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Rapid Analysis of NAD and Other Phosphorylated Metabolites in Complex Biological Samples by Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry
Abstract
Nucleotides and coenzymes play critical roles in energy metabolism and cellular signaling and as building blocks of nucleic acids. This work addresses the challenges in the measurement of the phosphorylated metabolites using hydrophilic interaction liquid chromatography coupled with mass spectrometry, which facilitates the separation and detection of polar metabolites. Here, we present optimized HILIC-MS/MS methods for rapid analysis of polar metabolites including nucleotides and their derivatives in complex biological matrices, such as murine adipose, skeletal, and liver tissues, human plasma, and bacteria. The developed methodologies enable separation of key nucleotides and other phosphorylated metabolites within 6 min and cofactors such as NAD+, NADH, NADP+, and NADPH within 4 min. Validation of these methods demonstrated high accuracy, precision, and sensitivity and stresses the substantial impact of matrix effects. The applicability of the methods was also tested on 13C-labeling experiments with mouse pluripotent stem cells. Additionally, sample pretreatment techniques, such as liquid-liquid extraction and solid-phase extraction, were evaluated as a tool to decrease the negative impact of matrix effects in complex samples. This work enhances the analytical capabilities for nucleotide quantification in metabolomics, facilitating the study of metabolic pathways and disease markers.
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Engineered Bacterial Ligand From Streptococcus pneumoniae on Macroporous Resins for Selective Affinity Capture of Secretory IgA
Abstract
Secretory immunoglobulin A (sIgA) is a promising emerging biopharmaceutical candidate, but it currently lacks a standardized platform for purification. To address this, a novel affinity chromatography resin was developed by immobilizing protein variants of the Streptococcus pneumoniae surface protein, SpsA, as an affinity ligand. SpsA targets the secretory component of sIgA, which increases selectivity and minimizes co-purification of product-related impurities. Directed attachment between a single cysteine residue and macroporous epoxy-activated supports was employed as an immobilization strategy. Ligand orientation on the resin surface is of utmost importance, as the initial SpsA variant was unable to bind sIgA after immobilization. The cysteine residue was subsequently relocated from the C-terminus to either the N-terminus or loop region of the ligand, resulting in a functional resin. 177 nmol SpsA/mL resin were immobilized on the agarose-based resin, resulting in an equilibrium binding capacity of 9.1 mg sIgA/mL resin. SpsA resin was able to selectively purify sIgA from cell culture supernatant under dynamic conditions with free secretory component as the only product-related impurity. SpsA-based affinity chromatography may enable a scalable purification process for sIgA through an appropriate resin structure and improved selectivity.
Keywords: Affinity chromatography; downstream processing; ligand immobilization; resin development; secretory antibodies.
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Structural characterization of the Cu(II)-NTA spin label on α-helices by X-ray crystallography and electron paramagnetic resonance
Abstract
Site-directed Cu(II)-labelling in pulsed electron paramagnetic resonance (EPR) spectroscopy has demonstrated narrow Cu(II)-Cu(II) distance distributions suitable to resolve subtle protein conformational changes. The high precision derives from a double histidine (dHis) mutation that effectively locks a Cu(II)-nitrilotriacetic acid (Cu(II)-NTA) moiety in place. To date, no structures featuring the dHis-Cu(II)-NTA motif have been resolved. This work presents the atomic-resolution X-ray crystal structures of seven α-helical dHis sites of T4 lysozyme (T4L) in the presence and absence of Cu(II)-NTA. Our research captured the rigid octahedral coordination of the dHis-Cu(II)-NTA complex as well as non-conventional binding modes, which provide valuable insight into dHis site selection. Pulsed EPR experiments on double dHis T4L mutants displayed remarkable agreement to the crystallography-derived distances. This research showcases the rigid configuration of the dHis-Cu(II)-NTA motif, providing geometric constraints that can be leveraged in modeling and molecular dynamics programs to extract protein structural details from EPR experiments.
Keywords: Cu(II)-NTA; EPR; RIDME; X-ray crystallography; distance distributions; electron paramagnetic resonance; geometry constraints; pulsed dipolar EPR spectroscopy; relaxation-induced dipolar modulation enhancement; spin label; structural biology.
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Tetravalent antibodies are more potent and efficacious erythropoiesis-stimulating agents than erythropoietin in vivo
Abstract
Recent studies have shown that tetravalent antibodies are potent and efficacious agonists of the erythropoietin (EPO) receptor (EPOR) both in vitro and in vivo. To identify antibody-based erythropoiesis-stimulating agents (ESAs) with therapeutic potential, we evaluated various tetravalent antibody formats for EPOR agonism and key biophysical properties necessary for biologic drug development. We identified two distinct tetravalent antibody formats that strongly stimulated the growth of UT7/Epo cells, which rely on EPOR signaling for proliferation. Moreover, one of these formats exhibited ideal biophysical characteristics for drug development. This format consisted of a diabody (Db) and two antigen-binding fragment (Fab) arms fused to the N- and C-termini of an Fc domain, respectively, to form a tetravalent Db-Fc-Fab (EPRA-0322). In a mouse model expressing the human EPOR, EPRA-0322 induced erythropoiesis with greater potency, efficacy, and duration than darbepoetin, a hyperglycosylated EPO currently used in clinical practice. These findings highlight tetravalent antibodies, and the Db-Fc-Fab format in particular, as promising next-generation ESAs suitable for large-scale production and clinical use.
