111 results on '"Capelluto, Daniel"'
Search Results
2. Nanoflow valve for the removal of trapped air in microfluidic structures
- Author
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Cortes, Diego F., Tang, Tuo-Xian, Capelluto, Daniel G.S., and Lazar, Iulia M.
- Published
- 2017
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3. Structural, in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides
- Author
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Song, Wei, Gottschalk, Carter J., Tang, Tuo-Xian, Biscardi, Andrew, Ellena, Jeffrey F., Finkielstein, Carla V., Brown, Anne M., and Capelluto, Daniel G. S.
- Published
- 2020
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4. Preferential phosphatidylinositol 5-phosphate binding contributes to a destabilization of the VHS domain structure of Tom1
- Author
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Xiong, Wen, Tang, Tuo-Xian, Littleton, Evan, Karcini, Arba, Lazar, Iulia M., and Capelluto, Daniel G. S.
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- 2019
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5. Backbone 1H, 15N, and 13C resonance assignments of the Tom1 VHS domain
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Ellena, Jeffrey F., Xiong, Wen, Zhao, Xiaolin, Shanaiah, Narasimhamurthy, and Capelluto, Daniel G. S.
- Published
- 2017
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6. Chronotherapy: Intuitive, Sound, Founded…But Not Broadly Applied
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Selfridge, Julia M., Gotoh, Tetsuya, Schiffhauer, Samuel, Liu, JingJing, Stauffer, Philip E., Li, Andrew, Capelluto, Daniel G. S., and Finkielstein, Carla V.
- Published
- 2016
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7. Phafins Are More Than Phosphoinositide-Binding Proteins.
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Tang, Tuoxian, Hasan, Mahmudul, and Capelluto, Daniel G. S.
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COINCIDENCE circuits ,PROTEINS ,ASPARTIC acid ,ADAPTOR proteins ,PHOSPHOINOSITIDES - Abstract
Phafins are PH (Pleckstrin Homology) and FYVE (Fab1, YOTB, Vac1, and EEA1) domain-containing proteins. The Phafin protein family is classified into two groups based on their sequence homology and functional similarity: Phafin1 and Phafin2. This protein family is unique because both the PH and FYVE domains bind to phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide primarily found in endosomal and lysosomal membranes. Phafin proteins act as PtdIns(3)P effectors in apoptosis, endocytic cargo trafficking, and autophagy. Additionally, Phafin2 is recruited to macropinocytic compartments through coincidence detection of PtdIns(3)P and PtdIns(4)P. Membrane-associated Phafins serve as adaptor proteins that recruit other binding partners. In addition to the phosphoinositide-binding domains, Phafin proteins present a poly aspartic acid motif that regulates membrane binding specificity. In this review, we summarize the involvement of Phafins in several cellular pathways and their potential physiological functions while highlighting the similarities and differences between Phafin1 and Phafin2. Besides, we discuss research perspectives for Phafins. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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8. Tollip: a multitasking protein in innate immunity and protein trafficking
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Capelluto, Daniel G.S.
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- 2012
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9. The impact of sulfatide loss on the progress of Alzheimer's disease.
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Finkielstein, Carla V. and Capelluto, Daniel G. S.
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ALZHEIMER'S disease , *AMYLOID beta-protein precursor , *DISEASE risk factors - Published
- 2023
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10. The PH Domain and C-Terminal polyD Motif of Phafin2 Exhibit a Unique Concurrence in Animals.
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Hasan, Mahmudul and Capelluto, Daniel G. S.
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- 2022
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11. Backbone 1H, 15N, and 13C resonance assignments and secondary structure of the tollip CUE domain
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Azurmendi, Hugo F., Mitra, Sharmistha, Ayala, Iriscilla, Li, Liwu, Finkielstein, Carla V., and Capelluto, Daniel G. S.
- Published
- 2010
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12. Backbone 1H, 15N, and 13C resonance assignments of the Phafin2 pleckstrin homology domain.
- Author
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Ellena, Jeffrey F., Tang, Tuo-Xian, Shanaiah, Narasimhamurthy, and Capelluto, Daniel G. S.
