RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's ...disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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CIB1: a small protein with big ambitions Leisner, Tina M.; Freeman, Thomas C.; Black, Justin L. ...
The FASEB journal,
August 2016, Volume:
30, Issue:
8
Journal Article
Peer reviewed
Open access
ABSTRACT
Calcium‐ and integrin‐binding protein 1 (CIB1) is a small, ubiquitously expressed protein that was first identified as an intracellular binding partner of a platelet‐specific α‐integrin ...cytoplasmic tail. Although early studies revealed a role for CIB1 in regulating platelet integrin activity, recent studies have indicated a more diverse role for CIB1 in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. Increasing evidence also points to a novel role for CIB1 in cancer and cardiovascular disease. In addition, an array of CIB1 binding partners has been identified that provide important insight into how CIB1 may regulate these processes. Some of these binding partners include the serine/threonine kinases, p21‐activated kinase 1 (PAK1), apoptosis signal‐regulating kinase 1 (ASK1), and polo‐like kinase 3 (PLK3). Structural and mutational studies indicate that CIB1 binds most or all of its partners via a well‐defined hydrophobic cleft. Although CIB1 itself lacks known enzymatic activity, it supports the PI3K/AKT and MEK/ERK oncogenic signaling pathways, in part, by directly modulating enzymes in these pathways. In this review, we discuss our current understanding of CIB1 and key questions regarding structure and function and how this seemingly diminutive protein impacts important signaling pathways and cellular processes in human health and disease.—Leisner, T. M., Freeman, T. C., Black, J. L., Parise, L. V. CIB1: a small protein with big ambitions. FASEB J. 30, 2640‐2650 (2016). www.fasebj.org
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The primacy of lipids as essential components of cellular membranes is conserved across taxonomic domains. In addition to this crucial role as a semi-permeable barrier, lipids are also increasingly ...recognized as important signaling molecules with diverse functional mechanisms ranging from cell surface receptor binding to the intracellular regulation of enzymatic cascades. In this review, we focus on ether lipids, an ancient family of lipids having ether-linked structures that chemically differ from their more prevalent acyl relatives. In particular, we examine ether lipid biosynthesis in the peroxisome of mammalian cells, the roles of selected glycerolipids and glycerophospholipids in signal transduction in both prokaryotes and eukaryotes, and finally, the potential therapeutic contributions of synthetic ether lipids to the treatment of cancer.
Delivery of nucleic acids into solid tumor environments remains a pressing challenge. This study examines the ability of macrophages to horizontally transfer small interfering RNA (siRNA) lipoplexes ...to cancer cells. Macrophages are a natural candidate for a drug carrier because of their ability to accumulate at high densities into many cancer types, including, breast, prostate, brain, and colon cancer. Here, it is demonstrated that macrophages can horizontally transfer siRNA to cancer cells during in vitro coculture. The amount of transfer can be dosed depending on the amount of siRNA loaded and total number of macrophages delivered. Macrophages loaded with calcium integrin binding protein‐1 (CIB1)‐siRNA result in decreased tumorsphere growth and decreased mRNA expression of CIB1 and KI67 in MDA‐MB‐468 human breast cancer cells. Adoptive transfer of macrophages transfected with CIB1‐siRNA localizes to the orthotopic MDA‐MB‐468 tumor. Furthermore, it is reported that macrophage activation can modulate this transfer process as well as intracellular trafficking protein Rab27a. As macrophages are heavily involved in tumor progression, understanding how to use macrophages for drug delivery can substantially benefit the treatment of tumors.
Macrophage mediated horizontal siRNA transfer provides an incredible opportunity to deliver therapeutic oligonucleotides (i.e., siRNA, mRNA, microRNA, pDNA) to macrophage‐rich disease environments such as cancer. This work provides a basis for the mechanism of macrophage gene transfer via activation dependent extravesicular vesicle release and thus lays the groundwork for development of nanocomplexes that can control macrophage activation and support gene delivery.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Abstract Chemical probes are an indispensable tool for translating biological discoveries into new therapies, though are increasingly difficult to identify since novel therapeutic targets are often ...hard-to-drug proteins. We introduce FRASE-based hit-finding robot (FRASE-bot), to expedite drug discovery for unconventional therapeutic targets. FRASE-bot mines available 3D structures of ligand-protein complexes to create a database of FRAgments in Structural Environments (FRASE). The FRASE database can be screened to identify structural environments similar to those in the target protein and seed the target structure with relevant ligand fragments. A neural network model is used to retain fragments with the highest likelihood of being native binders. The seeded fragments then inform ultra-large-scale virtual screening of commercially available compounds. We apply FRASE-bot to identify ligands for Calcium and Integrin Binding protein 1 (CIB1), a promising drug target implicated in triple negative breast cancer. FRASE-based virtual screening identifies a small-molecule CIB1 ligand (with binding confirmed in a TR-FRET assay) showing specific cell-killing activity in CIB1-dependent cancer cells, but not in CIB1-depletion-insensitive cells.
Patients with epidermodysplasia verruciformis (EV) and biallelic null mutations of
(encoding EVER1) or
(EVER2) are selectively prone to disseminated skin lesions due to keratinocyte-tropic human ...β-papillomaviruses (β-HPVs), which lack E5 and E8. We describe EV patients homozygous for null mutations of the
gene encoding calcium- and integrin-binding protein-1 (CIB1). CIB1 is strongly expressed in the skin and cultured keratinocytes of controls but not in those of patients. CIB1 forms a complex with EVER1 and EVER2, and CIB1 proteins are not expressed in EVER1- or EVER2-deficient cells. The known functions of EVER1 and EVER2 in human keratinocytes are not dependent on CIB1, and CIB1 deficiency does not impair keratinocyte adhesion or migration. In keratinocytes, the CIB1 protein interacts with the HPV E5 and E8 proteins encoded by α-HPV16 and γ-HPV4, respectively, suggesting that this protein acts as a restriction factor against HPVs. Collectively, these findings suggest that the disruption of CIB1-EVER1-EVER2-dependent keratinocyte-intrinsic immunity underlies the selective susceptibility to β-HPVs of EV patients.
Peptides have historically been underutilized for covalent inhibitor discovery, despite their unique abilities to interact with protein surfaces and interfaces. This is in part due to a lack of ...methods for screening and identifying covalent peptide ligands. Here, we report a method to identify covalent cyclic peptide inhibitors in mRNA display. We combine co- and post-translational library diversification strategies to create cyclic libraries with reactive dehydroalanines (Dhas), which we employ in selections against two model targets. The most potent hits exhibit low nanomolar inhibitory activities and disrupt known protein–protein interactions with their selected targets. Overall, we establish Dhas as electrophiles for covalent inhibition and showcase how separate library diversification methods can work synergistically to dispose mRNA display to novel applications like covalent inhibitor discovery.
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IJS, KILJ, NUK, PNG, UL, UM
Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module ...strongly linked to Alzheimer’s disease (AD) traits and microglia. These proteins are more abundant in Alzheimer’s patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN–CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein–protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN–CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN–CD44 interaction.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We introduce machine learning (ML) to perform classification and quantitation of images of nuclei from human blood neutrophils. Here we assessed the use of convolutional neural networks (CNNs) using ...free, open source software to accurately quantitate neutrophil NETosis, a recently discovered process involved in multiple human diseases. CNNs achieved >94% in performance accuracy in differentiating NETotic from non-NETotic cells and vastly facilitated dose-response analysis and screening of the NETotic response in neutrophils from patients. Using only features learned from nuclear morphology, CNNs can distinguish between NETosis and necrosis and between distinct NETosis signaling pathways, making them a precise tool for NETosis detection. Furthermore, by using CNNs and tools to determine object dispersion, we uncovered differences in NETotic nuclei clustering between major NETosis pathways that is useful in understanding NETosis signaling events. Our study also shows that neutrophils from patients with sickle cell disease were unresponsive to one of two major NETosis pathways. Thus, we demonstrate the design, performance, and implementation of ML tools for rapid quantitative and qualitative cell analysis in basic science.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Patients diagnosed with triple negative breast cancer (TNBC) have limited treatment options and often suffer from resistance and toxicity due to chemotherapy. We previously found that depleting ...calcium and integrin-binding protein 1 (CIB1) induces cell death selectively in TNBC cells, while sparing normal cells. Therefore, we asked whether CIB1 depletion further enhances tumor-specific killing when combined with either the commonly used chemotherapeutic, docetaxel, or the cell death-inducing ligand, TRAIL.
We targeted CIB1 by RNA interference in MDA-MB-436, MDA-MB-231, MDA-MB-468, docetaxel-resistant MDA-MB-436 TNBC cells and ME16C normal breast epithelial cells alone or combination with docetaxel or TRAIL. Cell death was quantified via trypan blue exclusion using flow cytometry and cell death mechanisms were analyzed by Western blotting. Cell surface levels of TRAIL receptors were measured by flow cytometry analysis.
CIB1 depletion combined with docetaxel significantly enhanced tumor-specific cell death relative to each treatment alone. The enhanced cell death strongly correlated with caspase-8 activation, a hallmark of death receptor-mediated apoptosis. The death receptor TRAIL-R2 was upregulated in response to CIB1 depletion, which sensitized TNBC cells to the ligand TRAIL, resulting in a synergistic increase in cell death. In addition to death receptor-mediated apoptosis, both combination treatments activated a non-apoptotic mechanism, called paraptosis. Interestingly, these combination treatments also induced nearly complete death of docetaxel-resistant MDA-MB-436 cells, again via apoptosis and paraptosis. In contrast, neither combination treatment induced cell death in normal ME16C cells.
Novel combinations of CIB1 depletion with docetaxel or TRAIL selectively enhance naive and docetaxel-resistant TNBC cell death while sparing normal cell. Therefore, combination therapies that target CIB1 could prove to be a safe and durable strategy for treatment of TNBC and potentially other cancers.
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