T. Tettey: None. S. Kim: None. K. Wagh: None. Q. Lao: None. L.R. Schiltz: None. D.A. Stavreva: None. D.M. Presman: None. D.P. Merke: None. G.L. Hager: None. The glucocorticoid receptor (GR) is a hormone regulated transcription factor which binds chromatin to regulate many genes, including stress-response and inflammatory genes. Dexamethasone, a GR agonist, is widely used in the treatment of severe inflammation, including COVID-19. GR is vital for life and several GR mutations have been linked to severe pathophysiological conditions. Here we report a novel mutation in the DNA binding domain (DBD) of GR (T437I) in a patient with partial glucocorticoid resistance, a rare genetic disorder characterized by generalized insensitivity to glucocorticoids and a consequent hyperactivation of the hypothalamic pituitary adrenal axis. While the clinical manifestations of this hT437I GR mutation are apparent, its molecular mechanisms remain unexplored. Employing a comprehensive approach that integrates biophysical methods, molecular biology, and genomics, we investigate the molecular underpinnings of this genetic anomaly. Using CRISPR/Cas9 gene editing, we introduce GFP or Halo-tagged hT437I GR mutant into GR null mouse cells. Our findings indicate that the T437I GR variant exhibits normal hormone binding and nuclear translocation. Live-cell imaging using number and brightness analysis reveals that the mutant receptor maintains a wildtype-like oligomeric state but adopts a heterodimeric conformation in the presence of endogenous GR. Global transcriptomics analyses of the mouse hT437I mutant show that most dexamethasone-regulated genes are unresponsive following hormone treatment. We hypothesize that this mutation within the DBD compromises the binding of GR to regulatory sites of target genes. Leveraging fluorescence high-resolution microscopy, we employ single molecule tracking to study the dynamic behavior of Halo-tagged hT437I GR in mouse cells. Our results indicate that the GR mutant has a higher dwell-time on chromatin compared to the wild-type receptor. This suggests that the hT437I mutation impairs the receptor's binding to genomic sites, potentially leading to a failure in regulating GR target genes. This ongoing exploration of the molecular intricacies aims to deepen our understanding of the clinical phenotype observed in the patient. Presentation: 6/2/2024

Reversal of insulin resistance to combat type 2 diabetes mellitus by newer thiazolidinedione's in fructose induced insulin resistant rats

In our pursuit of discovering new antidiabetic agents to manage type 2 diabetes mellitus (T2DM), our approach aimed to identify the bioactive feature/pharmacophore responsible for PPAR-γ expression, as it is accountable for the glucose homeostasis and lipid metabolism. This was achieved by pharmacophore model generation, screening of rationally designed newer thiazolidinedione's library, identifying synthesizing and characterizing the top ten molecules (5a-5j) for their (Invitro &invivo) antidiabetic activity. Preliminary screening of all the ligands by Invitro glucose uptake assay in L6 myotubes (skeletal muscle cell line of rats) revealed compound 5b and 5f stimulated the glucose uptake with 79.29 ± 1.02 % and 74.58 ± 1.02 % respectively compared to pioglitazone with 82.36 ± 0.98 %. This was validated by PPAR-γ TF expression assay, which highlighted a dose dependent increase in transactivation of PPAR-γ. These compounds 5b and 5f were evaluated in fructose induced insulin resistance rat model. Where the treatment with 5b and 5f markedly increased the exogenous clearance of glucose and exogenous insulin via OGTT and ITT respectively, also improved the glucose utilization by significantly increasing content of glycogen and uptake of glucose in rat hemidiaphragm and reversed insulin resistance. Likewise a significant decreased in the VLDL and triglyceride levels was seen in 5b and 5f treated groups compared to insulin resistant (IR) group. It improved glycogenesis by catabolism of glucose and maintained glycaemic control. Similarly it had marked action on enzymatic oxidative biomarkers. Compound 5b displayed better, improved T1/2 (half-life) of 4.21 h and Kel (elimination constant) of 0.381 was noticed in comparison to compound 5f indicating the pharmacokinetic profile. Insilico studies like DFT calculations refined the geometry of 5b and 5f ligands, docking and molecular simulation provided the insights in binding affinity, dynamic behaviour and stability of ligands in PPAR-γ ligand binding domain. MM/GBSA provided the energetics of 5b and 5f in binding pocket. Finally network pharmacology identified ADIPOQ (adiponectin), NR1C3 (PPAR-γ), SLC2A4 (GLUT4), and LEP (leptin) proteins associate with compound 5b and 5f and enriched in Adipocytokine pathway, and PPAR-γ signaling pathway.

