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Molecules (Basel, Switzerland) Dec 2021To improve the tumor-targeting efficacy of photodynamic therapy, biotin was conjugated with chlorin e6 to develop a new tumor-targeting photosensitizer, Ce6-biotin. The...
To improve the tumor-targeting efficacy of photodynamic therapy, biotin was conjugated with chlorin e6 to develop a new tumor-targeting photosensitizer, Ce6-biotin. The Ce6-biotin had good water solubility and low aggregation. The singlet-oxygen generation rate of Ce6-biotin was slightly increased compared to Ce6. Flow cytometry and confocal laser scanning microscopy results confirmed Ce6-biotin had higher binding affinity toward biotin-receptor-positive HeLa human cervical carcinoma cells than its precursor, Ce6. Due to the BR-targeting ability of Ce6-biotin, it exhibited stronger cytotoxicity to HeLa cells upon laser irradiation. The IC50 against HeLa cells of Ce6-biotin and Ce6 were 1.28 µM and 2.31 µM, respectively. Furthermore, both Ce6-biotin and Ce6 showed minimal dark toxicity. The selectively enhanced therapeutic efficacy and low dark toxicity suggest that Ce6-biotin is a promising PS for BR-positive-tumor-targeting photodynamic therapy.
Topics: Antineoplastic Agents; Biotin; Cell Survival; Chlorophyllides; HeLa Cells; Humans; Neoplasms; Photochemotherapy; Photosensitizing Agents
PubMed: 34885922
DOI: 10.3390/molecules26237342 -
Nano Letters May 2023The effect of an externally applied directional force on molecular friction is so far poorly understood. Here, we study the force-driven dissociation of the...
The effect of an externally applied directional force on molecular friction is so far poorly understood. Here, we study the force-driven dissociation of the ligand-protein complex biotin-streptavidin and identify anisotropic friction as a not yet described type of molecular friction. Using AFM-based stereographic single molecule force spectroscopy and targeted molecular dynamics simulations, we find that the rupture force and friction for biotin-streptavidin vary with the pulling angle. This observation holds true for friction extracted from Kramers' rate expression and by dissipation-corrected targeted molecular dynamics simulations based on Jarzynski's identity. We rule out ligand solvation and protein-internal friction as sources of the angle-dependent friction. Instead, we observe a heterogeneity in free energy barriers along an experimentally uncontrolled orientation parameter, which increases the rupture force variance and therefore the overall friction. We anticipate that anisotropic friction needs to be accounted for in a complete understanding of friction in biomolecular dynamics and anisotropic mechanical environments.
Topics: Biotin; Streptavidin; Friction; Ligands; Molecular Dynamics Simulation; Microscopy, Atomic Force
PubMed: 36948207
DOI: 10.1021/acs.nanolett.2c04632 -
Annales de Biologie Clinique Aug 2017
Topics: Artifacts; Biotin; Diagnostic Errors; Diagnostic Techniques, Endocrine; Dietary Supplements; False Positive Reactions; Humans; Hyperthyroidism; Immunoassay; Self Medication
PubMed: 28751281
DOI: 10.1684/abc.2017.1252 -
Journal of the American Society For... Sep 2021Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications....
Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications. However, the popular on-bead digestion method faces major challenges of streptavidin contamination, overwhelming signals from endogenous biotinylated proteins, the lost information on biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated in addition to the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency, which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiol-cleavable biotin-assisted TurboID proteomics achieved remarkable intraorganelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.
Topics: Biotin; Biotinylation; HEK293 Cells; HeLa Cells; Humans; Mitochondria; Mitochondrial Proteins; Proteomics; Sulfhydryl Compounds
PubMed: 33909971
DOI: 10.1021/jasms.1c00079 -
Biochemistry and Cell Biology =... Feb 2020RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A... (Review)
Review
RNA is involved in all domains of life, playing critical roles in a host of gene expression processes, host-defense mechanisms, cell proliferation, and diseases. A critical component in many of these events is the ability for RNA to interact with proteins. Over the past few decades, our understanding of such RNA-protein interactions and their importance has driven the search and development of new techniques for the identification of RNA-binding proteins. In determining which proteins bind to the RNA of interest, it is often useful to use the approach where the RNA molecule is the "bait" and allow it to capture proteins from a lysate or other relevant solution. Here, we review a collection of methods for modifying RNA to capture RNA-binding proteins. These include small-molecule modification, the addition of aptamers, DNA-anchoring, and nucleotide substitution. With each, we provide examples of their application, as well as highlight their advantages and potential challenges.
