Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Mycobacterial biotin synthases require an auxiliary protein to convert dethiobiotin into biotin.

Lipid biosynthesis in the pathogen Mycobacterium tuberculosis depends on biotin for posttranslational modification of key enzymes. However, the mycobacterial biotin synthetic pathway is not fully understood. Here, we show that rv1590, a gene of previously unknown function, is required by M. tuberculosis to synthesize biotin. Chemical-generic interaction experiments mapped the function of rv1590 to the conversion of dethiobiotin to biotin, which is catalyzed by biotin synthases (BioB). Biochemical studies confirmed that in contrast to BioB of Escherichia coli, BioB of M. tuberculosis requires Rv1590 (which we named "biotin synthase auxiliary protein" or BsaP), for activity. We found homologs of bsaP associated with bioB in many actinobacterial genomes, and confirmed that BioB of Mycobacterium smegmatis also requires BsaP. Structural comparisons of BsaP-associated biotin synthases with BsaP-independent biotin synthases suggest that the need for BsaP is determined by the [2Fe-2S] cluster that inserts sulfur into dethiobiotin. Our findings open new opportunities to seek BioB inhibitors to treat infections with M. tuberculosis and other pathogens.

Advances and prospects in microbial production of biotin.

Biotin, serving as a coenzyme in carboxylation reactions, is a vital nutrient crucial for the natural growth, development, and overall well-being of both humans and animals. Consequently, biotin is widely utilized in various industries, including feed, food, and pharmaceuticals. Despite its potential advantages, the chemical synthesis of biotin for commercial production encounters environmental and safety challenges. The burgeoning field of synthetic biology now allows for the creation of microbial cell factories producing bio-based products, offering a cost-effective alternative to chemical synthesis for biotin production. This review outlines the pathway and regulatory mechanism involved in biotin biosynthesis. Then, the strategies to enhance biotin production through both traditional chemical mutagenesis and advanced metabolic engineering are discussed. Finally, the article explores the limitations and future prospects of microbial biotin production. This comprehensive review not only discusses strategies for biotin enhancement but also provides in-depth insights into systematic metabolic engineering approaches aimed at boosting biotin production.

Super-resolution proximity labeling with enhanced direct identification of biotinylation sites.

Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.

Redox-Responsive "Catch and Release" Cryogels: A Versatile Platform for Capture and Release of Proteins and Cells.

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.

Redox proteomics in melanoma cells: An optimized protocol.

Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins.

Chloroflexus aurantiacus acetyl-CoA carboxylase evolves fused biotin carboxylase and biotin carboxyl carrier protein to complete carboxylation activity.

Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded ß-barrel of the partially resolved BCCP domain. Disruption of ß-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTß-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTß to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC.

CAR T cell therapies face challenges in combating solid tumors due to their single-target approach, which becomes ineffective if the targeted antigen is absent or lost. Universal CAR T cells (UniCAR Ts) provide a promising solution by utilizing molecular tags (linkers), such as biotin conjugated to monoclonal antibodies, enabling them to target a variety of tumor antigens. Recently, we showed that conventional CAR T cells could penetrate the extracellular matrix (ECM) of ADCC-resistant tumors, which forms a barrier to therapeutic antibodies. This finding led us to investigate whether UniCAR T cells, targeted by soluble antibody-derived linkers, could similarly tackle ADCC-resistant tumors where ECM restricts antibody penetration. We engineered UniCAR T cells by incorporating a biotin-binding monomeric streptavidin 2 (mSA2) domain for targeting HER2 via biotinylated trastuzumab (BT). The activation and cytotoxicity of UniCAR T cells in the presence or absence of BT were evaluated in conventional immunoassays. A 3D spheroid coculture was set up to test the capability of UniCAR Ts to access ECM-masked HER2+ cells. For in vivo analysis, we utilized a HER2+ xenograft model in which intravenously administered UniCAR T cells were supplemented with intraperitoneal BT treatments. In vitro, BT-guided UniCAR T cells showed effective activation and distinct anti-tumor response. Upon target recognition, IFNγ secretion correlated with BT concentration. In the presence of BT, UniCAR T cells effectively penetrated HER2+ spheroids and induced cell death in their core regions. In vivo, upon intravenous administration of UniCAR Ts, circulating BT linkers immediately engaged the mSA2 domain and directed effector cells to the HER2+ tumors. However, these co-treated mice died early, possibly due to the lung infiltration of UniCAR T cells that could recognize both native biotin and HER2. Our results suggest that UniCAR T cells guided with soluble linkers present a viable alternative to conventional CAR T cells, especially for patients resistant to antibody therapy and those with solid tumors exhibiting high antigenic variability. Critical to their success, however, is the choice of an appropriate binding domain for the CAR and the corresponding soluble linker, ensuring both efficacy and safety in therapeutic applications.

Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Affinity Purification of Membrane ß-Barrel Proteins via Biotin-Tagged Peptidiscs.

ß-barrel membrane proteins play a crucial role in bacterial pathogenesis and antibiotic resistance, making them a prime focus for the development of new antibiotics and therapeutics. However, their inherent hydrophobic nature and limited presence pose challenges for their high-throughput characterization using conventional methods. In this context, we present a simple but efficacious approach using peptidisc, a membrane mimetic, to overcome the low abundance and hydrophobicity of these proteins. Our methodology, illustrated here using Escherichia coli (E. coli) as a model organism, covers the entire process from outer membrane fraction preparation to data analysis. This detailed protocol outlines the purification of a diverse collection of ß-barrel membrane proteins, rendering them water-soluble and readily amenable to mass spectrometry and downstream drug screening strategies.

ICLAMP: a novel technique to explore adenosine deamination via inosine chemical labeling and affinity molecular purification.

Recent developments in sequencing and bioinformatics have advanced our understanding of adenosine-to-inosine (A-to-I) RNA editing. Surprisingly, recent analyses have revealed the capability of adenosine deaminase acting on RNA (ADAR) to edit DNA:RNA hybrid strands. However, edited inosines in DNA remain largely unexplored. A precise biochemical method could help uncover these potentially rare DNA editing sites. We explore maleimide as a scaffold for inosine labeling. With fluorophore-conjugated maleimide, we were able to label inosine in RNA or DNA. Moreover, with biotin-conjugated maleimide, we purified RNA and DNA containing inosine. Our novel technique of inosine chemical labeling and affinity molecular purification offers substantial advantages and provides a versatile platform for further discovery of A-to-I editing sites in RNA and DNA.

Identification of G-quadruplex-interacting proteins in living cells using an artificial G4-targeting biotin ligase.

G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4's biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins. Implementing this innovative strategy in live cells, we unveiled a cohort of 149 potential G4-interacting proteins, which exhibiting multifaceted functionalities. We then substantiate the directly binding affinity of 7 candidate G4-interacting-proteins (SF3B4, FBL, PP1G, BCL7C, NDUV1, ILF3, GAR1) in vitro. Remarkably, we verified that splicing factor 3B subunit 4 (SF3B4) binds preferentially to the G4-rich 3' splice site and the corresponding splicing sites are modulated by the G4 stabilizer PDS, indicating the regulating role of G4s in mRNA splicing procedure. The PLGPB strategy could biotinylate multiple proteins simultaneously, which providing an opportunity to map G4-interacting proteins network in living cells.

CYP1B1 affects the integrity of the blood-brain barrier and oxidative stress in the striatum: An investigation of manganese-induced neurotoxicity.

