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Journal of Inorganic Biochemistry Jun 2024Over the last 50 years resonance Raman spectroscopy has become an invaluable tool for the exploration of chromophores in biological macromolecules. Among them, heme... (Review)
Review
Over the last 50 years resonance Raman spectroscopy has become an invaluable tool for the exploration of chromophores in biological macromolecules. Among them, heme proteins and metal complexes have attracted considerable attention. This interest results from the fact that resonance Raman spectroscopy probes the vibrational dynamics of these chromophores without direct interference from the surrounding. However, the indirect influence via through-bond and through-space chromophore-protein interactions can be conveniently probed and analyzed. This review article illustrates this point by focusing on class 1 cytochrome c, a comparatively simple heme protein generally known as electron carrier in mitochondria. The article demonstrates how through selective excitation of resonance Raman active modes information about the ligation, the redox state and the spin state of the heme iron can be obtained from band positions in the Raman spectra. The investigation of intensities and depolarization ratios emerged as tools for the analysis of in-plane and out-of-plane deformations of the heme macrocycle. The article further shows how resonance Raman spectroscopy was used to characterize partially unfolded states of oxidized cytochrome c. Finally, it describes its use for exploring structural changes due to the protein's binding to anionic surfaces like cardiolipin containing membranes.
PubMed: 38901065
DOI: 10.1016/j.jinorgbio.2024.112641 -
Journal of the Science of Food and... Jun 2024Walnut protein (WP) is recognized as a valuable plant protein. However, the poor solubility and functional properties limit its application in the food industry. It is a...
BACKGROUND
Walnut protein (WP) is recognized as a valuable plant protein. However, the poor solubility and functional properties limit its application in the food industry. It is a great requirement to improve the physicochemical properties of WP.
RESULTS
Following a 90 min restricted enzymatic hydrolysis period, the solubility of WP significantly increased from 3.24% to 54.54%, with the majority of WP hydrolysates (WPHs) possessing a molecular weight exceeding 50 kDa. Circular dichroism spectra showed that post-hydrolysis, the structure of the protein became more flexible, while the hydrolysis time did not significantly alter the protein's secondary structure. After hydrolysis, WP's surface hydrophobicity significantly increased from 2279 to 6100. Furthermore, WPHs exhibited a strong capacity for icariin loading and micelle formation with critical micelle concentration values of 0.71, 0.99 and 1.09 mg mL, respectively. Moreover, similar immuno-enhancement activities were observed in WPHs. After exposure to WPHs, the pinocytosis of RAW264.7 macrophages was significantly improved. WPH treatment also increased the production of nitric oxide, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in macrophages. Up-regulation of mRNA expressions of IL-6, inducible nitric oxide synthase (iNOS) and TNF-α was observed in a dose-dependent manner.
CONCLUSION
The enhancement of functionality and bioactivity in WP can be achieved through the application of limited enzyme digestion with trypsin. This process effectively augments the nutritional value and utility of the protein, making it a valuable component in various dietary applications. © 2024 Society of Chemical Industry.
PubMed: 38899487
DOI: 10.1002/jsfa.13666 -
BioRxiv : the Preprint Server For... Jun 2024Proteins are dynamic systems whose structural preferences determine their function. Unfortunately, building atomically detailed models of protein structural ensembles...
Proteins are dynamic systems whose structural preferences determine their function. Unfortunately, building atomically detailed models of protein structural ensembles remains challenging, limiting our understanding of the relationships between sequence, structure, and function. Combining single molecule Förster resonance energy transfer (smFRET) experiments with molecular dynamics simulations could provide experimentally grounded, all-atom models of a protein's structural ensemble. However, agreement between the two techniques is often insufficient to achieve this goal. Here, we explore whether accounting for important experimental details like averaging across structures sampled during a given smFRET measurement is responsible for this apparent discrepancy. We present an approach to account for this time-averaging by leveraging the kinetic information available from Markov state models of a protein's dynamics. This allows us to accurately assess which timescales are averaged during an experiment. We find this approach significantly improves agreement between simulations and experiments in proteins with varying degrees of dynamics, including the well-ordered protein T4 lysozyme, the partially disordered protein apolipoprotein E (ApoE), and a disordered amyloid protein (Aβ40). We find evidence for hidden states that are not apparent in smFRET experiments because of time averaging with other structures, akin to states in fast exchange in NMR, and evaluate different force fields. Finally, we show how remaining discrepancies between computations and experiments can be used to guide additional simulations and build structural models for states that were previously unaccounted for. We expect our approach will enable combining simulations and experiments to understand the link between sequence, structure, and function in many settings.
PubMed: 38895430
DOI: 10.1101/2024.06.03.597137 -
BioRxiv : the Preprint Server For... Jun 2024Knowledge of the structures formed by proteins and small ligands is of fundamental importance for understanding molecular principles of chemotherapy and for designing...