6813 Des-Ciclesonide: A Novel Selective Glucocorticoid Receptor Modulator With Therapeutic Potential for Bronchopulmonary Dysplasia, a Chronic Lung Disease of Prematurity

Abstract Disclosure: N. El Khoury: None. J. Jaumotte: None. C. Min: None. C. Madigan: None. J. Wang: None. U. Chandran: None. V. Sampath: None. T.J. Cole: None. P. Monaghan-Nichols: None. R. Houtman: None. K.W. Nettles: None. D.B. DeFranco: None. Infants born before 30 weeks gestation are at risk of developing bronchopulmonary dysplasia (BPD), a prevalent chronic lung disease of prematurity. Glucocorticoids (GCs) such as dexamethasone (DEX) have been a mainstay of BPD prevention as they reduce lung inflammation and injury. However, their use is encumbered with several adverse effects including hyperglycemia, impaired somatic and brain growth and cerebral palsy. These major concerns have resulted in the AAP recommending caution in GC use in preterm infants.Ciclesonide (CIC), an inhaled GC prodrug approved for the treatment of asthma and allergic rhinitis in children, is converted to the potent glucocorticoid receptor (GR) agonist des-ciclesonide (DES) by enzymes enriched in the airways. We previously reported that s.c. delivery of CIC in neonatal rats activated GR transcriptional responses in lung but did not trigger the adverse effects on somatic growth, brain weight or cerebral white matter loss caused by DEX leading us to propose CIC as a potential new pharmacotherapy for BPD. To determine whether limited systemic metabolism of CIC in neonatal rats was solely responsible for its enhanced safety profile, we assessed various outcomes in neonatal rats followed s.c. delivery of its active metabolite DES. Consistent with previous studies, five daily s.c. injections of DEX led to a reduction in body and brain weight as well as reduced serum levels of insulin-like growth factor-1 and chronic hyperglycemia. Surprisingly, analogous s.c. injection of neonatal rats with DES, which has a higher binding affinity for GR than DEX, did not trigger any of these adverse effects. However, DES is as effective as DEX in reducing the expression of various proinflammatory cytokine mRNAs in neonatal rat lung induced by an 11-day systemic treatment with bleomycin. Bulk RNAseq analyses revealed transcriptional responses of neonatal lung and liver to DES, although less robust than that observed to DEX (i.e., 25% and 13% in lung and liver, respectively). Nonetheless, a NAPing assay for coregulator peptide binding showed increased binding of all the DES responsive peptide interactions relative to DEX and cortisol. All-atom molecular dynamics simulations revealed site-specific H-bonding changes within the GR ligand binding domain (LBD) caused by the bulky C16/C17substitution within DES, which was translated into broader changes in LBD structure, specifically revealed as differences in RMSF in helix 7/8 and the surfaces interacting with the NCOA2 peptide. Furthermore, network pathway analysis illustrated that DES mobility at helix 7/8 uniquely generated a meaningful allosteric signaling pathway between Q642 and NCOA2 peptide. In conclusion, the pharmacokinetic properties of the GR prodrug CIC coupled with the tissue-selective pharmacodynamic properties of its active metabolite DES make this FDA approved drug a potential new therapeutic agent for BPD. Presentation: 6/3/2024

