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1
Pilely, Katrine, Martin Rask Johansen, Rikke Raaen Lund, Thomas Kofoed, Thomas Kjærsgaard Jørgensen, Lars Skriver, and Ejvind Mørtz. "Monitoring process-related impurities in biologics–host cell protein analysis." Analytical and Bioanalytical Chemistry 414, no. 2 (October 1, 2021): 747–58. http://dx.doi.org/10.1007/s00216-021-03648-2.
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AbstractDuring biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC–MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC–MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC–MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC–MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs.
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Gillespie, Paul F., Yanjie Wang, Kuo Yin, Emily Groegler, Nicholas Cunningham, Alyssa Q. Stiving, Jessica Raffaele, et al. "Automated, Quantitative Capillary Western Blots to Analyze Host Cell Proteins in COVID-19 Vaccine Produced in Vero Cell Line." Vaccines 12, no. 12 (December 5, 2024): 1373. https://doi.org/10.3390/vaccines12121373.
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Background/Objectives: Host cell protein (HCP) content is a major attribute for biological and vaccine products that must be extensively characterized prior to product licensure. Enzyme Linked Immunosorbent Assay (ELISA) and Mass Spectrometry (MS) are conventional methods for quantitative host cell protein analysis in biologic and vaccine products. Both techniques are usually very tedious, labor-intensive, and challenging to transfer to other laboratories. In addition, the ELISA methodology requires 2D SDS PAGE and 2D western blot antibody reagent validation to establish reagent coverage. This reagent coverage provides a rather weak link that is currently accepted, as the western blot is run under denaturing conditions and the ELISA is run under native conditions. Simple Western™ is a relatively new, automated, capillary western blot-based technology that allows for the separation, blotting, and detection of proteins. But, unlike traditional western blots, Simple Western™ is quantitative, allowing for the quantification of HCP content in biologic and vaccine samples. Antibody reagent validation is much more straightforward, as the reagent coverage can be directly linked between the 2D methodology and Simple Western™, as they are both run under denatured and reduced conditions. Methods: Herein we describe the development of a capillary western blot method to quantify the HCP content in samples generated using a Vero cell line for the production of an investigational live virus vaccine candidate (V590) for Coronavirus Disease-2019 (COVID-19). The HCP content in COVID-19 vaccine samples was evaluated using three methods: the new capillary western, the gold standard ELISA, and SDS-PAGE. Results/Conclusions: Strong agreement was observed in the HCP content data between the capillary western and SDS PAGE methods, whereas the ELISA HCP data were outliers, suggesting that the capillary western is generating HCP concentrations closer to the true concentration. This is the first report of using capillary western technology in analyzing HCP in vaccine samples.
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Lavoie, R., Alice di Fazio, R. Blackburn, Michael Goshe, Ruben Carbonell, and Stefano Menegatti. "Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery." International Journal of Molecular Sciences 20, no. 7 (April 8, 2019): 1729. http://dx.doi.org/10.3390/ijms20071729.
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The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.
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Van Manen-Brush, Kathleen, Jacob Zeitler, John R. White, Paul Younge, Samantha Willis, and Marisa Jones. "Improving Chinese hamster ovary host cell protein ELISA using Ella®: an automated microfluidic platform." BioTechniques 69, no. 3 (September 2020): 186–92. http://dx.doi.org/10.2144/btn-2020-0074.
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Chinese hamster ovary (CHO) cells are a mammalian cell line used in the production of therapeutic proteins. Host cell proteins (HCPs) are process-related impurities that are derived from the host cell expression system. During biopharmaceutical drug development, removal of HCPs is required. Enzyme-linked immunosorbent assay (ELISA) is a common technique to quantitate HCPs, but is a labor-intensive process that takes up to 7 h. Ella® is an automated instrument that utilizes microfluidics and glass nanoreactors to quantitate HCPs in 75 min using similar ELISA reagents. The antibodies and antigens are captured on three distinct glass nanoreactors, resulting in sensitive reproducible data. Our results indicate that Ella quantitates CHO HCPs with precision, accuracy, sensitivity and trends comparable with our traditional CHO HCP ELISA.
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Gonzaga, Zennia Jean C., Christina Merakou, Antonio DiGiandomenico, Gregory P. Priebe, and Bernd H. A. Rehm. "A Pseudomonas aeruginosa-Derived Particulate Vaccine Protects against P. aeruginosa Infection." Vaccines 9, no. 7 (July 20, 2021): 803. http://dx.doi.org/10.3390/vaccines9070803.
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Despite numerous efforts to develop an effective vaccine against Pseudomonas aeruginosa, no vaccine has yet been approved for human use. This study investigates the utility of the P. aeruginosa inherently produced polyhydroxyalkanaote (PHA) inclusions and associated host–cell proteins (HCP) as a particulate vaccine platform. We further engineered PHA inclusions to display epitopes derived from the outer membrane proteins OprF/OprI/AlgE (Ag) or the type III secretion system translocator PopB. PHA and engineered PHA beads induced antigen-specific humoral, cell-mediated immune responses, anti-HCP and anti-polysaccharide Psl responses in mice. Antibodies mediated opsonophagocytic killing and serotype-independent protective immunity as shown by 100% survival upon challenge with P. aeruginosa in an acute pneumonia murine model. Vaccines were stable at 4 °C for at least one year. Overall, our data suggest that vaccination with subcellular empty PHA beads was sufficient to elicit multiple immune effectors that can prevent P. aeruginosa infection.
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Wu, Hung-Yi, Pei-Che Chung, Hsiao-Wei Shih, Sy-Ray Wen, and Erh-Min Lai. "Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System in Agrobacterium tumefaciens." Journal of Bacteriology 190, no. 8 (February 8, 2008): 2841–50. http://dx.doi.org/10.1128/jb.01775-07.
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ABSTRACT Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.
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Zoued, Abdelrahim, Eric Durand, Cecilia Bebeacua, Yannick R. Brunet, Badreddine Douzi, Christian Cambillau, Eric Cascales, and Laure Journet. "TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System." Journal of Biological Chemistry 288, no. 38 (August 6, 2013): 27031–41. http://dx.doi.org/10.1074/jbc.m113.499772.
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The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.
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Alhuthali, Sakhr, and Cleo Kontoravdi. "Population balance modelling captures host cell protein dynamics in CHO cell cultures." PLOS ONE 17, no. 3 (March 23, 2022): e0265886. http://dx.doi.org/10.1371/journal.pone.0265886.
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Monoclonal antibodies (mAbs) have been extensively studied for their wide therapeutic and research applications. Increases in mAb titre has been achieved mainly by cell culture media/feed improvement and cell line engineering to increase cell density and specific mAb productivity. However, this improvement has shifted the bottleneck to downstream purification steps. The higher accumulation of the main cell-derived impurities, host cell proteins (HCPs), in the supernatant can negatively affect product integrity and immunogenicity in addition to increasing the cost of capture and polishing steps. Mathematical modelling of bioprocess dynamics is a valuable tool to improve industrial production at fast rate and low cost. Herein, a single stage volume-based population balance model (PBM) has been built to capture Chinese hamster ovary (CHO) cell behaviour in fed-batch bioreactors. Using cell volume as the internal variable, the model captures the dynamics of mAb and HCP accumulation extracellularly under physiological and mild hypothermic culture conditions. Model-based analysis and orthogonal measurements of lactate dehydrogenase activity and double-stranded DNA concentration in the supernatant show that a significant proportion of HCPs found in the extracellular matrix is secreted by viable cells. The PBM then served as a platform for generating operating strategies that optimise antibody titre and increase cost-efficiency while minimising impurity levels.
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Jeon, Hyung Jin, Bo Kyoung Choi, Seo In Hwang, Soo Hyun Kim, Gil Jung Kim, Jae Chan Park, Zung Yoon Yang, and Kwang Yeon Hwang. "Optimization for Simultaneous Removal of Product/Process-Related Impurities of Peptide Fc-Fusion Protein Using Cation Exchange Chromatography." Processes 10, no. 11 (November 11, 2022): 2359. http://dx.doi.org/10.3390/pr10112359.