- Abstract
Phafin2 is a peripheral protein that triggers cellular signaling from endosomal and lysosomal compartments. The specific subcellular localization of Phafin2 is mediated by the presence of a tandem of phosphatidylinositol 3-phosphate (PtdIns3P)-binding domains, the pleckstrin homology (PH) and the Fab-1, YOTB, Vac1, and EEA1 (FYVE) domains. The requirement for both domains for binding to PtdIns3P still remains unclear. To understand the molecular interactions of the Phafin2 PH domain in detail, we report its nearly complete
1 H,15 N, and13 C backbone resonance assignments. [ABSTRACT FROM AUTHOR]- Published
- 2022
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13. The DIX domain targets dishevelled to actin stress fibres and vesicular membranes
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Capelluto, Daniel G. S., Kutateladze, Tatiana G., Habas, Raymond, Finkielstein, Carla V., He, Xi, and Overduin, Michael
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Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
Author(s): Daniel G. S. Capelluto [1]; Tatiana G. Kutateladze [1, 2]; Raymond Habas [2, 3]; Carla V. Finkielstein [2, 4]; Xi He [5]; Michael Overduin (corresponding author) [1] Colorectal cancer [...]
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- 2002
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14. Disabled-2 modulates homotypic and heterotypic platelet interactions by binding to sulfatides
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Welsh, John D., Charonko, John J., Salmanzadeh, Alireza, Drahos, Karen E., Shafiee, Hadi, Stremler, Mark A., Davalos, Rafael V., Capelluto, Daniel G. S., Vlachos, Pavlos P., and Finkielstein, Carla V.
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- 2011
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15. Phosphoinositide, Phosphopeptide and Pyridone Interactions of the Abl SH2 Domain
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Tokonzaba, Etienne, Capelluto, Daniel G. S., Kutateladze, Tatiana G., and Overduin, Michael
- Published
- 2006
16. Unconventional mechanism of autoregulation of membrane binding by the PH-FYVE tandem domains of Phafin2
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Hasan, Mahmudul, Tang, Tuo-Xian, Spruill, Bridget, Ellena, Jeffrey F., and Capelluto, Daniel G.S.
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- 2022
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17. Molecular mechanism for the commitment of TOM1 to cargo trafficking
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Roach, Tiffany G., Xiong, Wen, Beyer, Josephine H., and Capelluto, Daniel G.
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- 2022
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18. Modeling the relationship between the p53 C-terminal domain and its binding partners using molecular dynamics
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Allen, William J., Capelluto, Daniel G. S., Finkielstein, Carla V., and Bevan, David R.
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Hydrophobic effect -- Analysis ,Molecular dynamics -- Usage ,Protein binding -- Analysis ,Tumor suppressor genes -- Research ,Chemicals, plastics and rubber industries - Published
- 2010
19. Structural and membrane binding properties of the Prickle PET domain
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Sweede, Matthew, Ankem, Gayatri, Chutvirasakul, Boonta, Azurmendi, Hugo F., Chbeir, Souhad, Watkins, Justin, Helm, Richard F., Finkielstein, Carla V., and Capelluto, Daniel G.S.
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Circular dichroism -- Usage ,Alcohol -- Chemical properties ,Alcohol, Denatured -- Chemical properties ,Membrane proteins -- Structure ,Membrane proteins -- Chemical properties ,Protein binding -- Analysis ,Biological sciences ,Chemistry - Abstract
The structural and functional characterization of the regions in Prickle (Pk) protein that regulates cell movements through its association with the Dishevelled (Dsh) protein and PET domain is presented. The results revealed that the three LIM domains of Pk physically interact to regulate membrane penetration of the PET domain that is engaged in Pk membrane insertion.
- Published
- 2008
20. Structural, thermodynamic, and phosphatidylinositol 3-phosphate binding properties of Phafin2.
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Tang, Tuo‐Xian, Jo, Ami, Deng, Jingren, Ellena, Jeffrey F., Lazar, Iulia M., Davis, Richey M., and Capelluto, Daniel G. S.