7222 Structural and Mechanistic Studies of the Bioactive Anti-Müllerian Hormone Procomplex

Abstract Disclosure: J.A. Howard: None. L. Hok: None. N.J. Sanford: None. R.L. Cate: None. K. Hart: Employee; Self; Regeneron Pharmaceuticals. E. Leach: None. D. Pepin: None. P.K. Donahoe: None. T.B. Thompson: Advisory Board Member; Self; Oviva Therapeutics, Keros Therapeutics. The Anti-Mullerian hormone (AMH), a divergent member of the TGF-beta signaling superfamily, plays crucial roles in reproductive development and maintenance. Owing to its key role as a negative feedback regulator in folliculogenesis, AMH has garnered significant interest for therapeutic applications in contraception and oncofertility. The bioactive form of AMH is a noncovalent complex, comprising the TGFβ-like signaling domain and a dimeric prodomain whose structure remains largely unexplored. The prodomain component is necessary for the dimerization and secretion of the signaling domain, shuttling the ligand to autocrine, paracrine, and endocrine targets. In contrast, the structure and biochemistry of this domain remain poorly characterized, and its role in enhancing AMH signaling remains unclear. This gap in understanding significantly limits the enhancement and wider application of AMH-based protein therapeutics. Molecular modeling unveiled an unexpected AMH prodomain structure: an N-terminal dimerizing domain (NTD) linked to a novel C-terminal binding domain (CTD). This atypical design was affirmed by both small-angle X-ray scattering and negative-stain EM. The CTD introduces a unique TGFβ superfamily fold consisting of a four-helix bundle and an unstructured binding “belt”. The more prodomain-like NTD does not interact with the signaling ligand, nor does it consistently interact with the CTD. Both domains exhibit high levels of interspecies conservation as well as disease-causing mutations, such as those found in PMDS, PCOS, and POI. Using single-particle Cryo-EM we have elucidated the molecular mechanisms of the interaction between the AMH prodomain and signaling domain bound by mAb 6E11. Our 3.2Å cryo-EM structure confirmed the predicted architecture and binding function of the CTD and demonstrated a new dimension of conformational plasticity within the signaling ligand. We observed movements up to 5Å at the fingertips and 10Å in the outer helices of the CTD. More unexpectedly, the conformation of the AMH signaling ligand in the procomplex form is far more open and extended than has been observed in any other structure of a TGF-beta superfamily ligand, including the AMH ligand bound to its receptor AMHR2. We see that transitioning from a prodomain-bound to a receptor-bound state involves significant conformational remodeling of the signaling domain. This reorganization supports a conformational shift mechanism for AMH signaling in which bivalent binding of AMHR2 at the cell surface induces a >15Å and 38° closing of the ligand fingertips, leading to prodomain displacement and subsequent recruitment of type I receptors. Our findings correct earlier structural misconceptions and build a comprehensive model for interpreting existing functional data. This structure provides a foundation for understanding AMH dysregulation and developing advanced AMH therapeutics. Presentation: 6/1/2024

Exploring the ability of the MD+FoldX method to predict SARS-CoV-2 antibody escape mutations using large-scale data

Antibody escape mutations pose a significant challenge to the effectiveness of vaccines and antibody-based therapies. The ability to predict these escape mutations with computer simulations would allow us to detect threats early and develop effective countermeasures, but a lack of large-scale experimental data has hampered the validation of these calculations. In this study, we evaluate the ability of the MD+FoldX molecular modeling method to predict escape mutations by leveraging a large deep mutational scanning dataset, focusing on the SARS-CoV-2 receptor binding domain. Our results show a positive correlation between predicted and experimental data, indicating that mutations with reduced predicted binding affinity correlate moderately with higher experimental escape fractions. We also demonstrate that higher precision can be achieved using affinity cutoffs tailored to distinct SARS-CoV-2 antibodies from four different classes rather than a one-size-fits-all approach. Further, we suggest that the quartile values of optimized cutoffs reported for each class in this study can serve as a valuable guide for future work on escape mutation predictions. We find that 70% of the systems surpass the 50% precision mark, and demonstrate success in identifying mutations present in significant variants of concern and variants of interest. Despite promising results for some systems, our study highlights the challenges in comparing predicted and experimental values. It also emphasizes the need for new binding affinity methods with improved accuracy that are fast enough to estimate hundreds to thousands of antibody-antigen binding affinities.

Enzymatic Production of Trehalose and Trehalulose by Immobilized Thermostable Trehalose Synthase.

Trehalose, a versatile disaccharide renowned for its unique physical and chemical properties, finds extensive application in the food, pharmaceutical, and cosmetic industries. While conventional extraction methods face challenges, enzymatic conversion offers a promising avenue for the industrial production of trehalose. This study delves into a novel synthetic approach utilizing a recombinant enzyme, merging the thermostable trehalose synthase domain from Thermus thermophiles with a cellulose binding domain. Immobilization of this enzyme on cellulose matrices enhances stability and facilitates product purification, opening avenues for efficient enzymatic synthesis. Notably, the engineered enzyme demonstrates additional activity, converting sucrose into trehalulose. This dual functionality, combined with immobilization strategies, holds immense potential for scalable and cost-effective production of trehalose and trehalulose, offering promising prospects in various industrial and biomedical applications.