Keywords: EPO; EPOR; agonist; antibody; antibody polymer; diabody; dimer; drug design; erythropoiesis; helical ultrastructure; protein engineering.
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AbDesign: database of point mutants of antibodies with associated structures reveals poor generalization of binding predictions from machine learning models
Abstract
Antibodies are naturally evolved molecular recognition scaffolds that can bind a variety of surfaces. Their designability is crucial to the development of biologics, with computational methods holding promise in accelerating the delivery of medicines to the clinic. Modeling antibody-antigen recognition is prohibitively difficult, with data paucity being one of the biggest hurdles. Current affinity datasets comprise a small number of experimental measurements, which are often not standardized between molecules. Here, we address these issues by creating a dataset of seven antigens with two antibodies each, for which we introduce a heterogeneous set of mutations to the CDR-H3 measured by ELISA. Each of the parental complexes has a known crystal structure. We perform benchmarking of state-of-the-art affinity prediction algorithms to gauge their effectiveness. Current computational methods exhibit substantial limitations in accurately predicting the effects of single-point mutations. In contrast, the older empirical, physics-based method FoldX performs well in identifying mutants that retain binding. These findings highlight the need for more resources like the one presented here, i.e. large, molecularly diverse, and experimentally consistent datasets.
Keywords: Affinity maturation; database; machine learning; protein design.
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Allostery Links hACE2 Binding, Pan-variant Neutralization and Helical Extension in the SARS-CoV-2 Spike Protein
Abstract
The SARS-CoV-2 spike protein is highly antigenic, with epitopes in three distinct regions of the receptor binding domain (RBD) alone that have known mechanisms of neutralization. In previous work, we predicted a fourth RBD epitope based on allosteric conformational perturbations measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS) upon complexation with the canonical spike protein target, human angiotensin-converting enzyme 2 (hACE2). We subsequently identified a pan-neutralizing antibody (ICO-hu104) with the predicted epitope, however, as the epitope was somewhat distant from the hACE2 binding interface, and our previous work limited to the spike RBD, the neutralization mechanism was unclear. Using HDX-MS, we investigated the binding of ICO-hu104 to the full-length SARS-CoV-2 spike protein from Wuhan, Delta and Omicron variants. We demonstrate that binding of ICO-hu104 results in an increase in deuterium uptake in the distant HR1 domain in latter variants, which in a biological context could be indicative of destabilisation of the helices within this region, promoting premature S1 shedding or failure of helical extension during S2-mediated fusion. This is supported by our computational modelling, highlighting propagation of allosteric effects to the S2 coiled-coil region upon rigidification of the ICO-hu104 epitope. Collectively, this work demonstrates an alternative neutralization mechanism for ICO-hu104 which is distinct from its first-generation predecessors and thus opens alternative avenues targeting non-RBD epitopes through allosteric perturbations.
Keywords: SARS-CoV-2; antibody; epitope; hydrogen-deuterium exchange; spike protein.
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A conserved phosphorylation mechanism for regulating the interaction between the CMG replicative helicase and its forked DNA substrate.
Sandra Koit, Nele Tamberg, Allan Reinapae, Lauri Peil, Arnold Kristjuhan,
AbstractThe CMG helicase is a crucial enzyme complex that plays a vital role in the replication of genomic DNA in eukaryotes. Besides unwinding the DNA template and coordinating the replisome’s structure, it is also a key target for signaling pathways that regulate the replication process. We show that a specific serine/threonine residue in the MCM3 subunit of CMG, which has been previously linked to phosphorylation-dependent control mechanisms of genomic DNA replication in human cells, is a conserved phosphorylation site for Chk1 and potentially other protein kinases. This suggests a conserved regulatory mechanism associated with it in metazoans and several other eukaryotes, including budding yeast. Our in vitro analysis links this mechanism directly to the modulation of the CMG helicase activity by impacting its interactions with the forked DNA substrate. Further supporting its conserved role in regulation, we found that phosphomimetic substitution with aspartic acid and alanine knock-out of this conserved residue lead to opposite phenotypic defects in the growth of budding yeast cells. These findings outline a candidate conserved phosphorylation pathway for regulating genomic DNA replication in eukaryotes, which adjusts the interactions between the replicative helicase complex and its DNA substrate according to the specific needs of various physiological conditions.
Keywords: Eukaryote, DNA replication, DNA helicase, checkpoint control, protein phosphorylation.