Topics: Biotin; Humans; Molecular Structure; RNA; RNA-Binding Proteins; Small Molecule Libraries
PubMed: 30931575
DOI: 10.1139/bcb-2019-0041 -
Mutation Research May 2012Biotin serves as a covalently bound coenzyme in five human carboxylases; biotin is also attached to histones H2A, H3, and H4, although the abundance of biotinylated... (Review)
Review
Biotin serves as a covalently bound coenzyme in five human carboxylases; biotin is also attached to histones H2A, H3, and H4, although the abundance of biotinylated histones is low. Biotinylation of both carboxylases and histones is catalyzed by holocarboxylase synthetase. Human biotin requirements are unknown. Recommendations for adequate intake of biotin are based on the typical intake of biotin in an apparently healthy population, which is only a crude estimate of the true intake due to analytical problems. Importantly, intake recommendations do not take into account possible effects of biotin deficiency on impairing genome stability. Recent studies suggest that biotin deficiency causes de-repression of long terminal repeats, thereby causing genome instability. While it was originally proposed that these effects are caused by loss of biotinylated histones, more recent evidence suggests a more immediate role of holocarboxylase synthetase in forming multiprotein complexes in chromatin that are important for gene repression. Holocarboxylase synthetase appears to interact physically with the methyl-CpG-binding domain protein 2 and, perhaps, histone methyl transferases, thereby creating epigenetic synergies between biotinylation and methylation events. These observations might offer a mechanistic explanation for some of the birth defects seen in biotin-deficient animal models.
Topics: Biotin; Biotinylation; DNA Damage; Genomic Instability; Humans; Nutritional Requirements; Terminal Repeat Sequences
PubMed: 21871906
DOI: 10.1016/j.mrfmmm.2011.08.001 -
Microbiological Research Dec 2020The tangerine pathotype of Alternaria alternata affects many citrus cultivars, resulting in yield losses. The capability to produce the host-selective toxin and...
Biotin biosynthesis affected by the NADPH oxidase and lipid metabolism is required for growth, sporulation and infectivity in the citrus fungal pathogen Alternaria alternata.
The tangerine pathotype of Alternaria alternata affects many citrus cultivars, resulting in yield losses. The capability to produce the host-selective toxin and cell-wall-degrading enzymes and to mitigate toxic reactive oxygen species is crucial for A. alternata pathogenesis to citrus. Little is known about nutrient availability within citrus tissues to the fungal pathogen. In the present study, we assess the infectivity of a biotin deficiency mutant (ΔbioB) and a complementation strain (CP36) on citrus leaves to determine how biotin impacts A. alternata pathogenesis. Growth and sporulation of ΔbioB are highly dependent on biotin. ΔbioB retains its ability to acquire and transport biotin from the surrounding environment. Growth deficiency of ΔbioB can also be partially restored by the presence of oleic acid or Tween 20, suggesting the requirement of biotin in lipid metabolism. Experimental evidence indicates that de novo biotin biosynthesis is regulated by the NADPH oxidase, implicating in the production of HO, and is affected by the function of peroxisomes. Three genes involved in the biosynthesis of biotin are clustered and co-regulated by biotin indicating a transcriptional feedback loop activation. Infectivity assays using fungal mycelium reveal that ΔbioB cultured on medium without biotin fails to infect citrus leaves; co-inoculation with biotin fully restores infectivity. The CP36 strain re-expressing a functional copy of bioB displays wild-type growth, sporulation and virulence. Taken together, we conclude that the attainability or accessibility of biotin is extremely restricted in citrus cells. A. alternata must be able to synthesize biotin in order to utilize nutrients for growth, colonization and development within the host.
Topics: Alternaria; Biotin; Citrus; Lipid Metabolism; NADPH Oxidases; Oleic Acid; Peroxisomes; Plant Diseases; Polysorbates; Reactive Oxygen Species; Spores, Fungal
PubMed: 33032167
DOI: 10.1016/j.micres.2020.126566 -
Proceedings of the National Academy of... Apr 2019Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on...
Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information on the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin-biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket, while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and rate-dependent induced-fit motions for intermediates, which might be relevant for other receptor-ligand bonds.
Topics: Biotin; Models, Chemical; Molecular Dynamics Simulation; Protein Binding; Streptavidin
PubMed: 30890636
DOI: 10.1073/pnas.1816909116 -
PLoS Pathogens Jan 2023Pseudomonas aeruginosa is an opportunistic pathogen that predominantly causes nosocomial and community-acquired lung infections. As a member of ESKAPE pathogens,...
Pseudomonas aeruginosa is an opportunistic pathogen that predominantly causes nosocomial and community-acquired lung infections. As a member of ESKAPE pathogens, carbapenem-resistant P. aeruginosa (CRPA) compromises the limited therapeutic options, raising an urgent demand for the development of lead compounds against previously-unrecognized drug targets. Biotin is an important cofactor, of which the de novo synthesis is an attractive antimicrobial target in certain recalcitrant infections. Here we report genetic and biochemical definition of P. aeruginosa BioH (PA0502) that functions as a gatekeeper enzyme allowing the product pimeloyl-ACP to exit from fatty acid synthesis cycle and to enter the late stage of biotin synthesis pathway. In relative to Escherichia coli, P. aeruginosa physiologically requires 3-fold higher level of cytosolic biotin, which can be attributed to the occurrence of multiple biotinylated enzymes. The BioH protein enables the in vitro reconstitution of biotin synthesis. The repertoire of biotin abundance is assigned to different mouse tissues and/or organ contents, and the plasma biotin level of mouse is around 6-fold higher than that of human. Removal of bioH renders P. aeruginosa biotin auxotrophic and impairs its intra-phagosome persistence. Based on a model of CD-1 mice mimicking the human environment, lung challenge combined with systemic infection suggested that BioH is necessary for the full virulence of P. aeruginosa. As expected, the biotin synthesis inhibitor MAC13772 is capable of dampening the viability of CRPA. Notably, MAC13772 interferes the production of pyocyanin, an important virulence factor of P. aeruginosa. Our data expands our understanding of P. aeruginosa biotin synthesis relevant to bacterial infectivity. In particular, this study represents the first example of an extracellular pathogen P. aeruginosa that exploits biotin cofactor as a fitness determinant, raising the possibility of biotin synthesis as an anti-CRPA target.
Topics: Animals; Humans; Mice; Anti-Bacterial Agents; Bacterial Proteins; Biosynthetic Pathways; Biotin; Pseudomonas aeruginosa; Pseudomonas Infections
PubMed: 36689471
DOI: 10.1371/journal.ppat.1011110 -
Scientific Reports Jun 2022The streptavidin-biotin system is known to have a very high affinity and specificity and is widely used in biochemical immunoassays and diagnostics. However, this method...
The streptavidin-biotin system is known to have a very high affinity and specificity and is widely used in biochemical immunoassays and diagnostics. However, this method is affected by endogenous D-biotin in serum sample measurements (biotin interference). While several efforts using alternative high-affinity binding systems (e.g., genetically modified streptavidin and biotin derivatives) have been attempted, these efforts have all led to reduction in affinity. To solve this interference issue, the enantiomer of streptavidin was synthesized, which enabled specific binding to L-biotin. We successfully obtained a functional streptavidin molecule by peptide synthesis using D-amino acids and an in vitro folding technique. Several characterizations, including size exclusion chromatography (SEC), circular dichroism spectra (CD), and heat denaturation experiments collectively confirmed the higher-order enantiomer of natural streptavidin had been formed with comparable stability to the natural protein. L-biotin specific binding of this novel molecule enabled us to avoid biotin interference in affinity measurements using the Biacore system and enzyme-linked immunosorbent assay (ELISA). We propose the enantiomer of streptavidin as a potential candidate to replace the natural streptavidin-biotin system, even for in vivo use.
Topics: Biotin; Enzyme-Linked Immunosorbent Assay; Immunoassay; Stereoisomerism; Streptavidin
PubMed: 35688934
DOI: 10.1038/s41598-022-13763-4