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Fc fusion proteins are used as therapeutic agents with unique structures by combining the Fc domain of an antibody with other active proteins, cytokines, and enzymes. Peptide Fc-fusion proteins are complex fusion molecules that possess a structure different from that of monoclonal antibodies (mAbs) and are difficult to express, thereby affecting their quality. Many product/process-related impurities generated during the production of peptide Fc-fusion proteins pose a risk to the robustness of pre-existing three-column platforms for the purification of mAbs. Thus, we first evaluated the effect of pH, conductivity, and dynamic binding capacity (DBC; g of product per liter of resin) on the separation of host cell protein (HCP) and high molecular weight (HMW) and low molecular weight (LMW) proteins in strong cation exchange chromatography and then established an operating range using the design of experiments (DoE). Based on our studies, the optimal removal rates of HCP and HMW were achieved under the following conditions: 8 CV of wash buffer, 20–23 g/L of resin DBC, and an elution buffer conductivity of 63–66 mS/cm. The conductivity of the wash buffer used to remove the LMW was 50 mS/cm. In addition, reproducibility was confirmed by scaling up two batches using the Fractogel® EMD SO3− (M) resin. As a result of confirming with a validated test method in all batches, >55% yield, >98.2% purity, and >27% HCP reduction rate were satisfied. The cation exchanger exhibited an acceptable step yield and effectively reduced product/process-related impurities within the established range.
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Kiyonami, Reiko, Rafael Melani, Ying Chen, AI De Leon, and Min Du. "Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs." International Journal of Molecular Sciences 25, no. 17 (September 7, 2024): 9687. http://dx.doi.org/10.3390/ijms25179687.
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Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.
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Liu, Liyun, Shuai Hao, Ruiting Lan, Guangxia Wang, Di Xiao, Hui Sun, and Jianguo Xu. "The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells." Infection and Immunity 83, no. 7 (April 13, 2015): 2596–604. http://dx.doi.org/10.1128/iai.03071-14.
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The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.
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Misterova, A. A. V., V. A. Chicherin, and A. S. Gerasimov. "Purification Method Optimization of Recombinant Platelet-Derived Growth Factor rhPDGF-BB Expressed in Methylotrophic Yeast <i>Pichia pastoris</i>." Прикладная биохимия и микробиология 59, no. 4 (July 1, 2023): 383–91. http://dx.doi.org/10.31857/s0555109923040098.
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Recombinant human platelet-derived growth factor rhPDGF-BB is one of the major cytokines, which has been approved for medical use. Medical drug “becaplermin”, containing rhPDGF-BB has been approved for neuropathic ulcer and severe skin burns treatment, as well as in periodontal surgery (in combination with osteoconductive matrices). In this article, we sought to optimize purification process to obtain high purity rhPDGF-BB using methylotrophic yeast Pichia pastoris – a production host for rhPDGF-BB. A faster and simpler chromatography purification method has been suggested which allows to obtain rhPDGF-BB with purity 98% as determined by SDS-PAGE and containing host cell proteins (HCP) 33 ± 4 ng/mg, as measured by ELISA. The effective proliferative dose of rhPDGF-BB measured by WST-1 proliferative assay on 3T3 mouse fibroblast cell culture is 5.02 ± 2.64 ng/mL, which is comparable to commercially available analogues. The optimized method can be attractive for production scale use.
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Misaghi, Shahram, Søren Ottosen, Anita Izrael-Tomasevic, David Arnott, Mohamed Lamkanfi, James Lee, Jinfeng Liu, Karen O'Rourke, Vishva M. Dixit, and Angus C. Wilson. "Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1." Molecular and Cellular Biology 29, no. 8 (February 2, 2009): 2181–92. http://dx.doi.org/10.1128/mcb.01517-08.
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ABSTRACT Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins, and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. We show here that Bap1 interacts with host cell factor 1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 β-propeller using a variant of the HCF-binding motif found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines 1807 and 1808 in the HCF-1C subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using small interfering RNA results in a modest accumulation of HCF-1C, suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G1-S transition.
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Lavoie, R. Ashton, Alice di Fazio, Ruben G. Carbonell, and Stefano Menegatti. "Multiplexed Competitive Screening of One-Bead-One-Component Combinatorial Libraries Using a ClonePix 2 Colony Sorter." International Journal of Molecular Sciences 20, no. 20 (October 16, 2019): 5119. http://dx.doi.org/10.3390/ijms20205119.
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Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies—beyond mere library blocking and competitive screening—to ensure binding selectivity of selected leads. This work presents a method for multiplexed solid-phase peptide library screening using a ClonePix 2 Colony Picker that integrates (i) orthogonal fluorescent labeling for positive selection against a target protein and negative selection against competitor species with (ii) semi-quantitative tracking of target vs. competitor binding for every library bead. The ClonePix 2 technology enables global at-a-glance evaluation and customization of the parameters for bead selection to ensure high affinity and selectivity of the isolated leads. A case study is presented by screening a peptide library against green-labeled human immunoglobulin G (IgG) and red-labeled host cell proteins (HCPs) using ClonePix 2 to select HCP-binding ligands for flow-through chromatography applications. Using this approach, 79 peptide ligand candidates (6.6% of the total number of ligands screened) were identified as potential HCP-selective ligands, enabling a potential rate of >3,000 library beads screened per hour.
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Shi, Leitai, Jia Li, Xiaohong Wu, Shouchun Cao, Yunpeng Wang, Danhua Zhao, and Yuhua Li. "Novel Standard Substance from Primary Hamster Kidney Cells for Quality Control of Human Rabies Vaccines in China." Vaccines 13, no. 2 (February 13, 2025): 180. https://doi.org/10.3390/vaccines13020180.
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Background: Host cell proteins (HCPs) from primary hamster kidney cells (PHKCs) used to produce rabies vaccines may cause an allergic reaction in humans, so these residual HCPs must be controlled. Establishing a national standard for PHKC HCP is very important to ensure the consistency of HCPs between batches of the vaccine and to standardize the control of HCPs. Objectives: We aimed to establish a novel national standard substance to determine the HCP residue in rabies vaccines produced with PHKCs. Methods: A two-step multi-laboratory collaborative collaboration was undertaken. In the first step, the protein concentration of the standard substance stock solution was determined using Lowry’s method. In the second step, the concentration of the candidate standard substance was determined with an enzyme-linked immunosorbent assay. Results: The concentration of the PHKC protein standard was 4.0 μg/mL (95% confidence interval: 3.5–4.4 μg/mL). Conclusions: The PHKC protein standard was approved by the Chinese National Committee on Standards for the quality control of PHKC-based rabies vaccines for human use, and it plays an important role in controlling the quality of these human vaccines.
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Chen, Serene W., Zi Ying Zheng, Farouq Bin Mahfut, Yuansheng Yang, Masahiro Ogino, Kazuo Okada, Kohei Sato, and Wei Zhang. "Leveraging an advanced simulated moving bed approach to achieve 3-component separation for enhanced impurity removal in a non-affinity cation exchange capture step." PLOS ONE 18, no. 1 (January 25, 2023): e0280760. http://dx.doi.org/10.1371/journal.pone.0280760.
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One of the key challenges in downstream bioprocessing is to obtain products of high purity in a productive fashion through the effective removal of process and product related impurities. While a classical simulated moving bed (SMB) system operation can typically achieve a 2-component separation between the weakly bound impurities and target species, here we present an advanced SMB approach that can achieve a 3-component separation, including the removal of the strongly bound impurities from the target species. As a proof-of-concept, we demonstrate the enhanced removal of strongly bound host cell proteins (HCP) from the target monoclonal antibody (mAb) through the utilisation of the advanced SMB approach in a non-affinity cation exchange (CEX) capture step. In this way, 1 less polishing step was required to achieve the therapeutic requirements of < 100 ppm HCP and the overall process recovery was increased by ~ 6% compared to the corresponding process that utilised a batch CEX operation. The non-affinity CEX capture platform technology established through the utilisation of the advanced SMB approach presented here can potentially be further applied to address the downstream processing challenges presented by other challenging biotherapeutic modalities to yield a final target product with improved purity and recovery.
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Thompson, Jenny Heidbrink, Wai Keen Chung, Min Zhu, Liu Tie, Yali Lu, Nabila Aboulaich, Robert Strouse, and Wenjun David Mo. "Improved detection of host cell proteins (HCPs) in a mammalian cell-derived antibody drug using liquid chromatography/mass spectrometry in conjunction with an HCP-enrichment strategy." Rapid Communications in Mass Spectrometry 28, no. 8 (March 5, 2014): 855–60. http://dx.doi.org/10.1002/rcm.6854.