AIMS: Excessive influx of manganese (Mn) into the brain across the blood-brain barrier induces neurodegeneration. CYP1B1 is involved in the metabolism of arachidonic acid (AA) that affects vascular homeostasis. We aimed to investigate the effect of brain CYP1B1 on Mn-induced neurotoxicity. METHOD: Brain Mn concentrations and α-synuclein accumulation were measured in wild-type and CYP1B1 knockout mice treated with MnCl2 (30 mg/kg) and biotin (0.2 g/kg) for 21 continuous days. Tight junctions and oxidative stress were analyzed in hCMEC/D3 and SH-SY5Y cells after the treatment with MnCl2 (200 µM) and CYP1B1-derived AA metabolites (HETEs and EETs). RESULTS: Mn exposure inhibited brain CYP1B1, and CYP1B1 deficiency increased brain Mn concentrations and accelerated α-synuclein deposition in the striatum. CYP1B1 deficiency disrupted the integrity of the blood-brain barrier (BBB) and increased the ratio of 3, 4-dihydroxyphenylacetic acid (DOPAC) to dopamine in the striatum. HETEs attenuated Mn-induced inhibition of tight junctions by activating PPARγ in endothelial cells. Additionally, EETs attenuated Mn-induced up-regulation of the KLF/MAO-B axis and down-regulation of NRF2 in neuronal cells. Biotin up-regulated brain CYP1B1 and reduced Mn-induced neurotoxicity in mice. CONCLUSIONS: Brain CYP1B1 plays a critical role in both cerebrovascular and dopamine homeostasis, which might serve as a novel therapeutic target for the prevention of Mn-induced neurotoxicity.

Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth.

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h-1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.

Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX.

Aptamers have shown great promise as oligonucleotide-based affinity ligands for various medicinal and industrial applications. A critical step in the production of DNA aptamers via selective enhancement of ligands by exponential enrichment (SELEX) is the generation of ssDNA from dsDNA. There are a number of caveats associated with current methods for ssDNA generation, which can lower success rates of SELEX experiments. They often result in low yields thereby decreasing diversity or fail to eliminate parasitic PCR by-products leading to accumulation of by-products from round to round. Both contribute to the failure of SELEX protocols and therefore potentially limit the impact of aptamers compared to their peptide-based antibody counterparts. We have developed a novel method using ion pair reversed phase HPLC (IP RP HPLC) employed under denaturing conditions for the ssDNA re-generation stage of SELEX following PCR. We have utilised a range of 5' chemical modifications on PCR primers to amplify PCR fragments prior to separation and purification of the DNA strands using denaturing IP RP HPLC. We have optimised mobile phases to enable complete denaturation of the dsDNA at moderate temperatures that circumvents the requirement of high temperatures and results in separation of the ssDNA based on differences in their hydrophobicity. Validation of the ssDNA isolation and purity assessment was performed by interfacing the IP RP HPLC with mass spectrometry and fluorescence-based detection. The results show that using a 5' Texas Red modification on the reverse primer in the PCR stage enabled purification of the ssDNA from its complimentary strand via IP RP HPLC under denaturing conditions. Additionally, we have confirmed the purity of the ssDNA generated as well as the complete denaturation of the PCR product via the use of mass-spectrometry and fluorescence analysis therefore proving the selective elimination of PCR by-products and the unwanted complementary strand. Following lyophilisation, ssDNA yields of up to 80% were obtained. In comparison the streptavidin biotin affinity chromatography also generates pure ssDNA with a yield of 55%. The application of this method to rapidly generate and purify ssDNA of the correct size, offers the opportunity to improve the development of new aptamers via SELEX.

The Quantitative Biotinylproteomics Studies Reveal a WInd-Related Kinase 1 (Raf-Like Kinase 36) Functioning as an Early Signaling Component in Wind-Induced Thigmomorphogenesis and Gravitropism.