Knowledge of the structures formed by proteins and small ligands is of fundamental importance for understanding molecular principles of chemotherapy and for designing new and more effective drugs. Due to the still high costs and to the several limitations of experimental techniques, it is most often desirable to predict these ligand-protein complexes , particularly when screening for new putative drugs from databases of millions of compounds. While virtual screening based on molecular docking is widely used for this purpose, it generally fails in mimicking binding events associated with large conformational changes in the protein, particularly when the latter involve multiple domains. In this work, we describe a new methodology aimed at generating bound-like conformations of very flexible and allosteric proteins bearing multiple binding sites. Validation was performed on the enzyme adenylate kinase (ADK), a paradigmatic example of proteins that undergo very large conformational changes upon ligand binding. By only exploiting the unbound structure and the putative binding sites of the protein, we generated a significant fraction of bound-like structures, which employed in ensemble-docking calculations allowed to find native-like poses of substrates, inhibitors, and catalytically incompetent binders. Our protocol provides a general framework for the generation of bound-like conformations of flexible proteins that are suitable to host different ligands, demonstrating high sensitivity to the fine chemical details that regulate protein's activity. We foresee applications in virtual screening for difficult targets, prediction of the impact of amino acid mutations on structure and dynamics, and protein engineering.
PubMed: 38895346
DOI: 10.1101/2024.06.02.597018 -
Nutrients May 2024This study examined whey protein's impact on insulin resistance in a high-fat diet-induced pediatric obesity mouse model. Pregnant mice were fed high-fat diets, and male...
This study examined whey protein's impact on insulin resistance in a high-fat diet-induced pediatric obesity mouse model. Pregnant mice were fed high-fat diets, and male pups continued this diet until 8 weeks old, then were split into high-fat, whey, and casein diet groups. At 12 weeks old, their body weight, fasting blood glucose (FBG), blood insulin level (IRI), homeostatic model assessment for insulin resistance (HOMA-IR), liver lipid metabolism gene expression, and liver metabolites were compared. The whey group showed significantly lower body weight than the casein group at 12 weeks old ( = 0.034). FBG was lower in the whey group compared to the high-fat diet group ( < 0.01) and casein group ( = 0.058); IRI and HOMA-IR were reduced in the whey group compared to the casein group ( = 0.02, < 0.01, < 0.01, respectively). The levels of peroxisome proliferator-activated receptor α and hormone-sensitive lipase were upregulated in the whey group compared to the casein group ( < 0.01, = 0.03). Metabolomic analysis revealed that the levels of taurine and glycine, both known for their anti-inflammatory and antioxidant properties, were upregulated in the whey group in the liver tissue ( < 0.01, < 0.01). The intake of whey protein was found to improve insulin resistance in a high-fat diet-induced pediatric obesity mouse model.
Topics: Animals; Whey Proteins; Insulin Resistance; Diet, High-Fat; Male; Mice; Disease Models, Animal; Pediatric Obesity; Liver; Female; Blood Glucose; Insulin; Lipid Metabolism; Pregnancy; Mice, Inbred C57BL
PubMed: 38892554
DOI: 10.3390/nu16111622 -
International Journal of Molecular... Jun 2024Vitamin D-binding protein (DBP), also known as Gc-globulin, is a protein that affects several physiological processes, including the transport and regulation of vitamin... (Review)
Review
Vitamin D-binding protein (DBP), also known as Gc-globulin, is a protein that affects several physiological processes, including the transport and regulation of vitamin D metabolites. Genetic polymorphisms in the gene have a significant impact on vitamin D levels and may have implications for disease risk. polymorphisms are linked to differential immune responses, which could influence the onset of juvenile diseases. This narrative review examines the various roles of DBP, with a focus on bone health, immunological regulation, and lipid metabolism in children. Chronic disorders affected by polymorphisms include bone abnormalities, autoimmune diseases, cardiovascular issues, childhood asthma, allergies, cystic fibrosis, acute liver failure, celiac disease, inflammatory bowel disease, and chronic kidney disease. Future research should focus on identifying the processes that underpin the many roles that DBP plays and developing customized therapeutics to improve health outcomes in the juvenile population.
Topics: Humans; Vitamin D-Binding Protein; Child; Child Health; Vitamin D; Lipid Metabolism; Polymorphism, Genetic
PubMed: 38892458
DOI: 10.3390/ijms25116272 -
International Journal of Molecular... May 2024Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of all pancreatic cancers and is the most fatal of all cancers. The treatment response from... (Review)
Review
Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of all pancreatic cancers and is the most fatal of all cancers. The treatment response from combination chemotherapies is far from satisfactory and surgery remains the mainstay of curative strategies. These challenges warrant identifying effective treatments for combating this deadly cancer. PDAC tumor progression is associated with the robust activation of the coagulation system. Notably, cancer-associated thrombosis (CAT) is a significant risk factor in PDAC. CAT is a concept whereby cancer cells promote thromboembolism, primarily venous thromboembolism (VTE). Of all cancer types, PDAC is associated with the highest risk of developing VTE. Hypoxia in a PDAC tumor microenvironment also elevates thrombotic risk. Direct oral anticoagulants (DOACs) or low-molecular-weight heparin (LMWH) are used only as thromboprophylaxis in PDAC. However, a precision medicine approach is recommended to determine the precise dose and duration of thromboprophylaxis in clinical setting.