Neutralisation resistance of SARS-CoV-2 spike-variants is primarily mediated by synergistic receptor binding domain substitutions

ABSTRACT The evolution of SARS-CoV-2 has led to the emergence of numerous variants of concern (VOCs), marked by changes in the viral spike glycoprotein, the primary target for neutralising antibody (nAb) responses. Emerging VOCs, particularly omicron sub-lineages, show resistance to nAbs induced by prior infection or vaccination. The precise spike protein changes contributing to this resistance remain unclear in infectious cell culture systems. In the present study, a large panel of infectious SARS-CoV-2 mutant viruses, each with spike protein changes found in VOCs, including omicron JN.1 and its derivatives KP.2 and KP.3, was generated using a reverse genetic system. The susceptibility of these viruses to antibody neutralisation was measured using plasma from convalescent and vaccinated individuals. Synergistic roles of combined substitutions in the spike receptor binding domain (RBD) were observed in neutralisation resistance. However, recombinant viruses with the entire spike protein from a specific VOC showed enhanced resistance, indicating that changes outside the RBD are also significant. In silico analyses of spike antibody epitopes suggested that changes in neutralisation could be due to altered antibody binding affinities. Assessing ACE2 usage for entry through anti-ACE2 antibody blocking and ACE2 siRNA revealed that omicron BA.2.86 and JN.1 mutant viruses were less dependent on ACE2 for entry. However, surface plasmon resonance analysis showed increased affinity for ACE2 for both BA.2.86 and JN.1 compared to the ancestral spike. This detailed analysis of specific changes in the SARS-CoV-2 spike enhances understanding of coronavirus evolution, particularly regarding neutralising antibody evasion and ACE2 entry receptor dependence.

SARS-CoV-2 S protein harbors furin cleavage site located in a short loop between antiparallel β-strand

Furin cleavage site (FCS) of the SARS-CoV-2 S protein, which connects the S1/S2 junction, is essential for facilitating fusion with the host cells. Wild-type (Wt) SARS-CoV-2 S protein, PDB ID: 6yvb, lacks a sequence of amino acid residues, including the FCS that links the S1/S2 junction. For the first time, we demonstrated that a stretch of 14 amino acid residues (677QTNSPRRARSVASQ689) forms an antiparallel β-sheet comprising of PRRAR sequence in the FCS within a short loop. Upon comparing the loop content of the S1/S2 junction with that of Wt SARS-CoV-2 containing PRRAR in the FCS, we observed a decrease in antiparallel β-sheet content and an increase in loop content in the B.1.1.7 variant with HRRAR in the FCS. This short loop within antiparallel β-sheet can serve as a docking site for various proteases, including TMPRSS2 and α1AT. We performed a 300-ns simulation of the SARS-CoV-2 receptor binding domain (RBD) using several antibacterial and antiviral ligands commonly used to treat various infections. Our findings indicate that the receptor binding domain (RBD) comprising the receptor binding motif (RBM) utilizes β6 and a significant portion of the loop to bind with ligands, suggesting its potential for treating SARS-CoV-2 infections.

Differential effect of plakoglobin in restoring the tumor suppressor activities of p53-R273H vs. p53-R175H mutants.