Wind is one of the most prevalent environmental forces entraining plants to develop various mechano-responses, collectively called thigmomorphogenesis. Largely unknown is how plants transduce these versatile wind force signals downstream to nuclear events and to the development of thigmomorphogenic phenotype or anemotropic response. To identify molecular components at the early steps of the wind force signaling, two mechanical signaling-related phosphoproteins, identified from our previous phosphoproteomic study of Arabidopsis touch response, mitogen-activated protein kinase kinase 1 (MKK1) and 2 (MKK2), were selected for performing in planta TurboID (ID)-based quantitative proximity-labeling (PL) proteomics. This quantitative biotinylproteomics was separately performed on MKK1-ID and MKK2-ID transgenic plants, respectively, using the genetically engineered TurboID biotin ligase expression transgenics as a universal control. This unique PTM proteomics successfully identified 11 and 71 MKK1 and MKK2 putative interactors, respectively. Biotin occupancy ratio (BOR) was found to be an alternative parameter to measure the extent of proximity and specificity between the proximal target proteins and the bait fusion protein. Bioinformatics analysis of these biotinylprotein data also found that TurboID biotin ligase favorably labels the loop region of target proteins. A WInd-Related Kinase 1 (WIRK1), previously known as rapidly accelerated fibrosarcoma (Raf)-like kinase 36 (RAF36), was found to be a putative common interactor for both MKK1 and MKK2 and preferentially interacts with MKK2. Further molecular biology studies of the Arabidopsis RAF36 kinase found that it plays a role in wind regulation of the touch-responsive TCH3 and CML38 gene expression and the phosphorylation of a touch-regulated PATL3 phosphoprotein. Measurement of leaf morphology and shoot gravitropic response of wirk1 (raf36) mutant revealed that the WIRK1 gene is involved in both wind-triggered rosette thigmomorphogenesis and gravitropism of Arabidopsis stems, suggesting that the WIRK1 (RAF36) protein probably functioning upstream of both MKK1 and MKK2 and that it may serve as the crosstalk point among multiple mechano-signal transduction pathways mediating both wind mechano-response and gravitropism.

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

Mass Spectrometry-Based Precise Identification of Citrullinated Histones via Limited Digestion and Biotin Derivative Tag Enrichment.

Histone citrullination is an essential epigenetic post-translational modification (PTM) that affects many important physiological and pathological processes, but effective tools to study histone citrullination are greatly limited due to several challenges, including the small mass shift caused by this PTM and its low abundance in biological systems. Although previous studies have reported frequent occurrences of histone citrullination, these methods failed to provide a high-throughput and site-specific strategy to detect histone citrullination. Recently, we developed a biotin thiol tag that enabled precise identification of protein citrullination coupled with mass spectrometry. However, very few histone citrullination sites were identified, likely due to the highly basic nature of these proteins. In this study, we develop a novel method utilizing limited digestion and biotin derivative tag enrichment to facilitate direct in vivo identification of citrullination sites on histones. We achieve improved coverage of histone identification via partial enzymatic digestion and lysine block by dimethylation. With biotin tag-assisted chemical derivatization and enrichment, we also achieve precise annotation of histone citrullination sites with high confidence. We further compare different fragmentation methods and find that the electron-transfer-dissociation-based approach enables the most in-depth analysis and characterization. In total, we unambiguously identify 18 unique citrullination sites on histones in human astrocytoma U87 cells, including 15 citrullinated sites being detected for the first time. Some of these citrullination sites are observed to exhibit noticeable alterations in response to DNA damage, which demonstrates the superiority of our strategy in understanding the roles of histone citrullination in critical biological processes.

Streamlined Biotinylation, Enrichment and Analysis for Enhanced Plasma Membrane Protein Identification Using TurboID and TurboID-Start Biotin Ligases.

Plasma membrane proteins (PMPs) play pivotal roles in various cellular events and are crucial in disease pathogenesis, making their comprehensive characterization vital for biomedical research. However, the hydrophobic nature and low expression levels of PMPs pose challenges for conventional enrichment methods, hindering their identification and functional profiling. In this study, we presented a novel TurboID-based enrichment approach for PMPs that helped overcoming some of the existing limitations. We evaluated the efficacy of TurboID and its modified form, TurboID-START, in PMP enrichment, achieving efficient and targeted labelling of PMPs without the need for stable cell line generation. This approach resulted reduction in non-specific biotinylation events, leading to improved PMP enrichment and enabled assessment of the subcellular proteome associated with the plasma membrane. Our findings paved the way for studies targeting the dynamic nature of the plasma membrane proteome and aiming to capture transient associations of proteins with the plasma membrane. The novel TurboID-based enrichment approach presented here offers promising prospects for in-depth investigations into PMPs and their roles in cellular processes.