Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.

TurboID-mediated proximity labeling technologies to identify virus co-receptors.

Virus receptors determine the tissue tropism of viruses and have a certain relationship with the clinical outcomes caused by viral infection, which is of great importance for the identification of virus receptors to understand the infection mechanism of viruses and to develop entry inhibitor. Proximity labeling (PL) is a new technique for studying protein-protein interactions, but it has not yet been applied to the identification of virus receptors or co-receptors. Here, we attempt to identify co-receptor of SARS-CoV-2 by employing TurboID-catalyzed PL. The membrane protein angiotensin-converting enzyme 2 (ACE2) was employed as a bait and conjugated to TurboID, and a A549 cell line with stable expression of ACE2-TurboID was constructed. SARS-CoV-2 pseudovirus were incubated with ACE2-TurboID stably expressed cell lines in the presence of biotin and ATP, which could initiate the catalytic activity of TurboID and tag adjacent endogenous proteins with biotin. Subsequently, the biotinylated proteins were harvested and identified by mass spectrometry. We identified a membrane protein, AXL, that has been functionally shown to mediate SARS-CoV-2 entry into host cells. Our data suggest that PL could be used to identify co-receptors for virus entry.

Protocol for a semi-quantitative approach to identify protein S-palmitoylation in cultured cells by acyl biotin exchange assay.

Palmitoylation is a post-translational lipid modification in which palmitic acid is conjugated predominantly to cysteine residues of target proteins, allowing them to tether to cell membranes. Here, we describe a protocol to perform a stepwise acyl biotin exchange assay to identify protein S-palmitoylation. We describe steps for initial blocking of free thiols in protein lysates, subsequent replacement of thioester-linked palmitate groups with a biotin tag for affinity enrichment, and identification of palmitoylated proteins by SDS-PAGE. For complete details on the use and execution of this protocol, please refer to Leishman et al.1.

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.

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.

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.

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.

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.

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.

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.

Importancia del metabolismo de la biotina / Importance of biotin metabolism

La biotina pertenece al grupo de las vitaminas hidrosolubles del complejo B. En humanos la biotina está directamente involucrada en importantes procesos metabólicos como la gluconeogénesis, la síntesis de ácidos grasos y el catabolismo de algunos aminoácidos, debido a su papel como grupo prostético de las enzimas piruvato carboxilasa, propionil-CoA carboxilasa, b-metilcrotonil-CoA carboxilasa y de la acetil-CoA carboxilasa. La biotina se une al sitio activo de estas enzimas y funciona como acarreador de CO2. Las carboxilasas se sintetizan como apocarboxilasas, carentes de biotina y la forma activa se produce por la unión covalente de la biotina al grupo e-amino de un residuo de lisina de la apocarboxilasa, reacción catalizada por la holocarboxilasa sintetasa. El paso final de la degradación de las carboxilasas es el rompimiento de la fracción biotinil del grupo e-amino de la lisina que es catalizada por la biotinidasa y resulta en la liberación de la biotina libre, la cual puede ser nuevamente reciclada. La biotina regula, a nivel postranscripcional, la expresión de la propionil-CoA carboxilasa y, a nivel transcripcional, a la de la holocarboxilasa sintetasa. Además de su papel como cofactor y regulador de la biosíntesis de las carboxilasas, la biotina está involucrada en otras áreas del metabolismo, donde regula la síntesis de proteínas específicas entre las que se encuentran el receptor de la asialoglicoproteína, varias enzimas reguladoras del metabolismo de glucosa y proteínas que unen biotina en la yema de huevo, entre otras. La deficiencia de biotina se ha reportado en pacientes sometidos a una alimentación parenteral total, en personas que ingieren grandes cantidades de clara de huevo crudo, en niños con desnutrición energético proteínica severa y en personas con errores innatos del metabolismo. Entre estas últimas se encuentran las enfermedades autosómicas recesivas del metabolismo de biotina que resultan de la alteración de la actividad de la holocarboxilasa sintetasa o de la biotinidasa.

Efecto de la adición de Saccharomyces cerevisiae y melaza-urea sobre la digestibilidad in vivo e in situ en dietas para ovinos basadas en paja de cártamo / Effect of the addition of Saccharomyces cerevisiae and urea-molasses on in vivo and in situ digestibility in diets for sheep based on sesame straw

Se realizaron dos experimentos para evaluar los cambios en la digestibilidad de paja de cártamo (PAC) usando un suplemento melaza-urea (MU) y un probiótico (PRO; Saccharomyces cerevisiae). En el experimento 1, se determinó la degradabilidad in situ de fibra (FDN) con cánula ruminal en 12 ovinos (45 ñ 5.8 kg). Se incubaron in situ 4 g de muestra en base seca por periodos de 0, 6, 24, 48, 72 y 96 h, y se muestreó líquido ruminal a 0, 2, 4 y 6 h pospandrial para determinar pH y concentración de N amoniacal. Los resultados se analizaron usando un diseño completamente al azar con arreglo factorial de tratamiento (2 x 2 x 3), donde los factores fueron dos niveles de PRO (o vs .5 g/d intrarruminal), MU (0 vs 10 por ciento) y tres niveles de PAC (50, 60 y 70). La digestibilidad de FDN se incrementó (P< .05) por PRO (3.2 por ciento promedio) y MU (3 por ciento promedio) en todos los tiempos de incubación; también hubo diferencias (P< .05) en la digestión de FDN para 50 por ciento de PAC (35.9 por ciento) con respeco a 60 y 70 por ciento (31.82 y 30.75 por ciento, respectivamente). En el experimento 2 se midió la digestibilidad in vivo (DIV) usando 70 por ciento de PAC, dos niveles de pro (0 vs .05 g/animal/d) y MU (0 vs .10 por ciento) y una ración testigo (T5:40 por ciento PAC, 30 por ciento heno de alfalfa y 30 por ciento concentrado) en un diseño completamente al azar con arreglo factorial (2 x 2 + 1). Los tratamientos fueron: T1, MU 0 por ciento + PRO 0 por ciento; T2, MU 0 por ciento + PRO .5 por ciento; T3, MU 10 por ciento + PRO 0 por ciento y T4, MU 10 por ciento + PRO .5 por ciento. La DIVFDN (por ciento) fue diferente (P< .05) entre tratamientos: 66.8a (T4), 61.9ab (T5),52.2bc (T3),52.1bc (T2) y 41.5c (T1). No hubo cambios en comsumo y pH ruminal (P>.05). El número de protozoarios/ml x 10 3 fue diferente (P< .05) entre tratamientos: 1128a (T4), 950ab (T5), 900ab (T3), 764b (T2) y 771b (T1). Los resultados indican que el probiótico y el suplemento melaza-urea incrementaron la digestibilidad de la fibra y la población de protozoarios, los cuales pudieran haber influido en la digestibilidad de la fibra

Studies on the mechanism of biotin uptake by brusch-border membrane vesicles of hamster enterocytes

Several studies on biotin intestinal transport in the hamster have shown a biotin-specific carrier, but there are conflicting reports on whether it is transported actively, or by facilitated diffusion and on its Na+ dependence. We have studied it for the first time using brush-border membrane vesicles (BBMV), with concentrations in a more physiological (nanomolar)range and found an overshoot component, evidencing a carrier-mediated active process, driving the vitamin against a concentration gradient. Uptake was substantially reduced when potassium substituted for sodium. When the vesicles were treated with trypsin, Na+-dependent uptake was markedly reduced and the overshoot phenomenon was abolished, providing additional evidence for the carrier-mediated trasnport. The amount of uptake in a K+ gradient was considered due to passive diffusion and was about 30 percent of that observed in a Na+ gradient. A similar amount was observed when trypsinized vesicle were incubated in this latter gradient. Our results indicate that in the hamster's brush border intestinal epithelium, Na+-dependent active transport is the most important component in the intestinal uptake of biotin at nanomolar concentrations