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Suppressing Tymovirus replication in plants using a variant of ubiquitin
AbstractRNA viruses have evolved numerous strategies to overcome host resistance and immunity, including the use of multifunctional proteases that not only cleave viral polyproteins during virus replication but also deubiquitinate cellular proteins to suppress ubiquitin (Ub)-mediated antiviral mechanisms. Here, we report an approach to attenuate the infection of Arabidopsis thaliana by Turnip Yellow Mosaic Virus (TYMV) by suppressing the polyprotein cleavage and deubiquitination activities of the TYMV protease (PRO). Performing selections using a library of phage-displayed Ub variants (UbVs) for binding to recombinant PRO yielded several UbVs that bound the viral protease with nanomolar affinities and blocked its function. The strongest binding UbV (UbV3) candidate had a EC50 of 0.3 nM and inhibited both polyprotein cleavage and DUB activity of PRO in vitro. X-ray crystal structures of UbV3 alone and in complex with PRO reveal that the inhibitor exists as a dimer that binds two copies of PRO. Consistent with our biochemical and structural findings, transgenic expression of UbV3 in the cytosol of A. thaliana suppressed TYMV replication in planta, with the reduction in viral load being correlated to UbV3 expression level. Our results demonstrate the potential of using UbVs to protect plants from tymovirus infection, a family of viruses that contain numerous members of significant agricultural concern, as well as other plant viruses that express functionally related proteases with deubiquitinating activity.
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Anti-SARS-CoV-2 antibodies in a nasal spray efficiently block viral transmission between ferrets.
Gildemann K, Tsernant ML, Liivand L, Ennomäe R, Poikalainen V, Lepasalu L, Rom S, Kavak A, Cox RM, Wolf JD, Lieber CM, Plemper RK, Männik A, Ustav M Jr, Ustav M, Gerhold JM.
AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread in the population. We recently reported the production of bovine colostrum-derived antibodies that can neutralize the virus. These have been formulated into a nasal spray. The immunoglobulin preparation is capable of blocking interaction of the trimeric spike protein (Tri S) of SARS-CoV-2 with the cellular receptor angiotensin-converting enzyme 2 (ACE2), entry of a pseudovirus carrying the Tri S into ACE2 over-expressing human embryonic kidney (HEK) cells, and entry of the virus into live Vero E6 cells. Using an ELISA assay, we demonstrate here that this holds true for different SARS-CoV-2 variants of concern. Using the ferret transmission model, we show that the nasal spray formulation of anti-SARS-CoV-2 immunoglobulins efficiently blocks transmission of SARS-CoV-2 from infected to uninfected ferrets. The results indicate that the use of the nasal spray in humans can add an effective additional layer of protection against the virus, and might be applicable for other viruses of the upper respiratory tract.
Keywords: Immunology; Virology.
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Artesunate treats obesity in male mice and non-human primates through GDF15/GFRAL signalling axis.
Guo X, Asthana P, Zhai L, Cheng KW, Gurung S, Huang J, Wu J, Zhang Y, Mahato AK, Saarma M, Ustav M, Kwan HY, Lyu A, Chan KM, Xu P, Bian ZX, Wong HLX.
AbstractObesity, a global health challenge, is a major risk factor for multiple life-threatening diseases, including diabetes, fatty liver, and cancer. There is an ongoing need to identify safe and tolerable therapeutics for obesity management. Herein, we show that treatment with artesunate, an artemisinin derivative approved by the FDA for the treatment of severe malaria, effectively reduces body weight and improves metabolic profiles in preclinical models of obesity, including male mice with overnutrition-induced obesity and male cynomolgus macaques with spontaneous obesity, without inducing nausea and malaise. Artesunate promotes weight loss and reduces food intake in obese mice and cynomolgus macaques by increasing circulating levels of Growth Differentiation Factor 15 (GDF15), an appetite-regulating hormone with a brainstem-restricted receptor, the GDNF family receptor α-like (GFRAL). Mechanistically, artesunate induces the expression of GDF15 in multiple organs, especially the liver, in mice through a C/EBP homologous protein (CHOP)-directed integrated stress response. Inhibition of GDF15/GFRAL signalling by genetic ablation of GFRAL or tissue-specific knockdown of GDF15 abrogates the anti-obesity effect of artesunate in mice with diet-induced obesity, suggesting that artesunate controls bodyweight and appetite in a GDF15/GFRAL signalling-dependent manner. These data highlight the therapeutic benefits of artesunate in the treatment of obesity and related comorbidities.
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Structural and functional validation of a highly specific Smurf2 inhibitor.
Tessier TM, Chowdhury A, Stekel Z, Fux J, Sartori MA, Teyra J, Jarvik N, Chung J, Kurinov I, Sicheri F, Sidhu SS, Singer AU, Zhang W.
AbstractSmurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross-reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein-based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2-E3 protein interaction interface.
Keywords: E3 ligases; HECT domain; crystal structure; inhibitor; phage display; protein engineering; ubiquitin variants.
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