- Abstract
Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments. Here, we show that human Phafin2 is a moderately elongated monomer of ∼28 kDa with an intensity-average hydrodynamic diameter of ∼7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an α/β structure and predicts ∼40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.4°C. The folding-unfolding curves, obtained using urea- and guanidine hydrochloride-mediated denaturation, indicate that Phafin2 undergoes a two-state native-to-denatured transition. Analysis of these transitions shows that the free energy change for urea- and guanidine hydrochloride-induced Phafin2 denaturation in water is ∼4 kcal mol
−1 . PtdIns(3)P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns(3)P-binding domains of the protein in endomembrane compartments. [ABSTRACT FROM AUTHOR]- Published
- 2017
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21. Backbone H, N, and C resonance assignments of the Tom1 VHS domain.
- Author
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Ellena, Jeffrey, Xiong, Wen, Zhao, Xiaolin, Shanaiah, Narasimhamurthy, and Capelluto, Daniel
- Abstract
Efficient trafficking of ubiquitinated receptors (cargo) to endosomes requires the recruitment of adaptor proteins that exhibit ubiquitin-binding domains for recognition and transport. Tom1 is an adaptor protein that not only associates with ubiquitinated cargo but also represents a phosphoinositide effector during specific bacterial infections. This phosphoinositide-binding property is associated with its N-terminal Vps27, Hrs, STAM (VHS) domain. Despite its biological relevance, there are no resonance assignments of Tom1 VHS available that can fully characterize its molecular interactions. Here, we report the nearly complete H, N, and C backbone resonance assignments of the VHS domain of human Tom1. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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22. New Insights into the Dynamics of Zwitterionic Micelles and Their Hydration Waters by Gigahertz-to-Terahertz Dielectric Spectroscopy.
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George, Deepu K., Charkhesht, Ali, Hull, Olivia A., Mishra, Archana, Capelluto, Daniel G. S., Mitchell-Koch, Katie R., and Vinh, Nguyen Q.
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- 2016
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23. Disabled-2: A modular scaffold protein with multifaceted functions in signaling.
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Finkielstein, Carla V. and Capelluto, Daniel G. S.
- Subjects
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ADAPTOR proteins , *ENDOCYTOSIS , *PHOSPHATIDYLINOSITOLS , *SULFATIDES , *TUMOR suppressor proteins , *DOWNREGULATION , *OVARIAN cancer treatment - Abstract
Disabled-2 (Dab2) is a multimodular scaffold protein with signaling roles in the domains of cell growth, trafficking, differentiation, and homeostasis. Emerging evidences place Dab2 as a novel modulator of cell-cell interaction; however, its mode of action has remained largely elusive. In this review, we highlight the relevance of Dab2 function in cell signaling and development and provide the most recent and comprehensive analysis of Dab2's action as a mediator of homotypical and heterotypical interactions. Accordingly, Dab-2 controls the extent of platelet aggregation through various motifs within its N-terminus. Dab2 interacts with the cytosolic tail of the integrin receptor blocking inside-out signaling, whereas extracellular Dab2 competes with fibrinogen for integrin αIIbβ3 receptor binding and, thus, modulates outside-in signaling. An additional level of regulation results from Dab2's association with cell surface lipids, an event that defines the extent of cell-cell interactions. As a multifaceted regulator, Dab2 acts as a mediator of endocytosis through its association with the [FY]xNPx[YF] motifs of internalized cell surface receptors, phosphoinositides, and clathrin. Other emerging roles of Dab2 include its participation in developmental mechanisms required for tissue formation and in modulation of immune responses. This review highlights the various novel mechanisms by which Dab2 mediates an array of signaling events with vast physiological consequences. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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24. Opening the Debate: How to Fulfill the Need for Physicians' Training in Circadian-Related Topics in a Full Medical School Curriculum.
- Author
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Selfridge, Julia M., Moyer, Kurtis, Capelluto, Daniel G. S., and Finkielstein, Carla V.