Topics: Humans; Venous Thromboembolism; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Anticoagulants; Risk Factors; Animals; Tumor Microenvironment
PubMed: 38891849
DOI: 10.3390/ijms25115661 -
Molecular Neurobiology Jun 2024Alzheimer's disease, characterized by the accumulation of abnormal protein aggregates and neuronal damage in the brain, leads to a gradual decline in cognitive function... (Review)
Review
Alzheimer's disease, characterized by the accumulation of abnormal protein aggregates and neuronal damage in the brain, leads to a gradual decline in cognitive function and memory. As a complex neurodegenerative disorder, it involves disruptions in various biochemical pathways and neurotransmitter systems, contributing to the progressive loss of neurons and synaptic connections. The complexity of Alzheimer's signaling pathways complicates treatment, presenting a formidable challenge in the quest for effective therapeutic interventions. A member of the Src family of kinases (SFKs), Fyn, is a type of non-receptor tyrosine kinase that has been linked to multiple essential CNS processes, such as myelination and synaptic transmission. Fyn is an appealing target for AD treatments because it is uniquely linked to the two major pathologies in AD by its interaction with tau, in addition to being activated by amyloid-beta (Aβ) through PrP. Fyn mediates neurotoxicity and synaptic impairments caused by Aβ and is involved in regulating the process of Aβ synthesis.Additionally, the tau protein's tyrosine phosphorylation is induced by Fyn. Fyn is also a challenging target because of its widespread body expression and strong homology with other kinases of the Src family, which could cause unintentional off-target effects. This review emphasizes signaling pathways mediated by Fyn that govern neuronal development and plasticity while also summarizing the most noteworthy recent research relevant to Fyn kinase's function in the brain. Additionally, the therapeutic inhibition of Fyn kinase has been discussed, with a focus on the Fyn kinase inhibitors that are in clinical trials, which presents a fascinating opportunity for targeting Fyn kinase in the creation of possible therapeutic approaches for the management of Alzheimer's disease.
PubMed: 38890236
DOI: 10.1007/s12035-024-04286-2 -
Journal of Bacteriology Jun 2024Sequence differences among the subtypes of toxin TcdB (2,366 amino acids) are broadly distributed across the entire protein, with the notable exception of 76 residues...
UNLABELLED
Sequence differences among the subtypes of toxin TcdB (2,366 amino acids) are broadly distributed across the entire protein, with the notable exception of 76 residues at the protein's carboxy terminus. This sequence invariable region (SIR) is identical at the DNA and protein level among the TcdB variants, suggesting this string of amino acids has undergone selective pressure to prevent alterations. The functional role of the SIR domain in TcdB has not been determined. Analysis of a recombinantly constructed TcdB mutant lacking the SIR domain did not identify changes in TcdB's enzymatic or cytopathic activities. To further assess the SIR region, we constructed a strain with the final 228 bp deleted from the gene, resulting in the production of a truncated form of TcdB lacking the SIR (TcdB2). Using a combination of approaches, we found in the absence of the SIR sequence TcdB2 retained cytotoxic activity but was not secreted from . TcdB2 was not released from the cell under autolytic conditions, indicating the SIR is involved in a more discrete step in toxin escape from the bacterium. Fractionation experiments combined with antibody detection found that TcdB2 accumulates at the cell membrane but is unable to complete steps in secretion beyond this point. These data suggest conservation of the SIR domain across variants of TcdB could be influenced by the sequence's role in efficient escape of the toxin from .
IMPORTANCE
is a leading cause of antibiotic associated disease in the United States. The primary virulence factors produced by are two large glucosylating toxins TcdA and TcdB. To date, several sequence variants of TcdB have been identified that differ in various functional properties. Here, we identified a highly conserved region among TcdB subtypes that is required for release of the toxin from . This study reveals a putative role for the longest stretch of invariable sequence among TcdB subtypes and provides new details regarding toxin release into the extracellular environment. Improving our understanding of the functional roles of the conserved regions of TcdB variants aids in the development of new, broadly applicable strategies to treat CDI.
PubMed: 38888328
DOI: 10.1128/jb.00096-24 -
Journal of Extracellular Vesicles Jun 2024Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent...
Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.
Topics: Microglia; Extracellular Vesicles; Nitric Oxide; Animals; Lipopolysaccharides; Signal Transduction; Mice; Proteomics; Protein Processing, Post-Translational; Cysteine; Nitric Oxide Synthase Type II
PubMed: 38887871
DOI: 10.1002/jev2.12455