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Nature Communications Apr 2024Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural...
Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa-5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20-100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.
Topics: Cross-Linking Reagents; Protein Denaturation; Photometry; Proteins; Electrophoresis, Polyacrylamide Gel; Protein Interaction Mapping; Mass Spectrometry; Humans
PubMed: 38664367
DOI: 10.1038/s41467-024-47732-4 -
BioRxiv : the Preprint Server For... Apr 2024Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on...
Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP and RNA (TSAR) as a template for proteome-wide discovery of substrate-dependent ligand binding. Using proteomic thermal shift assays, we show that simple biochemical additives can facilitate detection of target engagement in native cell lysates. We apply our approach to rocaglates, a family of molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) family of RNA helicases. To identify unexpected interactions, we optimized a target class-specific thermal denaturation window and evaluated ATP analog and RNA probe dependencies for key rocaglate-DDX interactions. We report novel DDX targets of the rocaglate clamping spectrum, confirm that DDX3X is a common target of several widely studied analogs, and provide structural insights into divergent DDX3X affinities between synthetic rocaglates. We independently validate novel targets of high-profile rocaglates, including the clinical candidate Zotatifin (), using limited proteolysis-mass spectrometry and fluorescence polarization experiments. Taken together, our study provides a model for screening uncompetitive inhibitors using a systematic chemical-proteomics approach to uncover actionable DDX targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicase targets.
PubMed: 38659867
DOI: 10.1101/2024.04.19.590252 -
Scientific Reports Apr 2024Fluorinated graphene, a two-dimensional nanomaterial composed of three atomic layers, a central carbon layer sandwiched between two layers of fluorine atoms, has...
Fluorinated graphene, a two-dimensional nanomaterial composed of three atomic layers, a central carbon layer sandwiched between two layers of fluorine atoms, has attracted considerable attention across various fields, particularly for its potential use in biomedical applications. Nonetheless, scant effort has been devoted to assessing the potential toxicological implications of this nanomaterial. In this study, we scrutinize the potential impact of fluorinated graphene on a protein model, HP35 by utilizing extensive molecular dynamics (MD) simulation methods. Our MD results elucidate that upon adsorption to the nanomaterial, HP35 undergoes a denaturation process initiated by the unraveling of the second helix of the protein and the loss of the proteins hydrophobic core. In detail, substantial alterations in various structural features of HP35 ensue, including alterations in hydrogen bonding, Q value, and RMSD. Subsequent analyses underscore that hydrophobic and van der Waals interactions (predominant), alongside electrostatic energy (subordinate), exert influence over the adsorption of HP35 on the fluorinated graphene surface. Mechanistic scrutiny attests that the unrestrained lateral mobility of HP35 on the fluorinated graphene nanomaterial primarily causes the exposure of HP35's hydrophobic core, resulting in the eventual structural denaturation of HP35. A trend in the features of 2D nanostructures is proposed that may facilitate the denaturation process. Our findings not only substantiate the potential toxicity of fluorinated graphene but also unveil the underlying molecular mechanism, which thereby holds significance for the prospective utilization of such nanomaterials in the field of biomedicine.
Topics: Graphite; Molecular Dynamics Simulation; Protein Conformation, alpha-Helical; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Protein Unfolding; Halogenation; Adsorption; Nanostructures; Peptide Fragments; Neurofilament Proteins
PubMed: 38649777
DOI: 10.1038/s41598-024-59780-3 -
BioRxiv : the Preprint Server For... Apr 2024Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies....
Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-minute and 1-hour postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.
PubMed: 38645137
DOI: 10.1101/2023.11.20.567854 -
Journal of Hazardous Materials Jun 2024Alachlor, a widely used chloroacetanilide herbicide for controlling annual grasses in crops, has been reported to rapidly trigger protein denaturation and aggregation in...
Alachlor, a widely used chloroacetanilide herbicide for controlling annual grasses in crops, has been reported to rapidly trigger protein denaturation and aggregation in the eukaryotic model organism Saccharomyces cerevisiae. Therefore, this study aimed to uncover cellular mechanisms involved in preventing alachlor-induced proteotoxicity. The findings reveal that the ubiquitin-proteasome system (UPS) plays a crucial role in eliminating alachlor-denatured proteins by tagging them with polyubiquitin for subsequent proteasomal degradation. Exposure to alachlor rapidly induced an inhibition of proteasome activity by 90 % within 30 min. The molecular docking analysis suggests that this inhibition likely results from the binding of alachlor to β subunits within the catalytic core of the proteasome. Notably, our data suggest that nascent proteins in the endoplasmic reticulum (ER) are the primary targets of alachlor. Consequently, the unfolded protein response (UPR), responsible for coping with aberrant proteins in the ER, becomes activated within 1 h of alachlor treatment, leading to the splicing of HAC1 mRNA into the active transcription activator Hac1p and the upregulation of UPR gene expression. These findings underscore the critical roles of the protein quality control systems UPS and UPR in mitigating alachlor-induced proteotoxicity by degrading alachlor-denatured proteins and enhancing the protein folding capacity of the ER.
