-
Nature Communications Dec 2023The axon initial segment (AIS) is a specialized neuronal compartment required for action potential generation and neuronal polarity. However, understanding the...
The axon initial segment (AIS) is a specialized neuronal compartment required for action potential generation and neuronal polarity. However, understanding the mechanisms regulating AIS structure and function has been hindered by an incomplete knowledge of its molecular composition. Here, using immuno-proximity biotinylation we further define the AIS proteome and its dynamic changes during neuronal maturation. Among the many AIS proteins identified, we show that SCRIB is highly enriched in the AIS both in vitro and in vivo, and exhibits a periodic architecture like the axonal spectrin-based cytoskeleton. We find that ankyrinG interacts with and recruits SCRIB to the AIS. However, loss of SCRIB has no effect on ankyrinG. This powerful and flexible approach further defines the AIS proteome and provides a rich resource to elucidate the mechanisms regulating AIS structure and function.
Topics: Axon Initial Segment; Proteome; Biotinylation; Axons; Neurons
PubMed: 38081810
DOI: 10.1038/s41467-023-44015-2 -
Viruses Mar 2022The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how...
The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here, the cellular impact of expressing SARS-CoV-2 viral proteins was studied by global proteomic analysis, and proximity biotinylation (BioID) was used to map the SARS-CoV-2 virus-host interactome in human lung cancer-derived cells. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are associated with SARS-CoV-2 proteins. We have established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Furthermore, we identified 66 high-confidence interactions by comparing this study with previous reports, providing a strong foundation for future follow-up studies. Finally, we cross-referenced candidate interactors with the CLUE drug library to identify potential therapeutics for drug-repurposing efforts. Collectively, these studies provide a valuable resource to uncover novel SARS-CoV-2 biology and inform development of antivirals.
Topics: Biotinylation; COVID-19; Humans; Pandemics; Proteomics; SARS-CoV-2
PubMed: 35337019
DOI: 10.3390/v14030611 -
The Journal of Nutritional Biochemistry Jul 2005Histones H1, H2A, H2B, H3 and H4 are DNA-binding proteins that mediate the folding of DNA into chromatin. Various posttranslational modifications of histones regulate... (Review)
Review
Histones H1, H2A, H2B, H3 and H4 are DNA-binding proteins that mediate the folding of DNA into chromatin. Various posttranslational modifications of histones regulate processes such as transcription, replication and repair of DNA. Recently, a novel posttranslational modification has been identified: covalent binding of the vitamin biotin to lysine residues in histones, mediated by biotinidase and holocarboxylase synthetase. Here we describe a novel peptide-based technique, which was used to identify eight distinct biotinylation sites in histones H2A, H3 and H4. Biotinylation site-specific antibodies were generated to investigate biological functions of histone biotinylation. Evidence was provided that biotinylation of histones plays a role in cell proliferation, gene silencing and cellular response to DNA damage.
Topics: Animals; Biotin; Biotinylation; Chromatin; Enzymes; Histones; Humans
PubMed: 15992689
DOI: 10.1016/j.jnutbio.2005.03.025 -
ACS Applied Materials & Interfaces Dec 2021Biosensors and other biological platform technologies require the functionalization of their surface with receptors to enhance affinity and selectivity. Control over the...
Biosensors and other biological platform technologies require the functionalization of their surface with receptors to enhance affinity and selectivity. Control over the functionalization density is required to tune the platform's properties. Streptavidin (SAv) monolayers are widely used to immobilize biotinylated proteins, receptors, and DNA. The SAv density on a surface can be varied easily, but the predictability is dependent on the method by which the SAv is immobilized. In this study we show a method to quantitatively predict the SAv coverage on biotinylated surfaces. The method is validated by measuring the SAv coverage on supported lipid bilayers with a range of biotin contents and two different main phase lipids and by using quartz crystal microbalance and localized surface plasmon resonance. We explore a predictive model of the biotin-dependent SAv coverage without any fit parameters. Model and data allow to predict the SAv coverage based on the biotin coverage, in both the low- and high-density regimes. This is of special importance in applications with multivalent binding where control over surface receptor density is required, but a direct measurement is not possible.
Topics: Biomimetic Materials; Biotin; Biotinylation; Materials Testing; Streptavidin; Surface Properties
PubMed: 34813287
DOI: 10.1021/acsami.1c16446 -
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 -
G3 (Bethesda, Md.) May 2021The protein-protein interaction (PPI) is a basic strategy for life to operate. The analysis of PPIs in multicellular organisms is very important but extremely...
The protein-protein interaction (PPI) is a basic strategy for life to operate. The analysis of PPIs in multicellular organisms is very important but extremely challenging because PPIs are particularly dynamic and variable among different development stages, tissues, cells, and even organelles. Therefore, understanding PPI needs a good resolution of time and space. More importantly, understanding in vivo PPI needs to be realized in situ. Proximity-based biotinylation combined with mass spectrometry (MS) has emerged as a powerful approach to study PPI networks and protein subcellular compartmentation. TurboID, the newly engineered promiscuous ligase, has been reported to label proximate proteins effectively in various species. In Drosophila, we systematically apply TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and demonstrate the feasibility of TurboID-mediated labeling system in desired cell types. For a proof-of-principle, we use the TurboID-mediated biotinylation coupled with MS to distinguish CTP synthase with or without the ability to form filamentous cytoophidia, retrieving two distinct sets of proximate proteomes. Therefore, this makes it possible to map PPIs in vivo and in situ at a defined spatiotemporal resolution, and demonstrates a referable resource for cytoophidium proteome in Drosophila.
