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Determining ADCC activity of antibody-based therapeutc molecules using two bioluminescent reporter-based Bioassays.
Curr Protoc | 2021 | 查看原文 |
作者：Denise Garvin, Pete Stecha, Julia Gilden, Jun Wang, Jamison Grailer, Jim Hartnett, Frank Fan, Mei Cong, Zhijie Jey Cheng
- 摘要：Antibody Fc effector function is one of the main mechanisms of action (MoA) for therapeutic monoclonal antibodies. Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding the Fc effector function during monoclonal antibody development. This article covers two cell-based ADCC bioassays which can quantitatively measure the antibody potency in ADCC. Basic Protocol 1 describes the ADCC reporter bioassay using engineered ADCC effector cells which measures the FcγRIIIa-mediated luciferase reporter activation upon the binding of antibody-coated target cells. Basic Protocol 2 describes the PBMC ADCC bioassay using primary peripheral blood mononuclear cells (PBMC) as effector cells and engineered HiBiT target cells in an assay that measures the release of HiBiT from target cells upon antibody-mediated target lysis. Optimization of several key assay parameters including cell handling, effector:target (E:T) ratios, assay plate, and plate reader requirement, and how these parameters impact assay performance are discussed. © 2021 Promega Corporation. Current Protocols published by Wiley Periodicals LLC.展开
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Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions.
Cell Death Dis | 2022 | 查看原文 |
作者：Kristina Seiler, Magali Humbert, Petra Minder, Iris Mashimo, Anna M. Schläfli, Deborah Krauer, Elena A. Federzoni, Bich Vu, James J. Moresco, John R. Yates 3rd, Martin C. Sadowski, Ramin Radpour, Thomas Kaufmann, Jean-Emmanuel Sarry, Joern Dengjel, Mario P. Tschan & Bruce E. Torbett
- 摘要：The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.展开
关键词： N7200，hexokinases, HK1, HK2, HK3, glycolysis, acute myeloid leukemia AML, CD34+ hematopoietic stem and progenitor cells, myeloid differentiation, CRISPR/Cas9, RNA-seq, ATAC-seq, pulldown, ROS, DNA damage, oxidative stress, apoptosis, BIM, BCL-2 family, ATRA, cell survival
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Antibody targeting of E3 ubiquitin ligases for receptor degradation
Nature | 2022 | 查看原文 |
作者：Hadir Marei, Wen-Ting K. Tsai, Yee-Seir Kee, Karen Ruiz, Jieyan He, Chris Cox, Tao Sun, Sai Penikalapati, Pankaj Dwivedi, Meena Choi, David Kan, Pablo Saenz-Lopez, Kristel Dorighi, Pamela Zhang, Yvonne T. Kschonsak, Noelyn Kljavin, Dhara Amin, Ingrid Kim, Andrew G. Mancini, Thao Nguyen, Chunling Wang, Eric Janezic, Alexander Doan, Elaine Mai, …Felipe de Sousa e Melo
- 摘要：Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for ‘on-demand’ degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.展开
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Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike
Cell | 2022 | 查看原文 |
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Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells.
iScience | 2022 | 查看原文 |
作者：Lydia Ogrodzinski, et al.
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Programmable protein delivery with a bacterial contractile injection system.
Nature | 2023 | 查看原文 |
作者：Joseph Kreitz, Mirco J. Friedrich, Akash Guru, Blake Lash, Makoto Saito, Rhiannon K. Macrae & Feng Zhang
- 摘要：Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. One example, the extracellular contractile injection systems (eCISs), are syringe-like macromolecular complexes that inject protein payloads into eukaryotic cells by driving a spike through the cellular membrane. Recently, eCISs have been found to target mouse cells1,2,3, raising the possibility that these systems could be harnessed for therapeutic protein delivery. However, whether eCISs can function in human cells remains unknown, and the mechanism by which these systems recognize target cells is poorly understood. Here we show that target selection by the Photorhabdus virulence cassette (PVC)—an eCIS from the entomopathogenic bacterium Photorhabdus asymbiotica—is mediated by specific recognition of a target receptor by a distal binding element of the PVC tail fibre. Furthermore, using in silico structure-guided engineering of the tail fibre, we show that PVCs can be reprogrammed to target organisms not natively targeted by these systems—including human cells and mice—with efficiencies approaching 100%. Finally, we show that PVCs can load diverse protein payloads, including Cas9, base editors and toxins, and can functionally deliver them into human cells. Our results demonstrate that PVCs are programmable protein delivery devices with possible applications in gene therapy, cancer therapy and biocontrol.展开
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Platelet factors attenuate inflammation and rescue cognition in ageing
Nature | 2023 | 查看原文 |
作者：Adam B. Schroer, Patrick B. Ventura, Juliana Sucharov, Rhea Misra, M. K. Kirsten Chui, Gregor Bieri, Alana M. Horowitz, Lucas K. Smith, Katriel Encabo, Imelda Tenggara, Julien Couthouis, Joshua D. Gross, June M. Chan, Anthony Luke & Saul A. Villeda
- 摘要：Identifying therapeutics to delay, and potentially reverse, age-related cognitive decline is critical in light of the increased incidence of dementia-related disorders forecasted in the growing older population1. Here we show that platelet factors transfer the benefits of young blood to the ageing brain. Systemic exposure of aged male mice to a fraction of blood plasma from young mice containing platelets decreased neuroinflammation in the hippocampus at the transcriptional and cellular level and ameliorated hippocampal-dependent cognitive impairments. Circulating levels of the platelet-derived chemokine platelet factor 4 (PF4) (also known as CXCL4) were elevated in blood plasma preparations of young mice and humans relative to older individuals. Systemic administration of exogenous PF4 attenuated age-related hippocampal neuroinflammation, elicited synaptic-plasticity-related molecular changes and improved cognition in aged mice. We implicate decreased levels of circulating pro-ageing immune factors and restoration of the ageing peripheral immune system in the beneficial effects of systemic PF4 on the aged brain. Mechanistically, we identified CXCR3 as a chemokine receptor that, in part, mediates the cellular, molecular and cognitive benefits of systemic PF4 on the aged brain. Together, our data identify platelet-derived factors as potential therapeutic targets to abate inflammation and rescue cognition in old age.展开
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Structural insights into constitutive activity of 5-HT6 receptor
PNAS | 2023 | 查看原文 |
作者：Licong He, Qiaoyu Zhao, Jianzhong Qi, Yifan Wang, Wenyu Han, Zhangcheng Chen, Yao Cong and Sheng Wang
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An inverse agonist of orphan receptor GPR61 acts by a G proteincompetitive allosteric mechanism.
Nat Commun | 2023 | 查看原文 |
作者：Joshua A. Lees, João M. Dias, Francis Rajamohan, Jean-Philippe Fortin, Rebecca O’Connor, Jimmy X. Kong, Emily A. G. Hughes, Ethan L. Fisher, Jamison B. Tuttle, Gabrielle Lovett, Bethany L. Kormos, Rayomand J. Unwalla, Lei Zhang, Anne-Marie Dechert Schmitt, Dahui Zhou, Michael Moran, Kimberly A. Stevens, Kimberly F. Fennell, Alison E. Varghese, Andrew Maxwell, Emmaline E. Cote, Yuan Zhang & Seungil Han
- 摘要：GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, the lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study GPR61 structure and function. Here, we report a structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its allosteric binding site and mode of action. These findings offer mechanistic insights into an orphan GPCR while providing both a structural framework and tool compound to support further studies of GPR61 function and modulation.展开
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An improved pathway for autonomous bioluminescence imaging in eukaryotes
Nat Methods | 2024 | 查看原文 |
作者：Ekaterina S. Shakhova, Tatiana A. Karataeva, Nadezhda M. Markina, Tatiana Mitiouchkina, Kseniia A. Palkina, Maxim M. Perfilov, Monika G. Wood, Trish T. Hoang, Mary P. Hall, Liliia I. Fakhranurova, Anna E. Alekberova, Alena K. Malyshevskaia, Dmitry A. Gorbachev, Evgenia N. Bugaeva, Ludmila K. Pletneva, Vladislav V. Babenko, Daria I. Boldyreva, Andrey Y. Gorokhovatsky, Anastasia V. Balakireva, Feng Gao, Vladimir V. Choob, Lance P. Encell, Keith V. Wood, Ilia V. Yampolsky, …Alexander S. Mishin
- 摘要：The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.展开
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