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Islam, Tuhidul, Amith D. Naik, Yasuhiro Hashimoto, Stefano Menegatti, and Ruben G. Carbonell. "Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality." International Journal of Molecular Sciences 20, no. 1 (January 4, 2019): 161. http://dx.doi.org/10.3390/ijms20010161.
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This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.
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Martín-Sánchez, Esperanza, Camila Guerrero, Luis-Esteban Tamariz-Amador, Anastasiia Zherniakova, Aintzane Zabaleta, Catarina Maia, Laura Blanco, et al. "Deep Characterization of Immune Dysfunction in Patients with Multiple Myeloma (MM) and Identification of Cellular Biomarkers for Tailored Vaccination Strategies." Blood 142, Supplement 1 (November 28, 2023): 643. http://dx.doi.org/10.1182/blood-2023-181747.
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Background: Infection is the leading cause of death in MM. However, the extent of immune dysfunction compared to other B-cell lymphoproliferative disorders (B-CLPD) and healthy adults remains poorly studied. Greater knowledge is needed for tailored vaccination strategies and to improve outcomes upon new pathogens or breakthrough infections. Aim: Generate an atlas of the basal immune status and its response to vaccination in MM vs B-CLPD patients and age-matched health care practitioners (HCP), using the COVID-19 mRNA vaccines as a case study. Methods: A total of 1,099 blood and serum samples were collected from 177 individuals: 28 MM, 53 B-CLPD and 96 HCP older than 50. They were studied before COVID-19 mRNA vaccination, at days 7 and 14 after the first dose, at days 7 and 62 after the second dose, as well as before and at day 17 after the booster. Immune profiling was performed using multidimensional and computational flow cytometry that systematically analyzed 56 immune cell types per sample and time point: 17 B, 30 T, 6 antigen-presenting cell (APC) and 3 granulocytic subsets. Serum levels of IgM, IgG and IgA against the receptor-binding domain (RBD) of the spike (S) glycoprotein, S glycoprotein, nucleocapsid (N) and main protease were quantified using a multiplex-microsphere-based flow cytometry assay. SARS-CoV-2-specific CD8 T cells were quantified using a dextramer panel of S, N, membrane, and ORF3 proteins. Results: When compared to HCP and/or B-CLPD, MM patients showed abnormal distribution of 17/17 B, 22/30 T, 4/6 APC and 1/3 granulocytic cell subsets prior to vaccination. The most deviated cell types in MM were naïve CD21+ B-cells, naïve CD4 T-cells, PD1- and PD1+CD127low effector memory (EM) CD8 T cells, classical monocytes and neutrophils. The B cell compartment of MM patients showed impaired response to COVID-19 mRNA vaccination. While B-CLPD patients and HCP displayed significant expansions of naïve CD21-, IgM+IgD+CD27+CD21-, IgG+CD27-CD21-, and IgG+CD27+CD21- B-cell subsets, as well as of IgA+ circulating plasma cells, such expansions were not observed in MM patients. Accordingly, anti-RBD IgM, IgA and IgG titers were significantly reduced in MM compared to B-CLPD and HCP after the second dose ( P ≤.002). Importantly, the booster increased anti-RBD IgG levels in HCP (20,184 to 186,629 IU/mL, P <.001) but not in MM (1,991 to 5,998 IU/mL, P =.14) and B-CLPD (33,548 to 41,288 IU/mL, P =.19) patients. The immune response of the T-cell compartment was also altered in MM patients. The significant expansion of CD127lowPD1+ and CD127+CD25+ EM CD4, naïve CD4 and CD127lowPD1+ EM CD8 subsets observed in HCP was unnoted in MM and B-CLPD patients. Accordingly, the percentage of virus-specific CD8 T cells after the second dose did not increase in MM and B-CLPD patients. The booster increased virus-specific CD8 T cells in HCP (0.08 to 0.14%, P =.02), but not in MM (0.08 to 0.09%, P =.94) and B-CLPD (0.23 to 0.16%, P =.85). Furthermore, the booster induced virus-specific CD8 T cell differentiation into an EM phenotype in HCP but not in MM and B-CLPD patients. In addition to the dysfunctional B and T cell response to vaccination, MM patients showed abnormal kinetics of APC such as intermediateclassical and non-classical SLAN- and SLAN+ monocytes, as well as of granulocytic subsets such as basophils and neutrophils. Based on differences in immune-cell distribution using HCP as a reference, we calculated an immune dysregulation longitudinal cumulative score in each individual that included 48,496 immune parameters. Compared with HCP, up to 34% MM and 17% B-CLPD patients showed high immune dysregulation scores. Among them, 75% and 33%, respectively, had low seroconversion after the second dose. We found 7/30 T and 14/17 B-cell subsets associated with poor vaccine response in MM patients, namely CD127lowPD1+CXCR5+ EM CD8 T cells, as well as CD21- and CD21+ naïve, transitional, IgM+IgD+CD27+CD21+, IgM+CD27-CD21+, and IgG+CD27-CD21+ B cells. Conclusion: We provide an atlas of the immune dysfunction in MM patients and how it affects the efficacy of vaccination strategies such as for COVID-19. The schedule of vaccine doses may thus benefit from individualization according to patients' immune status, which could act as a surrogate of host, tumor and treatment-related immune dysfunction. Accordingly, we have identified key cell types for readily monitoring of patients' immune status using routinely available flow cytometry.
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González-Domínguez, Irene, Elianet Lorenzo, Alice Bernier, Laura Cervera, Francesc Gòdia, and Amine Kamen. "A Four-Step Purification Process for Gag VLPs: From Culture Supernatant to High-Purity Lyophilized Particles." Vaccines 9, no. 10 (October 9, 2021): 1154. http://dx.doi.org/10.3390/vaccines9101154.
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Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles.
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Gordan, John, Adriana Pitea, Rigney E. Turnham, Manon Eckhardt, Gwendolyn M. Jang, Huat Lim, Alex L. Choi, et al. "Abstract PO017: HBV alters YAP regulation in liver cancer by remodeling PP2A complexes." Clinical Cancer Research 28, no. 17_Supplement (September 1, 2022): PO017. http://dx.doi.org/10.1158/1557-3265.liverca22-po017.
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Abstract Viral hepatitis promotes hepatocellular carcinoma (HCC) initiation by inducing inflammation, cellular stress and cell death. However, the cell-intrinsic effects of viral infection in advanced HCC remain unclear. We used affinity purification mass spectrometry to develop a complete map of 145 interactions among Hepatitis B Virus (HBV) and host proteins in the HUH7 HCC tumor cell line, identifying known and novel HBV/host protein-protein interactions. We integrated this map with HCC genomes, identifying 61 proteins with preferential mutation in non-HBV HCC, suggesting that their interaction with HBV plays a role in cancer. Focusing on proteins that directly interact with the HBV oncoprotein X (HBx), we found that HBx rewires the effect of the PP2A phosphatase on HCC signaling. HBx binding excluded striatin-family regulatory subunits from the PP2A complex, causing Hippo kinase activation. This effect creates a requirement for integrin signaling to mTOR complex 2 to maintain expression of the YAP oncoprotein, critical for HCC growth. Thus, HBV rewires HCC signaling and may promote targetable dependencies. Citation Format: John Gordan, Adriana Pitea, Rigney E Turnham, Manon Eckhardt, Gwendolyn M Jang, Huat Lim, Alex L Choi, Sourav Bandyopadhyay, Danielle Swaney, Kevan Shokat, Trey Ideker, Nevan Krogan. HBV alters YAP regulation in liver cancer by remodeling PP2A complexes [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO017.
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Akhtar, Ayesha, Shivakumar Arumugam, and Shoaib Alam. "Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis." Current Chromatography 7, no. 2 (December 29, 2020): 121–33. http://dx.doi.org/10.2174/2213240607999201029204934.
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Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.
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Liu, Sixu, Jingqi Li, Qingtian Cheng, Kangyi Duan, Zhan Wang, Shuang Yan, Shuaishuai Tian, et al. "A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal." Viruses 16, no. 5 (May 13, 2024): 768. http://dx.doi.org/10.3390/v16050768.
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Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin–Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.
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Capotosti, Francesca, James J. D. Hsieh, and Winship Herr. "Species Selectivity of Mixed-Lineage Leukemia/Trithorax and HCF Proteolytic Maturation Pathways." Molecular and Cellular Biology 27, no. 20 (August 13, 2007): 7063–72. http://dx.doi.org/10.1128/mcb.00769-07.
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ABSTRACT Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1PRO repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.