The six most common missense mutations in the DNA binding domain of p53 are known as "hot spots" and include two of the most frequently occurring p53 mutations (p53-R175H and p53-R273H). p53 stability and function are regulated by various post-translational modifications such as phosphorylation, acetylation, sumoylation, methylation, and interactions with other proteins including plakoglobin. Previously, using various carcinoma cell lines we showed that plakoglobin interacted with wild-type and several endogenous p53 mutants (e.g., R280K, R273H, S241F, S215R, R175H) and restored their tumor suppressor activities in vitro. Since mutant p53 function is both mutant-specific and cell context-dependent, we sought herein, to determine if plakoglobin tumor suppressive effects on exogenously expressed p53-R273H and p53-R175H mutants are similarly maintained under the same genetic background using the p53-null and plakoglobin-deficient H1299 cell line. Functional assays were performed to assess colony formation, migration, and invasion while immunoblotting and qPCR were used to examine the subcellular distribution and expression of specific proteins and genes that are typically regulated by or regulate p53 function and are altered in mutant p53-expressing cell lines and tumors. We show that though, plakoglobin interacted with both p53-R273H and p53-R175H mutants, it had a differential effect on the transcription and subcellular distribution of their gene targets and their overall oncogenic properties in vitro. Notably, we found that plakoglobin's tumor suppressive effects were significantly stronger in p53-R175H expressing cells compared to p53-R273H cells. Together, our results indicate that exploring plakoglobin interactions with p53-R175H may be useful for the development of cancer therapeutics focused on the restoration of p53 function.

B-255 Identification of the protein expression of Androgen Receptor variants using Targeted proteomics in clinical Castration Resistant Prostate Cancer models

Abstract Background Castration Resistant Prostate Cancer (CRPC) is a treatment resistant form of prostate cancer (PCa). Currently, there is not a way to identify which patients will develop this resistance before full blown CRPC develops. Therefore, all PCa treatment approaches are similar yet this results in tumor regression in some cases and progression in others. Targeting the androgen receptor (AR) is still the main focus of current therapies even in CRPC. Emergence of AR splice variants (AR-Vs) after initial treatment is thought to be one of the primary mechanisms of resistance. AR-Vs lack the ligand binding domain rendering Androgen Deprivation Therapy (ADT) ineffective in tumors expressing these variants. Recent work has identified the DNA and RNA species of ARVs in CRPC but investigation into whether the protein is translated are unknown. One exception is the approval of an antibody test that detects a specific AR-v, AR-v7, from the blood of PC patients and predicts ADT response. However, there are instances where a patient may not express ARv7 and are still resistant to ADT. This may suggest that other AR-Vs, not currently detected at the protein level, are important predictors for ADT response. Methods The primary goal of this work is to expand the protein identification of known and unknown AR-Vs that may predict response to ADT using targeted liquid chromatography tandem mass spectrometry (LC-MS/MS). We developed the AR-V targeted LC-MS/MS assay by first designing AR splice variants from unique splice regions via RNA-guided sequences translated to amino acid sequences. The AR-variants full amino acid sequences were then in-silico trypsin digested using Expasy software tool that generated tryptic peptides. Blast analysis of the AR-V peptides was performed where no overlap with other human proteins was observed. These AR-V peptide sequences were commercially synthesized along with heavy isotopically analogs to be used as internal standards in the LC-MS/MS method. The method examined 9 peptides and included 2 to 3 MRM transitions per peptide and calibration curves were constructed using unlabeled peptides. Specimens examined consisted of a panel of PCa cell lines (n= 6), CRPC patient derived xenografts tumors (PDX) (n=4) and a negative control cell line (no androgen receptor expression), Cos-1, which served with a dual function and was used as matrix for peptide MRM optimization experiments. Linearity and sensitivity for each peptide included in the method was examined using calibration curves. Results The targeted MRM approached was successfully achieved for the quantification of 9 peptides from 8 proteins in 6 PCa cell lines and 4 PDX tumor specimens. Out of the 8 proteins evaluated, we observed 3 known targets (AR-exon1, AR-V7 and AR-V12) and 4 novel AR-Vs in 22Rv1 PCa cell lines. Additionally, CRPC PDX were assessed where 3 novel AR-Vs were detected. Conclusions These preliminary results using a LC-MS/MS platform show promising identification and quantification of novel AR-Vs that have not been measured before at the protein level. Subsequent analysis of larger cohorts will further elucidate AR-Vs role in PCa treatment resistance mechanisms and possibly assist in future treatment approaches.