Topics: Animals; Biotinylation; Drosophila; Mass Spectrometry; Protein Interaction Maps; Proteome
PubMed: 33724396
DOI: 10.1093/g3journal/jkab077 -
Journal of Microbiology and... Sep 2022To better understand the effects of PEGylation and biotinylation on the delivery efficiency of proteins, the cationic protein lysozyme (LZ) and anionic protein bovine...
To better understand the effects of PEGylation and biotinylation on the delivery efficiency of proteins, the cationic protein lysozyme (LZ) and anionic protein bovine serum albumin (BSA) were chemically conjugated with poly(ethylene glycol) (PEG) and biotin-PEG to primary amine groups of proteins using N-hydroxysuccinimide reactions. Four types of protein conjugates were successfully prepared: PEGylated LZ (PEG-LZ), PEGylated BSA (PEG-BSA), biotin-PEG-conjugated LZ (Bio-PEG-LZ), and biotin-PEG-conjugated BSA (Bio-PEG-BSA). PEG-LZ and Bio-PEG-LZ exhibited a lower intracellular uptake than that of LZ in A549 human lung cancer cells (in a two-dimensional culture). However, Bio-PEG-BSA showed significantly improved intracellular delivery as compared to that of PEG-BSA and BSA, probably because of favorable interactions with cells via biotin receptors. For A549/fibroblast coculture spheroids, PEG-LZ and PEG-BSA exhibited significantly decreased tissue penetration as compared with that of unmodified proteins. However, Bio-PEG-BSA showed tissue penetration comparable to that of unmodified BSA. In addition, citraconlyated LZ (Cit-LZ) showed reduced spheroid penetration as compared to that of LZ, probably owing to a decrease in protein charge. Taken together, chemical conjugation of targeting ligands-PEG to anionic proteins could be a promising strategy to improve intracellular delivery and in vivo activity, whereas modifications of cationic proteins should be more delicately designed.
Topics: Amines; Anions; Biotin; Biotinylation; Humans; Ligands; Muramidase; Neoplasms; Polyethylene Glycols; Serum Albumin, Bovine
PubMed: 36039388
DOI: 10.4014/jmb.2207.07058 -
Genes Aug 2022Proximity labeling employs modified biotin ligases or peroxidases that produce reactive radicals to covalently label proximate proteins with biotin in living cells. The...
Proximity labeling employs modified biotin ligases or peroxidases that produce reactive radicals to covalently label proximate proteins with biotin in living cells. The resulting biotinylated proteins can then be isolated and identified. A combination of programmable DNA targeting and proximity labeling that maps proteomic landscape at DNA elements with dCas9-APEX2 has been established in living cells. However, defining interactome at RNA elements has lagged behind. In combination with RNA-targeting CRISPR-Cas13, proximity labeling can also be used to identify proteins that interact with specific RNA elements in living cells. From this viewpoint, we briefly summarize the latest advances in CRISPR-guided proximity labeling in studying RNA-protein interactions, and we propose applying the most recent engineered proximity-labeling enzymes to study RNA-centric interactions in the future.
Topics: Biotin; Biotinylation; Ligases; Peroxidases; Proteins; Proteomics; RNA
PubMed: 36140717
DOI: 10.3390/genes13091549 -
Current Opinion in Neurobiology Jun 2018Understanding signaling pathways in neuroscience requires high-resolution maps of the underlying protein networks. Proximity-dependent biotinylation with engineered... (Review)
Review
Understanding signaling pathways in neuroscience requires high-resolution maps of the underlying protein networks. Proximity-dependent biotinylation with engineered enzymes, in combination with mass spectrometry-based quantitative proteomics, has emerged as a powerful method to dissect molecular interactions and the localizations of endogenous proteins. Recent applications to neuroscience have provided insights into the composition of sub-synaptic structures, including the synaptic cleft and inhibitory post-synaptic density. Here we compare the different enzymes and small-molecule probes for proximity labeling in the context of cultured neurons and tissue, review existing studies, and provide technical suggestions for the in vivo application of proximity labeling.
Topics: Animals; Biotinylation; Humans; Neurobiology; Neuroimaging; Proteome; Proteomics
PubMed: 29125959
DOI: 10.1016/j.conb.2017.10.015 -
The Journal of Nutrition Jul 2006Covalent modifications of histones are a crucial component of epigenetic events that regulate chromatin structures and gene function. Evidence exists that distinct... (Review)
Review
Covalent modifications of histones are a crucial component of epigenetic events that regulate chromatin structures and gene function. Evidence exists that distinct lysine residues in histones are modified by covalent attachment of the vitamin biotin, catalyzed by biotinidase and holocarboxylase synthetase. Biotinylation of histones appears to be conserved across species. The following biotinylation sites were identified using both MS and enzymatic biotinylation of synthetic peptides: K9, K13, K125, K127, and K129 in histone H2A; K4, K9, and K18 in histone H3; and K8 and K12 in histone H4. Evidence was provided that biotinylated histone H4 is enriched in pericentromeric heterochromatin, and that biotinylation of histone H4 participates in gene silencing, mitotic condensation of chromatin, and the cellular response to DNA damage. Biotinylation of histones is a reversible process and depends on the exogenous biotin supply, but the identities of histone debiotinylases remain uncertain. We propose that some effects of biotin deficiency can be attributed to abnormal chromatin structures.
Topics: Biotin; Biotinylation; Chromatin; Epigenesis, Genetic; Histones; Humans
PubMed: 16772434
DOI: 10.1093/jn/136.7.1763