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Zeisel, Mirjam B., Francesca Guerrieri, and Massimo Levrero. "Host Epigenetic Alterations and Hepatitis B Virus-Associated Hepatocellular Carcinoma." Journal of Clinical Medicine 10, no. 8 (April 16, 2021): 1715. http://dx.doi.org/10.3390/jcm10081715.
Full textAbstract:
Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and a leading cause of cancer-related deaths worldwide. Although much progress has been made in HCC drug development in recent years, treatment options remain limited. The major cause of HCC is chronic hepatitis B virus (HBV) infection. Despite the existence of a vaccine, more than 250 million individuals are chronically infected by HBV. Current antiviral therapies can repress viral replication but to date there is no cure for chronic hepatitis B. Of note, inhibition of viral replication reduces but does not eliminate the risk of HCC development. HBV contributes to liver carcinogenesis by direct and indirect effects. This review summarizes the current knowledge of HBV-induced host epigenetic alterations and their association with HCC, with an emphasis on the interactions between HBV proteins and the host cell epigenetic machinery leading to modulation of gene expression.
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Akhova, Oksana, Matthew Bainbridge, and Vikram Misra. "The Neuronal Host Cell Factor-Binding Protein Zhangfei Inhibits Herpes Simplex Virus Replication." Journal of Virology 79, no. 23 (December 15, 2005): 14708–18. http://dx.doi.org/10.1128/jvi.79.23.14708-14718.2005.
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ABSTRACT During lytic infection in epithelial cells the expression of herpes simplex virus type 1 (HSV-1) immediate-early (IE) genes is initiated by a multiprotein complex comprising the virion-associated protein VP16 and two cellular proteins, host cellular factor (HCF) and Oct-1. Oct-1 directly recognizes TAATGARAT elements in promoters of IE genes. The role of HCF is not clear. HSV-1 also infects sensory neurons innervating the site of productive infection and establishes a latent infection in these cells. It is likely that some VP16 is retained by the HSV-1 nucleocapsid as it reaches the neuronal nucleus. Its activity must therefore be suppressed for successful establishment of viral latency. Recently, we discovered an HCF-binding cellular protein called Zhangfei. Zhangfei, in an HCF-dependent manner, inhibits Luman/LZIP/CREB3, another cellular HCF-binding transcription factor. Here we show that Zhangfei is selectively expressed in human neurons. When delivered to cultured cells that do not normally express the protein, Zhangfei inhibited the ability of VP16 to activate HSV-1 IE expression. The inhibition was specific for HCF-dependent transcriptional activation by VP16, since a Gal4-VP16 chimeric protein was inhibited only on a TAATGARAT-containing promoter and not a on a Gal4-containing promoter. Zhangfei associated with VP16 and inhibited formation of the VP16-HCF-Oct-1 complex on TAATGARAT motifs. Zhangfei also suppressed HSV-1-induced expression of several cellular genes including topoisomerase IIα, suggesting that in addition to suppressing IE expression Zhangfei may have an inhibitory effect on HSV-1 DNA replication and late gene expression.
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Lu, Rui, Ping Yang, Sharmila Padmakumar, and Vikram Misra. "The Herpesvirus Transactivator VP16 Mimics a Human Basic Domain Leucine Zipper Protein, Luman, in Its Interaction with HCF." Journal of Virology 72, no. 8 (August 1, 1998): 6291–97. http://dx.doi.org/10.1128/jvi.72.8.6291-6297.1998.
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ABSTRACT In human cells infected with herpes simplex virus (HSV), viral gene expression is initiated by the virion protein VP16. VP16 does not bind DNA directly but forms a multiprotein complex on the viral immediate-early gene promoters with two cellular proteins: the POU domain protein Oct-1 and host cell factor (HCF; also called C1, VCAF, and CFF). Despite its apparent role in stabilizing the VP16-induced transcription complex, the natural biological role of HCF is unclear. Only recently HCF has been implicated in control of the cell cycle. To determine the role of HCF in cells and answer why HSV has evolved an HCF-dependent mechanism for the initiation of the lytic cycle, we identified the first human ligand for HCF (R. Lu et al., Mol. Cell. Biol. 17:5117–5126, 1997). This protein, Luman, is a member of the CREB/ATF family of transcription factors that can activate transcription from promoters containing cyclic AMP response elements (CRE). Here we provide evidence that Luman and VP16 share two important structural features: an acidic activation domain and a common mechanism for binding HCF. We found that Luman, its homolog in Drosophila, dCREB-A (also known as BBF-2), and VP16 bind to HCF by a motif, (D/E)HXY(S/A), present in all three proteins. In addition, a mutation (P134S) in HCF that prevents VP16 binding also abolishes its binding to Luman and dCREB-A. We also show that while interaction with HCF is not required for the ability of Luman to activate transcription when tethered to the GAL4 promoter, it appears to be essential for Luman to activate transcription through CRE sites. These data suggest that the HCF-Luman interaction may represent a conserved mechanism for transcriptional regulation in metazoans, and HSV mimics this interaction with HCF to monitor the physiological state of the host cell.
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Akil, Abdellah, Peixuan Song, Juan Peng, Claire Gondeau, Didier Samuel, and Ama Gassama-Diagne. "PIAS1 Regulates Hepatitis C Virus-Induced Lipid Droplet Accumulation by Controlling Septin 9 and Microtubule Filament Assembly." Pathogens 10, no. 10 (October 15, 2021): 1327. http://dx.doi.org/10.3390/pathogens10101327.
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Chronic hepatitis C virus (HCV) infection often leads to fibrosis and chronic hepatitis, then cirrhosis and ultimately hepatocellular carcinoma (HCC). The processes of the HVC life cycle involve intimate interactions between viral and host cell proteins and lipid metabolism. However, the molecules and mechanisms involved in this tripartite interaction remain poorly understood. Herein, we show that the infection of HCC-derived Huh7.5 cells with HCV promotes upregulation of the protein inhibitor of activated STAT1 (PIAS1). Reciprocally, PIAS1 regulated the expression of HCV core protein and HCV-induced LD accumulation and impaired HCV replication. Furthermore, PIAS1 controlled HCV-promoted septin 9 filament formation and microtubule polymerization. Subsequently, we found that PIAS1 interacted with septin 9 and controlled its assembly on filaments, which thus affected septin 9-induced lipid droplet accumulation. Taken together, these data reveal that PIAS1 regulates the accumulation of lipid droplets and offer a meaningful insight into how HCV interacts with host proteins.
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Słomka, Artur, Tudor Mocan, Bingduo Wang, Iuliana Nenu, Sabine Urban, Maria Gonzalez-Carmona, Ingo Schmidt-Wolf, et al. "EVs as Potential New Therapeutic Tool/Target in Gastrointestinal Cancer and HCC." Cancers 12, no. 10 (October 17, 2020): 3019. http://dx.doi.org/10.3390/cancers12103019.
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For more than a decade, extracellular vesicles (EVs) have been in focus of science. Once thought to be an efficient way to eliminate undesirable cell content, EVs are now well-accepted as being an important alternative to cytokines and chemokines in cell-to-cell communication route. With their cargos, mainly consisting of functional proteins, lipids and nucleic acids, they can activate signalling cascades and thus change the phenotype of recipient cells at local and systemic levels. Their substantial role as modulators of various physiological and pathological processes is acknowledged. Importantly, more and more evidence arises that EVs play a pivotal role in many stages of carcinogenesis. Via EV-mediated communication, tumour cells can manipulate cells from host immune system or from the tumour microenvironment, and, ultimately, they promote tumour progression and modulate host immunity towards tumour’s favour. Additionally, the role of EVs in modulating resistance to pharmacological and radiological therapy of many cancer types has become evident lately. Our understanding of EV biology and their role in cancer promotion and drug resistance has evolved considerably in recent years. In this review, we specifically discuss the current knowledge on the association between EVs and gastrointestinal (GI) and liver cancers, including their potential for diagnosis and treatment.
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Fan, Jinxin, Eduardo Barbieri, Shriarjun Shastry, Stefano Menegatti, Cristiana Boi, and Ruben G. Carbonell. "Purification of Adeno-Associated Virus (AAV) Serotype 2 from Spodoptera frugiperda (Sf9) Lysate by Chromatographic Nonwoven Membranes." Membranes 12, no. 10 (September 27, 2022): 944. http://dx.doi.org/10.3390/membranes12100944.
