Exploring the Influence of Zinc Ions on the Conformational Stability and Activity of Protein Disulfide Isomerase.

The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.

Tear nanoDSF Denaturation Profile Is Predictive of Glaucoma.

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.

Myotoxin-3 from the Pacific Rattlesnake Crotalus oreganus oreganus Venom Is a New Microtubule-Targeting Agent.

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling.

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.

Zinc Binds to RRM2 Peptide of TDP-43.

Transactive response DNA and RNA binding protein 43 kDa (TDP-43) is a highly conserved heterogeneous nuclear ribonucleoprotein (hnRNP), which is involved in several steps of protein production including transcription and splicing. Its aggregates are frequently observed in motor neurons from amyotrophic lateral sclerosis patients and in the most common variant of frontotemporal lobar degeneration. Recently it was shown that TDP-43 is able to bind Zn2+ by its RRM domain. In this work, we have investigated Zn2+ binding to a short peptide 256-264 from C-terminus of RRM2 domain using isothermal titration calorimetry, electrospray ionization mass spectrometry, QM/MM simulations, and NMR spectroscopy. We have found that this peptide is able to bind zinc ions with a Ka equal to 1.6 × 105 M-1. Our findings suggest the existence of a zinc binding site in the C-terminal region of RRM2 domain. Together with the existing structure of the RRM2 domain of TDP-43 we propose a model of its complex with Zn2+ which illustrates how zinc might regulate DNA/RNA binding.

Sequential binding of calcium ions to the B-repeat domain of SdrD from Staphylococcus aureus.

Biofilms of live bacteria forming on medical devices and implants contribute significantly to bacterial blood dissemination and to the spread of nosocomial infections. Cell surface SdrD protein plays a key role in the attachment of Staphylococcus aureus to the extracellular matrix (ECM) and in the formation of biofilm. SdrD binds calcium ions using its B1-B5 region bearing EF-hand Ca-binding sites, leading to conformational changes in the structure of SdrD. This alters the distance between the bacterial surface and the ECM-interacting domain of SdrD in a spring-like fashion, participating in bacterial attachment. In this study we investigated calcium binding to EF-hand sites of SdrD using isothermal titration calorimetry and determined the impact of this process on SdrD's thermodynamic stability. This allowed us to propose a model of B1-B5 reorganization upon binding of calcium and to get new insight into the molecular mechanism of SdrD's action.

A general framework to characterize inhibitors of calmodulin: use of calmodulin inhibitors to study the interaction between calmodulin and its calmodulin binding domains.

The prominent role of Ca(2+) in cell physiology is mediated by a whole set of proteins involved in Ca(2+)-signal generation, deciphering and arrest. Among these intracellular proteins, calmodulin (CaM) known as a prototypical calcium sensor, serves as a ubiquitous carrier of the intracellular calcium signal in all eukaryotic cell types. CaM is assumed to be involved in many diseases including Parkinson, Alzheimer, and rheumatoid arthritis. Defects in some of many reaction partners of CaM might be responsible for disease symptoms. Several classes of drugs bind to CaM with unwanted side effects rather than specific therapeutic use. Thus, it may be more promising to concentrate at searching for pharmacological interferences with the CaM target proteins, in order to find tools for dissecting and investigating CaM-regulatory and modulatory functions in cells. In the present study, we have established a screening assay based on fluorescence polarization (FP) to identify a diverse set of small molecules that disrupt the regulatory function of CaM. The FP-based CaM assay consists in the competition of two fluorescent probes and a library of chemical compounds for binding to CaM. Screening of about 5300 compounds (Strasbourg Academic Library) by displacement of the probe yielded 39 compounds in a first step, from which 6 were selected. Those 6 compounds were characterized by means of calorimetry studies and by competitive displacement of two fluorescent probes interacting with CaM. Moreover, those small molecules were tested for their capability to displace 8 different CaM binding domains from CaM. Our results show that these CaM/small molecules interactions are not functionally equivalent. The strategy that has been set up for CaM is a general model for the development and validation of other CaM interactors, to decipher their mode of action, or rationally design more specific CaM antagonists. Moreover, this strategy may be used for other protein binding assays intended to screen for molecules with preferred binding activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Properties of small rRNA methyltransferase RsmD: mutational and kinetic study.

Ribosomal RNA modification is accomplished by a variety of enzymes acting on all stages of ribosome assembly. Among rRNA methyltransferases of Escherichia coli, RsmD deserves special attention. Despite its minimalistic domain architecture, it is able to recognize a single target nucleotide G966 of the 16S rRNA. RsmD acts late in the assembly process and is able to modify a completely assembled 30S subunit. Here, we show that it possesses superior binding properties toward the unmodified 30S subunit but is unable to bind a 30S subunit modified at G966. RsmD is unusual in its ability to withstand multiple amino acid substitutions of the active site. Such efficiency of RsmD may be useful to complete the modification of a 30S subunit ahead of the 30S subunit's involvement in translation.

AERS spider: an online interactive tool to mine statistical associations in Adverse Event Reporting System.

BACKGROUND: Exploration of the Adverse Event Reporting System (AERS) data by a wide scientific community is limited due to several factors. First, AERS data must be intensively preprocessed to be converted into analyzable format. Second, application of the currently accepted disproportional reporting measures results in false positive signals. METHODS: We proposed a data mining strategy to improve hypothesis generation with respect to potential associations. RESULTS: By numerous examples, we illustrate that our strategy controls the false positive signals. We implemented a free online tool, AERS spider (www.chemoprofiling.org/AERS). CONCLUSIONS: We believe that AERS spider would be a valuable tool for drug safety experts.

Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process.

A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange.

Plasma nanoDSF Denaturation Profile at Baseline Is Predictive of Glioblastoma EGFR Status.

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Recently, we demonstrated that plasma denaturation profiles of glioblastoma patients obtained using Differential Scanning Fluorimetry can be automatically distinguished from healthy controls with the help of Artificial Intelligence (AI). Here, we used a set of machine-learning algorithms to automatically classify plasma denaturation profiles of glioblastoma patients according to their EGFR status. We found that Adaboost AI is able to discriminate EGFR alterations in GBM with an 81.5% accuracy. Our study shows that the use of these plasma denaturation profiles could answer the unmet neuro-oncology need for diagnostic predictive biomarker in combination with brain MRI and clinical data, in order to allow for a rapid orientation of patients for a definitive pathological diagnosis and then treatment. We complete this study by showing that discriminating another mutation, MGMT, seems harder, and that post-surgery monitoring using our approach is not conclusive in the 48 h that follow the surgery.

NMR solution structure of rat aß(1-16): toward understanding the mechanism of rats' resistance to Alzheimer's disease.

In an attempt to reveal the mechanism of rats' resistance to Alzheimer's disease, we determined the structure of the metal-binding domain 1-16 of rat ß-amyloid (rat Aß(1-16)) in solution in the absence and presence of zinc ions. A zinc-induced dimerization of the domain was detected. The zinc coordination site was found to involve residues His-6 and His-14 of both peptide chains. We used experimental restraints obtained from analyses of NMR and isothermal titration calorimetry data to perform structure calculations. The calculations employed an explicit water environment and a simulated annealing molecular-dynamics protocol followed by quantum-mechanical/molecular-mechanical optimization. We found that the C-tails of the two polypeptide chains of the rat Aß(1-16) dimer are oriented in opposite directions to each other, which hinders the assembly of rat Aß dimers into oligomeric aggregates. Thus, the differences in the structure of zinc-binding sites of human and rat Aß(1-16), their ability to form regular cross-monomer bonds, and the orientation of their hydrophobic C-tails could be responsible for the resistance of rats to Alzheimer's disease.

Identification of the three zinc-binding sites on tau protein.

Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, the precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on tau. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part, the repeat region and the C-terminal part. Further analysis enabled us to show that the N-terminal and the C-terminal sites are independent of each other. Using molecular simulations, we proposed a model of each site in a complex with zinc. Given the clinical importance of zinc in tau aggregation, our findings pave the way for designing potential therapies for tauopathies.

Binding of two zinc ions promotes liquid-liquid phase separation of Tau.

Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc.

APOE4 drives inflammation in human astrocytes via TAGLN3 repression and NF-κB activation.

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD.

Zinc Modulation of Neuronal Calcium Sensor Proteins: Three Modes of Interaction with Different Structural Outcomes.

Neuronal calcium sensors (NCSs) are the family of EF-hand proteins mediating Ca2+-dependent signaling pathways in healthy neurons and neurodegenerative diseases. It was hypothesized that the calcium sensor activity of NCSs can be complemented by sensing fluctuation of intracellular zinc, which could further diversify their function. Here, using a set of biophysical techniques, we analyzed the Zn2+-binding properties of five proteins belonging to three different subgroups of the NCS family, namely, VILIP1 and neurocalcin-δ/NCLD (subgroup B), recoverin (subgroup C), as well as GCAP1 and GCAP2 (subgroup D). We demonstrate that each of these proteins is capable of coordinating Zn2+ with a different affinity, stoichiometry, and structural outcome. In the absence of calcium, recoverin and VILIP1 bind two zinc ions with submicromolar affinity, and the binding induces pronounced conformational changes and regulates the dimeric state of these proteins without significant destabilization of their structure. In the presence of calcium, recoverin binds zinc with slightly decreased affinity and moderate conformational outcome, whereas VILIP1 becomes insensitive to Zn2+. NCALD binds Zn2+ with micromolar affinity, but the binding induces dramatic destabilization and aggregation of the protein. In contrast, both GCAPs demonstrate low-affinity binding of zinc independent of calcium, remaining relatively stable even at submillimolar Zn2+ concentrations. Based on these data, and the results of structural bioinformatics analysis, NCSs can be divided into three categories: (1) physiological Ca2+/Zn2+ sensor proteins capable of binding exchangeable (signaling) zinc (recoverin and VILIP1), (2) pathological Ca2+/Zn2+ sensors responding only to aberrantly high free zinc concentrations by denaturation and aggregation (NCALD), and (3) Zn2+-resistant, Ca2+ sensor proteins (GCAP1, GCAP2). We suggest that NCS proteins may therefore govern the interconnection between Ca2+-dependent and Zn2+-dependent signaling pathways in healthy neurons and zinc cytotoxicity-related neurodegenerative diseases, such as Alzheimer's disease and glaucoma.

An AI-Powered Blood Test to Detect Cancer Using NanoDSF.

Glioblastoma is the most frequent and aggressive primary brain tumor. Its diagnosis is based on resection or biopsy that could be especially difficult and dangerous in the case of deep location or patient comorbidities. Monitoring disease evolution and progression also requires repeated biopsies that are often not feasible. Therefore, there is an urgent need to develop biomarkers to diagnose and follow glioblastoma evolution in a minimally invasive way. In the present study, we described a novel cancer detection method based on plasma denaturation profiles obtained by a non-conventional use of differential scanning fluorimetry. Using blood samples from 84 glioma patients and 63 healthy controls, we showed that their denaturation profiles can be automatically distinguished with the help of machine learning algorithms with 92% accuracy. Proposed high throughput workflow can be applied to any type of cancer and could become a powerful pan-cancer diagnostic and monitoring tool requiring only a simple blood test.

Mechanism of Zn2+ and Ca2+ Binding to Human S100A1.

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 µm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.

Minimal Zn(2+) binding site of amyloid-ß.

Zinc-induced aggregation of amyloid-ß peptide (Aß) is a hallmark molecular feature of Alzheimer's disease. Here we provide direct thermodynamic evidence that elucidates the role of the Aß region 6-14 as the minimal Zn(2+) binding site wherein the ion is coordinated by His(6), Glu(11), His(13), and His(14). With the help of isothermal titration calorimetry and quantum mechanics/molecular mechanics simulations, the region 11-14 was determined as the primary zinc recognition site and considered an important drug-target candidate to prevent Zn(2+)-induced aggregation of Aß.

Stoichiometry of irreversible ligand binding to a one-dimensional lattice.

In this paper we investigate the problem of irreversible binding of a ligand that covers several identical binding sites on a macromolecule with a one-dimensional lattice. Due to steric constraints, irreversible binding or binding with slow kinetics results in partial saturation of the binding sites thus impacting the stoichiometry of the interaction. Here we present a recursive formula to calculate the exact fraction of the occupied binding sites for a ligand and macromolecule of arbitrary lengths. We also provide an analytical result for the exact fraction of the occupied sites in case of an infinitely long lattice. We conclude with a simplified empirical formula for the exact fraction of the occupied sites in case of an infinitely long lattice.