- Abstract
Background: Circadian rhythms are daily changes in our physiology and behavior that are manifested as patterns of brain wave activity, periodic hormone production, recurring cell regeneration, and other oscillatory biological activities. Their importance to human health is becoming apparent; they are deranged by shift work and jet-lag and in disparate conditions such as insomnia, sleep syndromes, coronary heart attacks, and depression, and are endogenous factors that contribute to cancer development and progression. Discussion: As evidence of the circadian connection to human health has grown, so has the number of Americans experiencing disruption of circadian rhythms due to the demands of an industrialized society. Today, there is a growing work force that experiences night shift work and time-zone shifts shaping the demands on physicians to best meet the needs of patients exposed to chronic circadian disruptions. The diverse range of illness associated with altered rhythms suggests that physicians in various fields will see its impact in their patients. However, medical education, with an already full curriculum, struggles to address this issue. Summary: Here, we emphasize the need for incorporating the topic of circadian rhythms in the medical curriculum and propose strategies to accomplish this goal. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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25. Ligand Binding Reveals a Role for Heme in Translationally-Controlled Tumor Protein Dimerization.
- Author
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Lucas, Andrew T., Fu, Xiangping, Liu, JingJing, Brannon, Mary K., Yang, Jianhua, Capelluto, Daniel G. S., and Finkielstein, Carla V.
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LIGAND binding (Biochemistry) ,TUMOR proteins ,PROTEIN expression ,CELL cycle ,CELL growth ,GENETIC transformation ,DIMERIZATION ,HEME - Abstract
The ranslationally-ontrolled umor rotein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a “buffer protein” controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His
76 His77 ) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP's cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the “buffer” condition to be dissociated from TCTP's role as a component of signal transduction mechanisms. [ABSTRACT FROM AUTHOR]- Published
- 2014
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26. A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif.
- Author
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Xiao, Shuyan, Zhao, Xiaolin, Finkielstein, Carla V., and Capelluto, Daniel G. S.
- Abstract
A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea-equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled-2 (Dab2) sulfatide-binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane-binding, recombinant peptides that co-purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S-transferase (GST) fusion protein using minimal media containing [
15 N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea-equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at −80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co-purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]- Published
- 2014
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27. Backbone H, N, and C resonance assignments and secondary structure of the tollip CUE domain.
- Author
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Azurmendi, Hugo, Mitra, Sharmistha, Ayala, Iriscilla, Li, Liwu, Finkielstein, Carla, and Capelluto, Daniel
- Abstract
The Toll-interacting protein (Tollip) is a negative regulator of the Toll-like receptor (TLR)-mediated inflammation response. Tollip is a modular protein that contains an Nterminal Tom1- binding domain (TBD), a central conserved domain 2 (C2), and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. Here, we report the sequence-specific backbone H, N, and C assignments of the human Tollip CUE domain. The CUE domain was found to be a stable dimer as determined by size-exclusion chromatography and molecular crosslinking studies. Analysis of the backbone chemical shift data indicated that the CUE domain exhibits three helical elements corresponding to 52% of the protein backbone. Circular dichroism spectrum analysis confirmed the helical nature of this domain. Comparison of the location of these helical regions with those reported for yeast CUE domains suggest differences in length for all helical elements. We expect the structural analysis presented here will be the foundation for future studies on the biological significance of the Tollip CUE domain, its molecular interactions, and the mechanisms that modulate its function during the inflammatory response. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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28. A Novel Heme-Regulatory Motif Mediates Heme-Dependent Degradation of the Circadian Factor Period 2.
- Author
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Jianhua Yang, Kim, Kevin D., Lucas, Andrew, Drahos, Karen E., Santos, Carlo S., Mury, Sean P., Capelluto, Daniel G. S., and Finkielstein, Carla V.
- Subjects
CIRCADIAN rhythms ,GENE expression ,CELL cycle regulation ,CELL death ,GENETIC regulation ,PHYSIOLOGICAL oxidation ,SPECTRUM analysis - Abstract
Although efforts have been made to identify circadian-controlled genes regulating cell cycle progression and cell death, little is known about the metabolic signals modulating circadian regulation of gene expression. We identify heme, an iron-containing prosthetic group, as a regulatory ligand controlling human Period-2 (hPer2) stability. Furthermore, we define a novel heme-regulatory motif within the C terminus of hPer2 (SC
841 PA) as necessary for heme binding and protein destabilization. Spectroscopy reveals that whereas the PAS domain binds to both the ferric and ferrous forms of heme, SC841 PA binds exclusively to ferric heme, thus acting as a redox sensor. Consequently, binding prevents hPer2 from interacting with its stabilizing counterpart cryptochrome. In vivo, hPer2 downregulation is suppressed by inhibitors of heme synthesis or proteasome activity, while SA841 PA is sufficient to stabilize hPer2 in transfected cells. Moreover, heme binding to the SC841 PA motif directly impacts circadian gene expression, resulting in altered period length. Overall, the data support a model where heme-mediated oxidation triggers hPer2 degradation, thus controlling heterodimerization and ultimately gene transcription. [ABSTRACT FROM AUTHOR]- Published
- 2008
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29. Multivalent Mechanism of Membrane Insertion by the FYVE Domain.