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The success of adeno-associated virus (AAV)-based therapeutics in gene therapy poses the need for rapid and efficient processes that can support the growing clinical demand. Nonwoven membranes represent an ideal tool for the future of virus purification: owing to their small fiber diameters and high porosity, they can operate at high flowrates while allowing full access to target viral particles without diffusional limitations. This study describes the development of nonwoven ion-exchange membrane adsorbents for the purification of AAV2 from an Sf9 cell lysate. A strong anion-exchange (AEX) membrane was developed by UV grafting glycidyl methacrylate on a polybutylene terephthalate nonwoven followed by functionalization with triethylamine (TEA), resulting in a quaternary amine ligand (AEX-TEA membrane). When operated in bind-and-elute mode at a pH higher than the pI of the capsids, this membrane exhibited a high AAV2 binding capacity (9.6 × 1013 vp·mL−1) at the residence time of 1 min, and outperformed commercial cast membranes by isolating AAV2 from an Sf9 lysate with high productivity (2.4 × 1013 capsids·mL−1·min−1) and logarithmic reduction value of host cell proteins (HCP LRV ~1.8). An iminodiacetic acid cation-exchange nonwoven (CEX-IDA membrane) was also prepared and utilized at a pH lower than the pI of capsids to purify AAV2 in a bind-and-elute mode, affording high capsid recovery and impurity removal by eluting with a salt gradient. To further increase purity, the CEX-IDA and AEX-TEA membranes were utilized in series to purify the AAV2 from the Sf9 cell lysate. This membrane-based chromatography process also achieved excellent DNA clearance and a recovery of infectivity higher that that reported using ion-exchange resin chromatography.
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Hayashi, Sanae, Katsuya Nagaoka, and Yasuhito Tanaka. "Blood-Based Biomarkers in Hepatitis B Virus-Related Hepatocellular Carcinoma, Including the Viral Genome and Glycosylated Proteins." International Journal of Molecular Sciences 22, no. 20 (October 13, 2021): 11051. http://dx.doi.org/10.3390/ijms222011051.
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Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) development and is a global public health issue. High performance biomarkers can aid the early detection of HCC development in HBV-infected individuals. In addition, advances in the understanding of the pathogenesis of HBV infection and in clinical laboratory techniques have enabled the establishment of disease-specific tests, prediction of the progression of liver diseases, including HCC, and auxiliary diagnosis of HCC, using blood-based methods instead of biopsies of liver or HCC tissues. Viral factors such as the HBV genotype, HBV genetic mutations, HBV DNA, and HBV-related antigens, as well as host factors, such as tumor-associated proteins and post-translational modifications, especially glycosylated proteins, can be blood-based, disease-specific biomarkers for HCC development in HBV-infected patients. In this review, we describe the clinical applications of viral biomarkers, including the HBV genome and glycosylated proteins, for patients at a risk of HBV-related HCC, based on their molecular mechanisms. In addition, we introduce promising biomarker candidates for practical use, including colony stimulating factor 1 receptor (CSF1R), extracellular vesicles, and cell-free, circulating tumor DNA. The clinical use of such surrogate markers may lead to a better understanding of the risk of disease progression and early detection of HCC in HBV-infected patients, thereby improving their prognosis.
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Gerresheim, Roeb, Michel, and Niepmann. "Hepatitis C Virus Downregulates Core Subunits of Oxidative Phosphorylation, Reminiscent of the Warburg Effect in Cancer Cells." Cells 8, no. 11 (November 8, 2019): 1410. http://dx.doi.org/10.3390/cells8111410.
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Hepatitis C Virus (HCV) mainly infects liver hepatocytes and replicates its single-stranded plus strand RNA genome exclusively in the cytoplasm. Viral proteins and RNA interfere with the host cell immune response, allowing the virus to continue replication. Therefore, in about 70% of cases, the viral infection cannot be cleared by the immune system, but a chronic infection is established, often resulting in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Induction of cancer in the host cells can be regarded to provide further advantages for ongoing virus replication. One adaptation in cancer cells is the enhancement of cellular carbohydrate flux in glycolysis with a reduction of the activity of the citric acid cycle and aerobic oxidative phosphorylation. To this end, HCV downregulates the expression of mitochondrial oxidative phosphorylation complex core subunits quite early after infection. This so-called aerobic glycolysis is known as the “Warburg Effect” and serves to provide more anabolic metabolites upstream of the citric acid cycle, such as amino acids, pentoses and NADPH for cancer cell growth. In addition, HCV deregulates signaling pathways like those of TNF-β and MAPK by direct and indirect mechanisms, which can lead to fibrosis and HCC.
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Angelova, Magdalena, Rodrigo F. Ortiz-Meoz, Suzanne Walker, and David M. Knipe. "Inhibition of O-LinkedN-Acetylglucosamine Transferase Reduces Replication of Herpes Simplex Virus and Human Cytomegalovirus." Journal of Virology 89, no. 16 (June 3, 2015): 8474–83. http://dx.doi.org/10.1128/jvi.01002-15.
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ABSTRACTO-linkedN-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies nuclear and cytoplasmic proteins via O-linked addition of a singleN-acetylglucosamine (GlcNAc) moiety. Among the many targets of OGT is host cell factor 1 (HCF-1), a transcriptional regulator that is required for transactivation of the immediate-early genes of herpes simplex virus (HSV). HCF-1 is synthesized as a large precursor that is proteolytically cleaved by OGT, which may regulate its biological function. In this study, we tested whether inhibition of the enzymatic activity of OGT with a small molecule inhibitor, OSMI-1, affects initiation of HSV immediate-early gene expression and viral replication. We found that inhibiting OGT's enzymatic activity significantly decreased HSV replication. The major effect of the inhibitor occurred late in the viral replication cycle, when it reduced the levels of late proteins and inhibited capsid formation. However, depleting OGT levels with small interfering RNA (siRNA) reduced the expression of HSV immediate-early genes, in addition to reducing viral yields. In this study, we identified OGT as a novel cellular factor involved in HSV replication. Our results obtained using a small molecule inhibitor and siRNA depletion suggest that OGT's glycosylation and scaffolding functions play distinct roles in the replication cycle of HSV.IMPORTANCEAntiviral agents can target viral or host gene products essential for viral replication. O-GlcNAc transferase (OGT) is an important cellular enzyme that catalyzes the posttranslational addition of GlcNAc sugar residues to hundreds of nuclear and cytoplasmic proteins, and this modification regulates their activity and function. Some of the known OGT targets are cellular proteins that are critical for the expression of herpes simplex virus (HSV) genes, suggesting a role for OGT in the replication cycle of HSV. In this study, we found that OGT is required for efficient expression of viral genes and for assembly of new virions. Thus, we identify OGT as a novel host factor involved in the replication of HSV and a potential target for antiviral therapy.
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El-Khobar, Korri E., Enoch Tay, Eve Diefenbach, Brian S. Gloss, Jacob George, and Mark W. Douglas. "Polo-like kinase-1 mediates hepatitis C virus-induced cell migration, a drug target for liver cancer." Life Science Alliance 6, no. 11 (August 30, 2023): e202201630. http://dx.doi.org/10.26508/lsa.202201630.
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Polo-like kinase 1 (PLK1) is a regulator of cell mitosis and cytoskeletal dynamics.PLK1overexpression in liver cancer is associated with tumour progression, metastasis, and vascular invasion. Hepatitis C virus (HCV) NS5A protein stimulates PLK1-mediated phosphorylation of host proteins, so we hypothesised that HCV–PLK1 interactions might be a mechanism for HCV-induced liver cancer. We used a HCV cell-culture model (Jc1) to investigate the effects of virus infection on the cytoskeleton. In HCV-infected cells, a novel posttranslational modification in β-actin was observed with phosphorylation at Ser239. Using in silico and in vitro approaches, we identified PLK1 as the mediating kinase. In functional experiments with a phosphomimetic mutant form of β-actin, Ser239 phosphorylation influences β-actin polymerization and distribution, resulting in increased cell motility. The changes were prevented by treating cells with the PLK1 inhibitor volasertib. In HCV-infected hepatocytes, increased cell motility contributes to cancer cell migration, invasion, and metastasis. PLK1 is an important mediator of these effects and early treatment with PLK1 inhibitors may prevent or reduce HCC progression, particularly in people with HCV-induced HCC.