A-235 Systemic Inflammation is associated with worse outcomes from SARS-CoV-2 infection but not neutralizing antibody

Abstract Background Numerous studies have demonstrated an association between systemic inflammation and mortality in patients with COVID-19. However, it is unclear how neutralizing antibody to severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) associates with outcomes. Further, how systemic inflammation impacts neutralizing antibody production is relatively poorly understood. The aim of this study was to determine the association between the neutralizing antibody response to SARS-CoV-2, inflammatory markers, and clinical outcomes in a cohort of hospitalized patients without prior exposure/vaccination. Methods This prospective observational study approved by the Washington University IRB included adults admitted through the ED at Barnes Jewish Hospital between 10/2020 and 10/2021 with symptomatic RT-PCR confirmed SARS-CoV-2 infection. A total of 395 serial samples were assessed from 208 patients without previous vaccination or documented exposure to SARS-CoV-2. Neutralization of SARS-CoV-2 was assessed using the Architect ACE2 binding inhibition assay (Abbott, Research Use only) which measures the % inhibition of interactions between antibodies and the SARS-COV-2 viral Spike Receptor Binding Domain. While 12.5% inhibition of ACE2 was considered positive >80% neutralization is the expected standard. CRP was assessed using the Architect Multigent CRP assay with 9.6mg/dL as the categorical cutoff for risk and interleukin 6 (IL-6) was assessed using the Alinity IL-6 assay (Abbott, Research Use Only) using 30 pg/mL as the threshold for risk in categorical analyses. The electronic medical record (EPIC) was interrogated for comorbidities, age, gender, 30-day mortality, 10-day intubation, and pharmacological interventions. Correlation between biomarkers was assessed using Spearman r. Kaplan-Meier curves and Cox proportional hazards models were constructed for 30-day mortality and 10-day intubation. Results 37 of 208 (18%) of patients died within thirty days of ED presentation and 59 (28%) required intubation within 10-days. There was a significant correlation between IL-6 and CRP (r=0.34) but not with ACE-2 binding (Spearman r <0.06 for both IL-6 and CRP). There was significantly higher CRP in those that died (median = 14 mg/dL, IQR = 8-21) than those that survived (5 mg/dL, 2-11). IL-6 was higher in patients that died (344 pg/mL, 0-870) than those that survived (65 pg/mL, 28-140). Median ACE-2 inhibition trended higher in those that survived (18%, 0-65%) than those that died (3%, 0-48%). Survival curves demonstrated that patients with IL-6 > 30 pg/mL, CRP > 9.6 mg/dL, and ACE2 binding <12.5% at baseline were more likely to die within 30-days. Multivariate modeling found that only IL-6 (Hazard Ratio, 1.28, 95%CI 1.08-1.52 for each doubling) and age (1.04, 1.01-1.08) were significantly associated with 30-day mortality. Conclusions Systemic inflammation measured by IL-6 and CRP upon admission is highly associated with 30-day mortality from SARS-CoV-2 infection. While ACE-2 inhibition was lower in those that died from COVID-19 at admission, it was not independently associated with mortality or correlated with inflammatory markers. This implies an importance of other aspects of the immune response for mediating inflammation and reducing risk of mortality from SARS-CoV-2.

Evaluation of an Affinity-Enhanced Anti-SARS-CoV2 Nanobody Design Workflow Using Machine Learning and Molecular Dynamics.

In silico optimization of protein binding has received a great deal of attention in the recent years. Since in silico prefiltering of strong binders is fast and cheap compared to in vitro library screening methods, the advent of powerful hardware and advanced machine learning algorithms has made this strategy more accessible and preferred. These advances have already impacted the global response to pandemic threats. In this study, we proposed and tested a workflow for designing nanobodies targeting the SARS-CoV-2 spike protein receptor binding domain (S-RBD) using machine learning techniques complemented by molecular dynamics simulations. We evaluated the feasibility of this workflow using a test set of 3 different nanobodies and 2 different S-RBD variants, from in silico design and bacterial expression to binding assays of the designed nanobody mutants. We successfully designed nanobodies that were subsequently tested against both the wild-type (Wuhan type) and the delta variant S-RBD and found 2 of them to be stronger binders compared to the wild-type nanobody. We use this case study to describe both the strengths and weaknesses of this in silico assisted nanobody design strategy.