- Author
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Kitaleladze, Tatiana G., Capelluto, Daniel G.s., Ferguson, Colin G., Cheever, Matthew L., Kutateladze, Andrei G., Prestwich, Glenn D., and Overduin, Micheal
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PROTEINS , *PHOSPHOLIPIDS , *CELL membranes , *MICELLES , *ANTIGENS , *ELECTROSTATICS - Abstract
Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with Ptdlns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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30. Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi.
- Author
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Capelluto, Daniel G.S., Hellman, Ulf, Cazzulo, Juan J., and Cannata, Joaquin J.B.
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TRYPANOSOMA cruzi , *SERINE , *METHANOL , *TRANSFERASES - Abstract
Studies the purification and properties of serine hydroxymethyltransferase (SHMT) extracted from Trypanosoma cruzi. List of chemicals used in the study; Method used for the purification of SHMT; Characteristics of SHMT observed in the study.
- Published
- 2000
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31. Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif.
- Author
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Zhao, Xiaolin, Xiong, Wen, Xiao, Shuyan, Tang, Tuo-Xian, Ellena, Jeffrey F., Armstrong, Geoffrey S., Finkielstein, Carla V., and Capelluto, Daniel G. S.
- Abstract
Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
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32. External Lipid PI3P Mediates Entry of Eukaryotic Pathogen Effectors into Plant and Animal Host Cells
- Author
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Kale, Shiv D., Gu, Biao, Capelluto, Daniel G.S., Dou, Daolong, Feldman, Emily, Rumore, Amanda, Arredondo, Felipe D., Hanlon, Regina, Fudal, Isabelle, Rouxel, Thierry, Lawrence, Christopher B., Shan, Weixing, and Tyler, Brett M.
- Published
- 2010
- Full Text
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33. The C-terminal acidic motif of Phafin2 inhibits PH domain binding to phosphatidylinositol 3-phosphate.
- Author
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Tang, Tuo-Xian, Finkielstein, Carla V., and Capelluto, Daniel G.S.
- Subjects
- *
SITE-specific mutagenesis , *SURFACE plasmon resonance , *ISOTHERMAL titration calorimetry - Abstract
Changes in membrane curvature are required to control the function of subcellular compartments; malfunctions of such processes are associated with a wide range of human diseases. Membrane remodeling often depends upon the presence of phosphoinositides, which recruit protein effectors for a variety of cellular functions. Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns3P)-binding effector involved in endosomal and lysosomal membrane-associated signaling. Both the Phafin2 PH and the FYVE domains bind PtdIns3P, although their redundant function in the protein is unclear. Through a combination of lipid-binding assays, we found that, unlike the FYVE domain, recognition of the PH domain to PtdIns3P requires a lipid bilayer. Using site-directed mutagenesis and truncation constructs, we discovered that the Phafin2 FYVE domain is constitutive for PtdIns3P binding, whereas PH domain binding to PtdIns3P is autoinhibited by a conserved C-terminal acidic motif. These findings suggest that binding of the Phafin2 PH domain to PtdIns3P in membrane compartments occurs through a highly regulated mechanism. Potential mechanisms are discussed throughout this report. Unlabelled Image • The Phafin2 FYVE domain constitutively binds free- and membrane-embedded PtdIns3P. • The Phafin2 PH domain only associates with PtdIns3P when the phosphoinositide is inserted into a lipid bilayer. • When isolated, the PH domain exhibits an apparent higher PtdIns3P binding affinity than that measured for the FYVE domain. • A conserved C-terminal acidic motif in Phafin2 intramolecularly inhibits binding of the PH domain to PtdIns3P. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
34. Tom1 Modulates Binding of Tollip to Phosphatidylinositol 3-Phosphate via a Coupled Folding and Binding Mechanism.
- Author
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Xiao, Shuyan, Brannon, Mary K., Zhao, Xiaolin, Fread, Kristen I., Ellena, Jeffrey F., Bushweller, John H., Finkielstein, Carla V., Armstrong, Geoffrey S., and Capelluto, Daniel G.S.