Topics: Saccharomyces cerevisiae; Acetamides; Herbicides; Endoplasmic Reticulum; Unfolded Protein Response; Saccharomyces cerevisiae Proteins; Proteasome Endopeptidase Complex; Cytosol; Molecular Docking Simulation; Proteotoxic Stress
PubMed: 38640676
DOI: 10.1016/j.jhazmat.2024.134270 -
Frontiers in Pharmacology 2024is an evergreen ornamental small fern, belonging to the family Pteridaceae, that grows in warm and rocky regions worldwide. Many species of genus are evidently...
is an evergreen ornamental small fern, belonging to the family Pteridaceae, that grows in warm and rocky regions worldwide. Many species of genus are evidently endowed with important phytochemicals and bioactivities. This study aimed to perform a preliminary phytochemical analysis of leaves alongside an evaluation of free radical scavenging, anti-inflammatory, antimicrobial, and clot lysis activities of extract fractions. A preliminary phytochemical analysis was done after fractionation of ethanolic extract (ECT) with -hexane (HCT) and chloroform (CCT). Then, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, egg albumin and RBC membrane stabilization tests, disc diffusion, and human blood clot lysis assays were performed. Phytochemical investigations suggested that the plant is rich in alkaloids, glycosides, tannins, and flavonoids. All obtained fractions exhibited concentration-dependent radical scavenging, inhibition of egg protein denaturation and RBC membrane lysis capacities. Except for antifungal tests, ECT exhibited better DPPH radical scavenging, anti-inflammatory, antibacterial, and clot lysis capacities than HCT and CCT fractions. However, all fractions exhibited a mild anti-inflammatory activity. might be a good source of antioxidant, anti-microbial, and anti-atherothrombotic agents. Further studies are required to isolate and characterize the active principles liable for each bioactivity, along with possible molecular interactions.
PubMed: 38638865
DOI: 10.3389/fphar.2024.1366889 -
Analytica Chimica Acta May 2024The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of...
Identification of heavily glycated proteoforms by hydrophilic-interaction liquid chromatography and native size-exclusion chromatography - High-resolution mass spectrometry.
BACKGROUND
The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of diseases and biopharmaceutical storage. The characterization of heavily glycated proteins at the intact level is of high interest as it allows to describe co-occurring protein modifications. However, the high heterogeneity of glycated protein makes this process challenging, and novel methods are required to accomplish this.
RESULTS
In this study, we investigated two novel LC-HRMS methods to study glycated reference proteins at the intact protein level: low-flow hydrophilic-interaction liquid chromatography (HILIC) and native size-exclusion chromatography (SEC). Model proteins were exposed to conditions that favored extensive glycation and the formation of AGEs. After glycation, complicated MS spectra were observed, along with a sharply reduced signal response, possibly due to protein denaturation and the formation of aggregates. When using HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides. Moreover, some positional glycated isomers were separated. The SEC-MS method under non-denaturing conditions provided insights into glycated aggregates but offered only a limited separation of glycated species based on molar mass. Overall, more than 25 different types of species were observed in both methods, differing in molar mass by 14-162 Da. 19 of these species have not been previously reported.
SIGNIFICANCE
The proposed strategies show great potential to characterize highly glycated intact proteins from native and denaturing perspectives and provide new opportunities for fast clinical diagnoses and investigating glycation-related diseases.
Topics: Mass Spectrometry; Chromatography, Liquid; Proteins; Chromatography, Gel; Protein Processing, Post-Translational
PubMed: 38637052
DOI: 10.1016/j.aca.2024.342543 -
Zeitschrift Fur Naturforschung. C,... Apr 2024Pancreatic cancer is a fatal illness caused by mutations in multiple genes. Pancreatic cancer damages the organ that helps in digestion, resulting in symptoms including...
Pancreatic cancer is a fatal illness caused by mutations in multiple genes. Pancreatic cancer damages the organ that helps in digestion, resulting in symptoms including fatigue, bloating, and nausea. The use of medicinal plants has been crucial in the treatment of numerous disorders. The medicinal plant has been widely exploited for its possibilities in biology and medicine. The current study aimed to assess the biopotential of biologically active substances against pancreatic cancer. The GC-MS data of these phytochemicals from were further subjected to computational approaches with pancreatic cancer genes to evaluate their potential as therapeutic candidates. Molecular docking analysis revealed that N-[Carboxymethyl] maleamic acid is the leading molecule responsible for protein denaturation inhibition, having the highest binding affinity of 6.8 kJ/mol among all other compounds with inflammatory proteins. Furthermore, ADMET analysis and Lipinski's rule validation were also performed revealing its higher absorption in the gastrointestinal tract. The results of the hepatotoxicity test demonstrated that phytochemicals are non-toxic, safe to use, and do not cause necrosis, fibrosis, or vacuolar degeneration even at excessive levels. has phytoconstituents that have a variety of pharmacological uses in consideration.
PubMed: 38635803
DOI: 10.1515/znc-2024-0057 -
Cureus Mar 2024Background Currently, nanotechnology is a rapidly advancing field of research. Because of their nanoscale dimensions, nanoparticles (NPs) find application in a wide...
Background Currently, nanotechnology is a rapidly advancing field of research. Because of their nanoscale dimensions, nanoparticles (NPs) find application in a wide range of industries, including engineering and medicine. The leaves of have anti-inflammatory qualities. The purpose of this study was to create SrO NPs isolated from the leaves of aqueous extract and to evaluate their anti-inflammatory efficacy. The . saltmarsh, commonly known as South-Indian Seepweed, is a mangrove-associated plant and has been used as traditional medicine for decades with multifunctional biological activity. Objectives The aim of our study is to biosynthesize strontium oxide NPs from saltmarsh and to see whether they have any anti-inflammatory properties. Materials and methods In the present study, the pharmacological significance was studied using crude extract and synthesized SrO NPs from The synthesized SrO NPs were characterized using UV spectrophotometry. The anti-inflammatory assay was analyzed using egg albumin denaturation. SrO NPs' peak observance was found at 630 nm, and a graph was plotted for the zone of inhibition vs concentration and compared with the standard. Results It was observed that the color of the SrO NPs deepened during the synthesis process. Furthermore, at a wavelength of 630 nm, the UV spectrum analysis showed a noteworthy absorption value of 1.4. The activity of inflammatory enzymes is significantly impacted by the anti-inflammatory properties of SrO NPs in the protein denaturation inhibition test. Conclusions The application of SrO NPs in the synthesis process has the potential to enhance the anti-inflammatory activity of as evidenced by the observed increase in anti-inflammatory capacity and defense against infections and injury.
PubMed: 38633965
DOI: 10.7759/cureus.56355 -
RSC Advances Apr 2024The two disaccharides, trehalose and sucrose, have been compared in many studies due to their structural similarity. Both possess the ability to stabilise and reduce...
The two disaccharides, trehalose and sucrose, have been compared in many studies due to their structural similarity. Both possess the ability to stabilise and reduce aggregation of proteins. Trehalose has also been shown to inhibit the formation of highly structured protein aggregates called amyloid fibrils. This study aims to compare how the thermal stability of the protein lysozyme at low pH (2.0 and 3.5) is affected by the presence of the two disaccharides. We also address the anti-aggregating properties of the disaccharides and their inhibitory effects on fibril formation. Differential scanning calorimetry confirms that the thermal stability of lysozyme is increased by the presence of trehalose or sucrose. The effect is slightly larger for sucrose. The inhibiting effects on protein aggregation are investigated using small-angle X-ray scattering which shows that the two-component system consisting of lysozyme and water (Lys/HO) at pH 2.0 contains larger aggregates than the corresponding system at pH 3.5 as well as the sugar containing systems. In addition, the results show that the particle-to-particle distance in the sugar containing systems (Lys/Tre/HO and Lys/Suc/HO) at pH 2.0 is longer than at pH 3.5, suggesting larger protein aggregates in the former. Finally, the characteristic distance separating β-strands in amyloid fibrils is observed for the Lys/HO system at pH 2.0, using wide-angle X-ray scattering, while it is not clearly observed for the sugar containing systems. This study further shows that the two disaccharides stabilise the native fold of lysozyme by increasing the denaturation temperature. However, other factors, such as a weakening of hydrophobic interactions and hydrogen bonding between proteins, might also play a role in their inhibitory effect on amyloid fibril formation.
PubMed: 38623289
DOI: 10.1039/d4ra01171f