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Sabitzki, Ricarda, Anna-Lena Roßmann, Marius Schmitt, Sven Flemming, Andrés Guillén-Samander, Hannah Michaela Behrens, Ernst Jonscher, Katharina Höhn, Ulrike Fröhlke, and Tobias Spielmann. "Role of Rabenosyn-5 and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites." PLOS Biology 22, no. 5 (May 31, 2024): e3002639. http://dx.doi.org/10.1371/journal.pbio.3002639.
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Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of Plasmodium falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasite’s food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the repurposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here, we identified a parasite Rbsn5-like protein and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5L resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall, this indicates that despite the low sequence conservation of PfRbsn5L and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC uptake consists of a highly parasite-specific part that feeds endocytosed material into an endosomal system containing more canonical elements, leading to the delivery of HCC to the food vacuole.
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Abusaliya, Abuyaseer, Se Hyo Jeong, Pritam Bhagwan Bhosale, Hun Hwan Kim, Min Yeong Park, Eunhye Kim, Chung Kil Won, et al. "Mechanistic Action of Cell Cycle Arrest and Intrinsic Apoptosis via Inhibiting Akt/mTOR and Activation of p38-MAPK Signaling Pathways in Hep3B Liver Cancer Cells by Prunetrin—A Flavonoid with Therapeutic Potential." Nutrients 15, no. 15 (July 31, 2023): 3407. http://dx.doi.org/10.3390/nu15153407.
Full textAbstract:
Hepatocellular carcinoma (HCC) has a poor prognosis and a low survival rate. Drugs without side effects are desperately needed since chemotherapy has a negative effect on the host cells. Previous research has firmly established that plant-based compounds have significant bioactivities without a negative impact on the host. Flavonoids, in particular, are a class of compounds with both anti-inflammatory and anti-cancer properties. Prunetrin (PUR) is a glycosyloxyisoflavone (Prunetin 4′-O-glucoside) derived from Prunus sp., and its other form, called prunetin, showed optimistic results in an anti-cancerous study. Hence, we aimed to discover the anti-cancer ability of prunetrin in liver cancer Hep3B cells. Our cytotoxicity results showed that PUR can decrease cell viability. The colony formation assay confirms this strongly and correlates with cell cytotoxicity results. Prunetrin, in a dose-dependent manner, arrested the cell cycle in the G2/M phase and decreased the expression of cyclin proteins such as Cyclin B1, CDK1/CDC2, and CDC25c. Prunetrin treatment also promoted the strong cleavage of two important apoptotic hallmark proteins called PARP and caspase-3. It also confirms that apoptosis occurs through the mitochondrial pathway through increased expression of cleaved caspase-9 and increased levels of the pro-apoptotic protein Bak. Bak was significantly increased with the declining expression of the anti-apoptotic protein Bcl-xL. Next, it inhibits the mTOR/AKT signaling pathways, proving that prunetrin includes apoptosis and decreases cell viability by suppressing these pathways. Further, it was also observed that the activation of p38-MAPK was dose-dependent. Taken together, they provide evidence that prunetrin has an anti-cancerous ability in Hep3B liver cancer cells by arresting the cell cycle via p38 and inhibiting mTOR/AKT.
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Budzinska, Magdalena A., Nicholas A. Shackel, Stephan Urban, and Thomas Tu. "Cellular Genomic Sites of Hepatitis B Virus DNA Integration." Genes 9, no. 7 (July 20, 2018): 365. http://dx.doi.org/10.3390/genes9070365.
Full textAbstract:
Infection with the Hepatitis B Virus (HBV) is one of the strongest risk-factors for liver cancer (hepatocellular carcinoma, HCC). One of the reported drivers of HCC is the integration of HBV DNA into the host cell genome, which may induce pro-carcinogenic pathways. These reported pathways include: induction of chromosomal instability; generation of insertional mutagenesis in key cancer-associated genes; transcription of downstream cancer-associated cellular genes; and/or formation of a persistent source of viral protein expression (particularly HBV surface and X proteins). The contribution of each of these specific mechanisms towards carcinogenesis is currently unclear. Here, we review the current knowledge of specific sites of HBV DNA integration into the host genome, which sheds light on these mechanisms. We give an overview of previously-used methods to detect HBV DNA integration and the enrichment of integration events in specific functional and structural cellular genomic sites. Finally, we posit a theoretical model of HBV DNA integration during disease progression and highlight open questions in the field.
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Fujiwara, Naoto, and Yujin Hoshida. "Hepatocellular Carcinoma Risk Stratification by Genetic Profiling in Patients with Cirrhosis." Seminars in Liver Disease 39, no. 02 (March 25, 2019): 153–62. http://dx.doi.org/10.1055/s-0039-1681031.
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AbstractPrediction of future hepatocellular carcinoma (HCC) risk in the sizable chronic liver disease population is an urgent unmet need to enable regular HCC screening for early detection. Germline deoxyribonucleic acid polymorphisms likely represent etiology-specific host factors that determine HCC susceptibility, including single nucleotide polymorphisms in EGF, IFNL3, MICA, and TLL1 in hepatitis C with or without active viral infection, and PNPLA3, TM6SF2, and MBOAT7 in metabolic liver diseases. Transcriptome-based prognostic liver signature in diseased liver tissue has been associated with long-term HCC risk in viral and metabolic etiologies. Transcriptomic signatures of hepatic injury and specific cell type such as aggregated lymphocytes also predict HCC development. Circulating factors such as proteins and their chemical modification, nucleotides, and metabolites may serve for less-invasive assessment of short- or long-term HCC risk. These biomarkers will enable individual HCC risk-based personalized clinical management for cost-effective early HCC detection and improvement of patient survival.
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Han, Qiuju, Huajun Zhao, Yu Jiang, Chunlai Yin, and Jian Zhang. "HCC-Derived Exosomes: Critical Player and Target for Cancer Immune Escape." Cells 8, no. 6 (June 8, 2019): 558. http://dx.doi.org/10.3390/cells8060558.
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Hepatocellular carcinoma (HCC) is a primary malignancy of the liver, and currently the second most common cause of cancer-related deaths worldwide with increasing incidence and poor prognosis. Exosomes are now considered as important mediators of host anti-tumor immune response as well as tumor cell immune escape. HCC-derived exosomes have been shown to attenuate the cytotoxicity of T-cells and NK cells, and promote the immuno-suppressive M2 macrophages, N2 neutrophils, and Bregs. These exosomes harbor several immune-related non-coding RNAs and proteins that drive immune-escape and tumor progression, and thus may serve as potential diagnostic biomarkers and therapeutic targets for HCC. In a previous study, we identified miR146a as an exosomal factor that promotes M2-polarization and suppresses the anti-HCC function of T-cells. In this review, we summarized the role of tumor-derived exosomes and their key components in mediating tumor immune escape during HCC development.
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Grapes, Matthew, and Peter O'Hare. "Differences in Determinants Required for Complex Formation and Transactivation in Related VP16 Proteins." Journal of Virology 74, no. 21 (November 1, 2000): 10112–21. http://dx.doi.org/10.1128/jvi.74.21.10112-10121.2000.
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ABSTRACT VP16-H is an essential structural protein of herpes simplex virus type 1 (HSV-1) and is also a potent activator of virus immediate-early (IE) gene expression. Current models of functional determinants within VP16-H indicate that it consists of two domains, an N-terminal domain involved in recruiting VP16-H to a multicomponent DNA binding complex with two host proteins, Oct-1 and host cell factor (HCF), and an acidic C-terminal domain exclusively involved in transactivation. VP16-E, from equine herpesvirus 1 (EHV-1), exhibits strong conservation with the N-terminal domain of VP16-H but, with the exception of a short segment at the extreme C terminus, lacks almost the entire acidic C-terminal domain. Studies of key activation determinants within the C terminus of VP16-H would predict that VP16-E may activate poorly, if at all. However, VP16-E is a potent activator of both EHV-1 and HSV-1 IE gene transcription. We show that VP16-E does not follow the simple two-domain model of VP16-H. Thus, despite the conservation in the N-terminal domains, this region in VP16-E is not sufficient for assembly into the DNA binding complex with Oct-1 and HCF. The short conserved determinant close to the C terminus is completely dispensable in VP16-H but is absolutely required in VP16-E. In activation studies, the potency of intact VP16-E was not recapitulated in chimeric proteins in which it was fused with a GAL4 DNA binding domain. Furthermore, a chimeric protein consisting of the C-terminal region of VP16-E fused to the N-terminal domain of VP16-H, while able to promote complex formation, nevertheless exhibited very weak activation. These results indicate that the mode of recruitment of the activation domain, i.e., through complex formation with Oct-1 and HCF, may be crucial for activation and that key determinants required for activation in VP16-E, and possibly VP16-H, may involve interactions between regions of the C terminus and the N terminus rather than discrete domains with independent functions.
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Padarath, Kiyasha, Aurélie Deroubaix, Previn Naicker, Stoyan Stoychev, and Anna Kramvis. "Comparative Proteomic Analysis of Huh7 Cells Transfected with Sub-Saharan African Hepatitis B Virus (Sub)genotypes Reveals Potential Oncogenic Factors." Viruses 16, no. 7 (June 29, 2024): 1052. http://dx.doi.org/10.3390/v16071052.
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In sub-Saharan Africa (SSA), the (sub)genotypes A1, D3, and E of the hepatitis B virus (HBV) prevail. Individuals infected with subgenotype A1 have a 4.5-fold increased risk of HCC compared to those infected with other (sub)genotypes. The effect of (sub)genotypes on protein expression and host signalling has not been studied. Mass spectrometry was used to analyse the proteome of Huh7 cells transfected with replication-competent clones. Proteomic analysis revealed significantly differentially expressed proteins between SSA (sub)genotypes. Different (sub)genotypes have the propensity to dysregulate specific host signalling pathways. Subgenotype A1 resulted in dysregulation within the Ras pathway. Ras-associated protein, RhoC, was significantly upregulated in cells transfected with subgenotype A1 compared to those transfected with other (sub)genotypes, on both a proteomic (>1.5-fold) and mRNA level (p < 0.05). Two of the main cellular signalling pathways involving RHOC, MAPK and PI3K/Akt/mTOR, regulate cell growth, motility, and survival. Downstream signalling products of these pathways have been shown to increase MMP2 and MMP9 expression. An extracellular MMP2 and MMP9 ELISA revealed a non-significant increase in MMP2 and MMP9 in the cells transfected with A1 compared to the other (sub)genotypes (p < 0.05). The upregulated Ras-associated proteins have been implicated as oncoproteins in various cancers and could contribute to the increased hepatocarcinogenic potential of A1.
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Bhattacharya, Ilina, Tejabhiram Yadavalli, David Wu, and Deepak Shukla. "Plasma Membrane-Derived Liposomes Exhibit Robust Antiviral Activity against HSV-1." Viruses 14, no. 4 (April 12, 2022): 799. http://dx.doi.org/10.3390/v14040799.
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Plasma membranes host a plethora of proteins and glycans on their outer surface that are exploited by viruses to enter the cells. In this study, we have utilized this property to limit a viral infection using plasma membrane-derived vesicles. We show that plasma membrane-derived liposomes are prophylactically and therapeutically competent at preventing herpes simplex virus type-1 (HSV-1) infection. Plasma membrane liposomes derived from human corneal epithelial (HCE) cells, which are natural targets of HSV-1 infection, as well as Vero and Chinese hamster ovary (CHO) cells were used in this study. Our study clearly demonstrates that HCE and Vero-derived cellular liposomes, which express the viral entry-specific cell surface protein receptors, exhibit robust antiviral activity especially when compared to CHO-derived liposomes, which lack the relevant HSV-1 entry receptors. Further experimentation of the plasma membrane-derived liposomes with HSV type-2 (HSV-2) and pseudorabies virus yielded similar results, indicating strong potential for the employment of these liposomes to study viral entry mechanisms in a cell free-environment.
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Pan, Wenting, Nasha Zhang, Wenjuan Liu, Jibing Liu, Liqing Zhou, Yang Liu, and Ming Yang. "The long noncoding RNA GAS8-AS1 suppresses hepatocarcinogenesis by epigenetically activating the tumor suppressor GAS8." Journal of Biological Chemistry 293, no. 44 (September 18, 2018): 17154–65. http://dx.doi.org/10.1074/jbc.ra118.003055.
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Long noncoding RNAs (lncRNAs) are vital players in cancers, including hepatocellular carcinoma (HCC). We previously identified an lncRNA, GAS8-AS1, that is located in intron 2 of GAS8. However, its involvement in HCC is still largely unknown. In this study, we report that both GAS8-AS1 and its host gene GAS8 act as HCC tumor suppressors. We found that expression of GAS8-AS1 or GAS8 is significantly decreased in HCC tissues and is associated with a poor prognosis among HCC patients. Interestingly, lncRNA GAS8-AS1 could promote GAS8 transcription. We detected a CpG island in the GAS8 promoter, but lncRNA GAS8-AS1 did not affect DNA methylation at this GAS8 promoter site. Moreover, we identified two GAS8-AS1–interacting proteins, mixed-lineage leukemia 1 (MLL1), a histone 3 Lys-4 (H3K4) methyltransferase, and its partner WD-40 repeat protein 5 (WDR5). RNA pulldown, ChIP, and RNA immunoprecipitation assays revealed that GAS8-AS1 is required for maintaining the GAS8 promoter in an open chromatin state by recruiting the MLL1/WDR5 complex and for enhancing RNA polymerase II activity and GAS8 transcription. Of note, GAS8-AS1–dependent GAS8 hyperactivation inhibited malignant transformation of hepatocytes. Our results provide important insights into how lncRNA GAS8-AS1 suppresses HCC development and suggest potential strategies for treating patients with liver cancer.
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Kapuria, Vaibhav, Ute F. Röhrig, Patrice Waridel, Fabienne Lammers, Vladimir S. Borodkin, Daan M. F. van Aalten, Vincent Zoete, and Winship Herr. "The conserved threonine-rich region of the HCF-1PRO repeat activates promiscuous OGT:UDP-GlcNAc glycosylation and proteolysis activities." Journal of Biological Chemistry 293, no. 46 (September 17, 2018): 17754–68. http://dx.doi.org/10.1074/jbc.ra118.004185.
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O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase–protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate—the so-called HCF-1PRO repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1PRO repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1PRO-repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor Rp-αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1PRO-repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1PRO-repeat sequences, we show that proposed OGT-based or UDP-GlcNAc–based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies.
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Colaianni, Francesco, Veronica Zelli, Chiara Compagnoni, Martina Sara Miscione, Mario Rossi, Davide Vecchiotti, Monica Di Padova, Edoardo Alesse, Francesca Zazzeroni, and Alessandra Tessitore. "Role of Circulating microRNAs in Liver Disease and HCC: Focus on miR-122." Genes 15, no. 10 (October 12, 2024): 1313. http://dx.doi.org/10.3390/genes15101313.
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miR-122 is the most abundant microRNA (miRNA) in the liver; it regulates several genes mainly involved in cell metabolism and inflammation. Host factors, diet, metabolic disorders and viral infection promote the development of liver diseases, including hepatocellular carcinoma (HCC). The downregulation of miR-122 in tissue is a common feature of the progression of liver injury. In addition, the release of miR-122 in the bloodstream seems to be very promising for the early diagnosis of both viral and non-viral liver disease. Although controversial data are available on the role of circulating miR-122 as a single biomarker, high diagnostic accuracy has been observed using miR-122 in combination with other circulating miRNAs and/or proteins. This review is focused on comprehensively summarizing the most recent literature on the potential role of circulating miR-122, and related molecules, as biomarker(s) of metabolic liver diseases, hepatitis and HCC.
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Tapper, Hans, Anna Karlsson, Matthias Mörgelin, Hans Flodgaard, and Heiko Herwald. "Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles." Blood 99, no. 5 (March 1, 2002): 1785–93. http://dx.doi.org/10.1182/blood.v99.5.1785.
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Human neutrophils have an important role in host defense against microbial infection. At different stages of an infectious process, neutrophils progressively up-regulate receptors and release various effector molecules. These are stored in several distinct types of granules with varying propensity to be secreted. Heparin-binding protein (HBP), also known as CAP37 or azurocidin, is a multifunctional, inactive serine-protease homologue. The present work shows that HBP is released from neutrophils on stimulation with secretagogues that do not trigger the secretion of azurophilic granule content. Therefore, the subcellular localization of HBP was investigated in more detail. Immunofluorescence microscopy revealed that HBP was localized close to the plasma membrane. Further analysis by fractionation of postnuclear supernatants from cavitated neutrophils showed that HBP is stored in azurophilic granules and secretory vesicles but that it is also detected to a minor extent in the plasma membrane. These findings were confirmed by immunoelectron microscopy showing that HBP colocalized with marker proteins of azurophilic granules and secretory vesicles. The presence of HBP in secretory vesicles possibly depends on the stage of cell differentiation, since the promyelocytic cell line HL-60 contains less HBP than mature neutrophils, stored exclusively in the less easily mobilized azurophilic granules. Our findings suggest that HBP can be synthesized or targeted to easily mobilized compartments at a late stage of neutrophil maturation. The ability of neutrophils to secrete HBP from secretory vesicles may be important for proinflammatory functions of this protein, such as the alteration of vascular permeability.
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Babb, Robert, C. Chris Huang, Deborah J. Aufiero, and Winship Herr. "DNA Recognition by the Herpes Simplex Virus Transactivator VP16: a Novel DNA-Binding Structure." Molecular and Cellular Biology 21, no. 14 (July 15, 2001): 4700–4712. http://dx.doi.org/10.1128/mcb.21.14.4700-4712.2001.
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ABSTRACT Upon infection, the herpes simplex virus (HSV) transcriptional activator VP16 directs the formation of a multiprotein-DNA complex—the VP16-induced complex—with two cellular proteins, the host cell factor HCF-1 and the POU domain transcription factor Oct-1, on TAATGARAT-containing sequences found in the promoters of HSV immediate-early genes. HSV VP16 contains carboxy-terminal sequences important for transcriptional activation and a central conserved core that is important for VP16-induced complex assembly. On its own, VP16 displays little, if any, sequence-specific DNA-binding activity. We show here that, within the VP16-induced complex, however, the VP16 core has an important role in DNA binding. Mutation of basic residues on the surface of the VP16 core reveals a novel DNA-binding surface with essential residues which are conserved among VP16 orthologs. These results illuminate how, through association with DNA, VP16 is able to interpret cis-regulatory signals in the DNA to direct the assembly of a multiprotein-DNA transcriptional regulatory complex.
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Gieske, Mary C., Gi Youn Na, Yongbum Koo, Misung Jo, Thomas E. Curry, and Chemyong Ko. "Decay-accelerating factor in the periovulatory rat ovary." Journal of Endocrinology 186, no. 2 (August 2005): 303–13. http://dx.doi.org/10.1677/joe.1.06218.
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One of the most prominent inflammatory reactions is the activation of the complement system. The activated complement system does not distinguish between pathogens and the host cell. In order to prevent autologous complement-mediated attack, host cells express a variety of both membrane-bound and fluid-phase complement regulatory proteins which control activity of the complement cascade by acting on convertase enzymes or the membrane-attack complex. Although the process of ovulation is facilitated by the inflammatory reaction, this reaction has the potential to cause serious damage to growing follicles, ovulated follicles, and other important ovarian tissues. This study was undertaken to characterize the expression and regulation of decay-accelerating factor (DAF), a complement regulator, as a potential mediator of ovarian tissue protection from ovulatory inflammation. DNA microarray and Northern blot analyses showed that an ovulatory gonadotropin stimulus dramatically yet transiently induced DAF mRNA expression in the immature rat ovary. Northern blot and PCR analyses revealed that of the three known DAF isoforms, glycosylphosphatidylinositol (GPI)-, soluble-, and transmembrane-(TM) DAF, GPI-DAF was the predominant form. In situ hybridization localized GPI-DAF mRNA expression in the theca-interstitial cells of the periovulatory ovary. Neither the anti-progestin RU486 nor the cyclooxygenase inhibitor indomethacin significantly inhibited human chorionic gonadotropin (hCG)-induced GPI-DAF mRNA expression in vivo. In vitro theca cell culture studies indicated that hCG induces GPI-DAF mRNA expression through the protein kinase A pathway. This study suggests that gonadotropin-induced GPI-DAF may be involved in the protection of ovarian tissues from the potential attack by the complement system activated by the inflammatory response associated with ovulation.
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Gromelsky Ljungcrantz, Emily, Sanna Askman, Fredrik Sjövall, and Magnus Paulsson. "Biomarkers in lower respiratory tract samples in the diagnosis of ventilator-associated pneumonia: a systematic review." European Respiratory Review 34, no. 176 (April 2025): 240229. https://doi.org/10.1183/16000617.0229-2024.
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BackgroundVentilator-associated pneumonia (VAP) is the most common intensive care unit-acquired infection, yet its diagnosis is complicated by the lack of reliable diagnostic criteria and validated biomarkers. Due to the compartmentalisation of the immune response, host proteins in respiratory tract samples are more likely than serum proteins to accurately identify VAP. However, a reliable biomarker is still missing and it is generally agreed that >90% sensitivity and specificity are required for the introduction of a VAP biomarker into clinical routine.MethodsA structured database search was performed to identify publications aimed at deriving or verifying human respiratory tract VAP biomarkers. The results were screened by two independent reviewers and summarised using statistical and narrative synthesis.Results40 articles were identified, focusing on 29 unique biomarkers with clinical and microbiological diagnoses of VAP as the reference standard. The most frequently studied biomarker was soluble triggering receptor expressed on myeloid cell 1 (sTREM-1) (n=16), followed by various interleukins (n=7), neutrophil-related proteins (n=8) and amylase as a surrogate for microaspiration (n=4). The target accuracy of >90% specificity and sensitivity for VAP was reported in four publications on sTREM-1, one on pentraxin-3 (PTX3) and one on heparin-binding protein (HBP). Meta-analysis of sTREM-1 resulted in a sensitivity of 78% (95% CI 61–89%) and specificity of 76% (95% CI 49–91%).DiscussionThis systematic review found that no biomarker can currently be recommended for clinical use due to performance below 90% specificity or sensitivity, or insufficient data (PTX3 and HBP). Accurate clinical phenotyping into VAP subcategories may enable the discovery of VAP biomarkers with higher accuracy.
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Gao, Biwei, Ruilian Xu, and Bo Hu. "A survey on the detection of hepatitis B virus (HBV) in blood samples of patients with hepatocellular carcinoma (HCC) in China." Journal of Clinical Oncology 41, no. 16_suppl (June 1, 2023): e16178-e16178. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.e16178.
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e16178 Background: China is the country with the highest occurrence of HCC, accounting for about 45.3% of the new cases in the world. HBV can cause chronic infection and potentially leads to the occurrence of HCC. Generally, HBV genomic segments are able to be integrated into the host’s genome, then affect the expression of host genes, form carcinogenic proteins and cause chromosomal aberrations in host cells. HCC tumor tissue samples have more integration sites of HBV on the genome compared with para-cancerous tissues. It suggests that HBV is likely to be continuously enriched and integrated in the host genome during the development of HCC, and promotes the occurrence of HCC. There is plenty of tumor molecular information in the blood plasma of patients. Features of virus integration patterns and relatively quantitative level of virus DNA in the blood plasma should be intriguing to study. Methods: 216 blood samples of 178 HCC Chinese patients were collected. Circulating cell-free DNA (cfDNA) was processed by hybridization-based target enrichment and sequenced by next-generation sequencing (NGS) technique using Haplox HapOnco 680 panel. Results: In the blood of 178 HCC patients, the cfDNA of a total of 58 patients was detected with HBV genome fragments integration. These HBV genome fragments mainly belong to two subtypes, HBV-B and HBV-C, of which single HBV-B type was detected in 18 cases, accounting for 31% of HBV integrated infections, single HBV-C type was detected in 39 cases, accounting for 67.2% of HBV integrated infections. Additionally, HBV-B and HBV-C were co-integrated in 1 case. In the integrated host genome, the average number of breakpoints produced by HBV-B infection was 3.158, and the most frequent integration site was ANKRD26P1 (6 times). The average number of breakpoints produced by HBV-C integration was 3.65, and the most frequent integration site was TERT (15 times). There was no significant difference in the number of breakpoints produced between HBV-B and HBV-C groups (p=0.49). In the total of 58 HBV-B datasets and 64 HBV-C datasets, the average abundance of integrated HBV-B reads was 5.6e-05, and the average abundance of HBV-C was 5.04e-0.5, and there was no significant difference between them (p = 0.71). Conclusions: Approximately one third of the 178 HCC patients were detected positive for HBV integrationin their blood samples, with the ratio of subtypes HBV-B and HBV-C roughly 1:2. HBV-B is slightly lower than HBV-C in the number of average breakpoint, but the difference is not significant. Similarly, the overall abundance of HBV-B and HBV-C of integrated reads in blood is also quite similar. However, the hotspot integration sites of HBV-B and HBV-C on the host genome are quite different.