B-050 A comparison of two semi-quantitative tests, Abbott’s Advise DX SARS-CoV-2 IgG II and Beckman Coulter’s Access SARS-COV-2 IgG II, for measuring IgG antibody levels after Sars-CoV-2 Pfizer-BioNTech vaccination

Abstract Background Using 219 samples collected from volunteer employees of our lab, obtained in different times after Sars-CoV-2 Pfizer-BioNTech vaccination, we compared two semi-quantitative tests: Methods Abbott's AdviseDx SARS-CoV-2 IgG II and Beckman Coulter’s Access SARS-CoV-2 IgG II assay. Both assays detect IgG antibodies to the receptor binding domain of the S1 subunit of the spike protein of SARS-CoV-2 in serum and plasma of tested individuals. For the Beckman test, AMR is from 0 to 450 AU/ml with cut off for positive test 10 AU/ml; for the Abbott test, AMR is from 0 to 25000 AU/ml with cut off for positive test 100 AU/ml. Results Using a fitted regression line, it appears that the best correlation between these two tests was found in the lower value range, below 25AU/ml for Beckman test and 4000 AU/ml for Abbott test (R-squared value of 0.842 and y=87.165x). For higher antibody levels, the result distribution resembles a binary pattern, which we find very interesting. We will discuss possible explanations of this finding. Conclusions

Mechanistic Basis for a Single Amino Acid Residue Mutation Causing Human DNA Ligase 1 Deficiency, A Rare Pediatric Disease

In mammalian cells, DNA ligase 1 (LIG1) functions as the primary DNA ligase in both genomic replication and single-strand break repair. Several reported mutations in human LIG1, including R305Q, R641L, and R771W, cause LIG1 syndrome, a primary immunodeficiency. While the R641L and R771W mutations, respectively located in the nucleotidyl transferase and oligonucleotide binding domains, have been biochemically characterized and shown to reduce catalytic efficiency, the recently reported R305Q mutation within the DNA binding domain (DBD) remains mechanistically unexplored. The R641L and R771W mutations are known to decrease the catalytic activity of LIG1 by affecting both interdomain interactions and DNA binding during catalysis, without significantly impacting overall DNA affinity. To elucidate the molecular basis of the LIG1 syndrome-causing R305Q mutation, we purified this single-residue mutant protein and investigated its secondary structure, protein stability, DNA binding affinity, and catalytic efficiency. Our findings reveal that the R305Q mutation significantly impairs the function of LIG1 by disrupting the DBD-DNA interactions, leading to a 7–21-fold lower DNA binding affinity and a 33–300-fold reduced catalytic efficiency of LIG1. Additionally, the R305Q mutation slightly decreases LIG1’s protein stability by 2 to 3.6 °C, on par with the effect observed previously with either the R641L or R771W mutant. Collectively, our results uncover a new mechanism whereby the R305Q mutation impairs LIG1-catalyzed nicked DNA ligation, resulting in LIG1 syndrome, and highlight the crucial roles of the DBD-DNA interactions in tight DNA binding and efficient LIG1 catalysis.

Neonatal SARS-CoV-2 mRNA Vaccination Efficacy Is Influenced by Maternal Antibodies.

Vaccination in pregnancy guards against infection. Maternal antibodies, however, can inhibit antibody production in neonates. We sought to determine the effects of maternal vaccination on neonatal immune response to a SARS-CoV-2 mRNA vaccine. We hypothesized that mRNA-lipid nanoparticles (LNP) vaccination allows for a de novo neonatal antibody response even in the presence of vertically transmitted maternal antibodies. Female mice were vaccinated with SARS-CoV-2 spike receptor binding domain (RBD) mRNA-LNPs. Mice were then bred, and 21-day-old pups were inoculated with the same mRNA-LNPs. Spike-specific IgG ELISAs were performed using mouse serum. A SARS-CoV-2 spike protein peptide library to perform peptide ELISAs characterized high affinity binding domains within the spike protein. Results were analyzed with one-way ANOVAs with Tukey's multiple comparisons tests. Compared to pups of unvaccinated dams, there were high levels of spike-specific IgG detected in the pups of vaccinated dams at 3 weeks of life (p < 0.0001). After neonatal vaccination, pups of unvaccinated dams had higher spike-specific serum IgG than pups of vaccinated dams at 12 weeks of life (p < 0.001). Antibody specificity to peptide moieties within spike RBD were similar when comparing a vaccinated dam to her pup at Week 3 of life, with different binding affinities observed in the pups by Week 15 of life. Pre-existing maternal antibodies may partially blunt the initial neonatal antibody response to mRNA-LNPs vaccination. This vaccine strategy, however, does not prohibit the subsequent development of a broad range of RBD antibody specificities that may be protective.

Monoclonal Antibodies Capable of Inhibiting the Interaction of the Receptor Binding Domain of SARS-CoV-2 Virus with the Angiotensin-Converting Receptor 2 of Human Cells

Monoclonal Antibodies Capable of Inhibiting the Interaction of the Receptor Binding Domain of SARS-CoV-2 Virus with the Angiotensin-Converting Receptor 2 of Human Cells

Epileptic encephalopathy linked to a DALRD3 missense variant that impairs tRNA modification

Epileptic encephalopathies are severe epilepsy syndromes characterized by early onset and progressive cerebral dysfunction. A nonsense variant in the DALR Anticodon Binding Domain Containing 3 (DALRD3) gene has been implicated in epileptic encephalopathy but no other disease-associated variants in DALRD3 have been described. In human cells, the DALRD3 protein forms a complex with the METTL2 methyltransferase to generate the 3-methylcytosine (m3C) modification in specific arginine tRNAs. Here, we identify an individual with a homozygous missense variant in DALRD3 who displays developmental delay, cognitive deficiencies, and multifocal epilepsy. The missense variant substitutes an arginine residue to cysteine (R517C) within the DALR domain of the DALRD3 protein that is required for binding tRNAs. Cells derived from the individual homozygous for the DALRD3-R517C variant exhibit reduced levels of m3C modification in arginine tRNAs, indicating that the R517C variant impairs DALRD3 function. Notably, the DALRD3-R517C protein displays reduced association with METTL2 and loss of interaction with substrate tRNAs. Our results uncover another loss-of-function variant in DALRD3 linked to epileptic encephalopathy disorders. Importantly, these findings underscore DALRD3-dependent tRNA modification as a key contributor to proper brain development and function.

High-resolution crystal structure of human coronavirus HKU1 receptor binding domain bound to TMPRSS2 receptor

High-resolution crystal structure of human coronavirus HKU1 receptor binding domain bound to TMPRSS2 receptor

Computational Insights into Kaempferol and Luteolin: Potential Mitigators of the Omicron B.1.1.529 SARS-CoV-2 Variant

Abstract The coronavirus disease 2019 (COVID-19) has been circulating in the local and global population. Among the SARS-CoV-2 variants, the Omicron strain tends to be detected in the recent genomic surveillance studies. Despite being a manageable disease, recovered patients may experience post-COVID-19 conditions such as cough (50.6%), fatigue (45.8%), and memory loss (37.4%). While developing an updated vaccine for COVID-19 is time-consuming, the in-silico discovery of phytoconstituents that bind to the SARS-CoV-2 receptor binding domain (RBD), may provide insights on the potential of edible plants as complementary treatments. This study aimed to investigate and compare the ligand-protein interactions of phytoconstituents and COVID-19 treatment drug, remdesivir, to the SARS-CoV-2 spike protein. The molecular docking results indicated that kaempferol and luteolin could potentially mitigate COVID-19, as they exhibited higher RBD-binding affinities than remdesivir.

Sec23IP recruits VPS13B/COH1 to ER exit site-Golgi interface for tubular ERGIC formation.

VPS13B/COH1 is the only known causative factor for Cohen syndrome, an early-onset autosomal recessive developmental disorder with intellectual inability, developmental delay, joint hypermobility, myopia, and facial dysmorphism as common features, but the molecular basis of VPS13B/COH1 in pathogenesis remains largely unclear. Here, we identify Sec23 interacting protein (Sec23IP) at the ER exit site (ERES) as a VPS13B adaptor that recruits VPS13B to ERES-Golgi interfaces. VPS13B interacts directly with Sec23IP via the VPS13 adaptor binding domain (VAB), and the interaction promotes the association between ERES and the Golgi. Disease-associated missense mutations of VPS13B-VAB impair the interaction with Sec23IP. Knockout of VPS13B or Sec23IP blocks the formation of tubular ERGIC, an unconventional cargo carrier that expedites ER-to-Golgi transport. In addition, depletion of VPS13B or Sec23IP delays ER export of procollagen, suggesting a link between procollagen secretion and joint laxity in patients with Cohen disease. Together, our study reveals a crucial role of VPS13B-Sec23IP interaction at the ERES-Golgi interface in the pathogenesis of Cohen syndrome.