- Subjects
- *
PHOSPHATIDYLINOSITOL 3-kinases , *CARRIER proteins , *PHOSPHOTRANSFERASES , *PHOSPHATES , *UBIQUITIN - Abstract
Summary Early endosomes represent the first sorting station for vesicular ubiquitylated cargo. Tollip, through its C2 domain, associates with endosomal phosphatidylinositol 3-phosphate (PtdIns(3)P) and binds ubiquitylated cargo in these compartments via its C2 and CUE domains. Tom1, through its GAT domain, is recruited to endosomes by binding to the Tollip Tom1-binding domain (TBD) through an unknown mechanism. Nuclear magnetic resonance data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N terminus when bound to Tom1 GAT through high-affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Tom1 GAT is also able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors the release of Tollip from endosomal membranes, allowing Tollip to commit to cargo trafficking. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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35. Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides.
- Author
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Mitra, Sharmistha, Traughber, C. Alicia, Brannon, Mary K., Gomez, Stephanie, and Capelluto, Daniel G. S.
- Subjects
- *
CELLULAR signal transduction , *PHYSIOLOGICAL control systems , *ENDOCYTOSIS , *LYSOSOMES , *PHOSPHOINOSITIDES - Abstract
A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endo-somal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endo-somal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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36. Structure, Sulfatide Binding Properties, and Inhibition of Platelet Aggregation by a Disabled-2 Protein-derived Peptide.
- Author
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Shuyan Xiao, Charonko, John J., Xiangping Fu, Salmanzadeh, Alireza, Davalos, Rafael V., Vlachos, Pavlos P., Finkielstein, Carla V., and Capelluto, Daniel G. S.
- Subjects
- *
SULFATIDES , *BLOOD platelet aggregation , *NUCLEAR magnetic resonance , *PHOSPHOTYROSINE , *INTEGRINS , *MICELLES , *FIBRINOGEN - Abstract
Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for αIIbβ3 integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide- binding motifs has been determined in dodecylphosphocholine micelles usingNMRspectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
37. Molecular Mechanisms Responsible for the Selective and Low-Grade Induction of Proinflammatory Mediators in Murine Macrophages by Lipopolysaccharide.
- Author
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Maitra, Urmila, Hui Deng, Glaros, Trevor, Baker, Bianca, Capelluto, Daniel G. S., Zihai Li, and Liwu Li
- Subjects
- *
MACROPHAGES , *INFLAMMATORY mediators , *LIPOPOLYSACCHARIDES , *LABORATORY mice , *ENDOTOXEMIA , *ATHEROSCLEROSIS , *DIABETES , *REACTIVE oxygen species - Abstract
Low-dose endotoxemia is prevalent in humans with adverse health conditions, and it correlates with the pathogenesis of chronic inflammatory diseases such as atherosclerosis, diabetes, and neurologic inflammation. However, the underlying molecular mechanisms are poorly understood. In this study, we demonstrate that subclinical low-dose LPS skews macrophages into a mild proinflammatory state, through cell surface TLR4, IL-1R-associated kinase-1, and the Toll-interacting protein. Unlike high-dose LPS, low-dose LPS does not induce robust activation of NF-κΒ, MAPKS, PI3K, or anti-inflammatory mediators. Instead, low-dose LPS induces activating transcription factor 2 through Toll-interacting protein-mediated generation of mitochondrial reactive oxygen species, allowing mild induction of proinflammatory mediators. Low-dose LPS also suppresses PI3K and related negative regulators of inflammatory genes. Our data reveal novel mechanisms responsible for skewed and persistent low-grade inflammation, a cardinal feature of chronic inflammatory diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
38. Lipid-mediated membrane binding properties of Disabled-2
- Author
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Alajlouni, Ruba, Drahos, Karen E., Finkielstein, Carla V., and Capelluto, Daniel G.S.
- