Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin α iib β 3 receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin α iib β 3 receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains. furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms. Abbreviations CD Circular dichroism Dab2 Disabled-2 DPC Dodecylphosphocholine GST Glutathione S-transferase N-PTB N-terminus containing the phosphotyrosine-binding domain SBP Sulfatide-binding peptide SPR Surface plasmon resonance The adaptor protein Disabled-2 (Dab2) is a multimodular signaling molecule involved in a variety of cellular processes including protein trafficking, cell growth and differentiation, cell adhesion, and modulation of platelet aggregation 1. Two Dab orthologs, Dab1 and Dab2, are present in mammals. Dab1 is primarily expressed in the brain 2 , whereas Dab2 is ubiquitously expressed in different tissues 3,4. Dab2 expression levels have been reported to exhibit a significant effect on cancer initiation and progression. Indeed, Dab2 expression is lost in breast, ovarian, prostate 5 , bladder 6 , and colorectal cancer cells 7 , suggesting that Dab2 can act as a tumor suppressor 8 .
Osteoarthritis (OA) is the most common form of arthritis and the fastest growing cause of chronic disability in the world. Formation of the ternary IL-1β /IL-1R1/IL-1RAcP protein complex and its downstream signaling has been implicated in osteoarthritis pathology. Current OA therapeutic approaches target either the cytokine IL-1β or the primary receptor IL-1RI but do not exploit the potential of the secondary receptor IL-1RAcP. Our previous work implicated the Arg286 residue of IL-1RAcP as a key mediator of complex formation. Molecular modeling confirmed Arg286 as a high-energy mediator of the ternary IL-1β complex architecture and interaction network. Anti-IL-1RAcP monoclonal antibodies (mAb) targeting the Arg286 residue were created and were shown to effectively reduce the influx of inflammatory cells to damaged joints in a mouse model of osteoarthritis. Inhibitory peptides based on the native sequence of IL-1RAcP were prepared and examined for efficacy at disrupting the complex formation. The most potent peptide inhibitor had an IC50 value of 304 pM in a pull-down model of complex formation, and reduced IL-1β signaling in a cell model by 90% at 2 μM. Overall, therapies that target the Arg286 region surface of IL-1RAcP, and disrupt subsequent interactions with subunits, have the potential to serve as next generation treatments for osteoarthritis.
25Disabled-2 (Dab2) is an adaptor protein that regulates numerous cellular processes. Among them, Dab2 26 modulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 27 intracellularly downregulates the integrin aIIbb3 receptor, converting it to a low affinity state for adhesion 28 and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with 29 both the integrin aIIbb3 receptor and sulfatides, both of which are known to be pro-aggregatory mediators, 30 blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 31 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two 32 potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using a combined 33 methodology including molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface 34 plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for 35 sulfatide binding. A hydrophilic region, primarily mediated by R42, is responsible for the interaction with 36 the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with the acyl 37 chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of 38 platelet P-selectin surface expression. The interacting Dab2 SBP residues with sulfatide resemble those 39 described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share 40 common binding mechanisms. 41 42 43 44 integrin receptor.45 46 3 INTRODUCTION 47 48The adaptor protein Disabled-2 (Dab2) is a multimodular signaling molecule involved in a variety of 49 cellular processes including protein trafficking, cell growth and differentiation, cell adhesion, and 50 modulation of platelet aggregation (1). Two Dab orthologs, Dab1 and Dab2, are present in mammals. Dab1 51 is primarily expressed in the brain (2), whereas Dab2 is ubiquitously expressed in different tissues (3, 4). 52Dab2 expression levels have been reported to exhibit a significant effect on cancer initiation and 53 progression. Indeed, Dab2 expression is lost in breast, ovarian, prostate (5), bladder (6), and colorectal 54 cancer cells (7), suggesting that Dab2 can act as a tumor suppressor (8). Two alternative spliced forms of 55 Dab2 are expressed in humans, p96 and p67, with the latter lacking a central exon (3). Dab2 contains a 56 phosphotyrosine-binding (PTB) domain located at the N-terminus, clathrin-binding sites, NPF and DPF 57 motifs, and a proline-rich SH3 domain located at the C-terminus, indicating that Dab2 primarily functions 58 as an adaptor protein. 60Recently, Dab2 was characterized as a negative regulator of platelet aggregation by modulating both pro-61 aggregatory inside-out and outside-in signaling pathways (1). Modulation of the inside-out signaling by 62 Dab2 is mediated by its cytosolic S24-phosphorylated form, which b...
Brown AM. Utilization of computational techniques and tools to introduce or reinforce knowledge of biochemistry and protein structure-function relationships.
Background: Acute telestroke consults rely on a rapid, accurate NIH Stroke Scale. An important sign in cortical-based ischemic strokes is a Visual Field Deficit (VFD) contributing up to 3 points on the NIHSS affecting treatment decisions. Remote confrontational VFD testing is challenging and requires a trained assistant. We created a simple, rapid, telecart peripheral device to remotely administer a standardized VF test . The “ V isual fields R apid A ssessment D evice” ( VRAD ) is a wearable eye-glasses device. The device flashes LED lights in the 4 quadrants of each eye via remote control by a teleprovider allowing real-time control and interpretation. Methods: The VRAD Phase I study examined FDA-related human design factors of comfort, speed, and ease of use with direct use of VRAD. The results guided optimization of the prototype in preparation for the Phase II validation clinical trial. Subjects were randomly-selected, consented adult UVA Stroke Unit inpatients, with confirmed ischemic strokes. Following each VF test, blinded teleproviders, the patient ,and the telepresenter (nurse) completed a brief binary response questionnaire with open-ended comments. Biostatistical analysis: N= 20 subjects; 95% confidence interval with precision of +/- 10%. Results: The vast majority of patients, presenters, and providers (95-100%) reported VRAD testing was easy to understand and rapid. Patients: The device was comfortable (90%); 10% felt the stimulus light was too bright and 5% too dim. All identified when the light was on and had enough time to respond. Presenters: All reported rapid testing; 75% felt the device was easy to don on patients. The device was “sturdy and durable” 90%, “easy to use” 75%, “easy to clean” 70%, and “easy to connect” 60%. Providers: Reported the device sufficiently screened standard VFs (90%). All agreed they could remotely administer the test in various settings. Conclusion: The VRAD Phase I trial found the device to be safe, effective, easy to use, and comfortable by patients, providers, and presenters. Based on the results, an upgraded prototype was manufactured for our upcoming Phase II Non-Inferiority Validation Trial (Fall 2021). This data is a proof-of-concept of the value of teleneurology peripheral tools in remote settings.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.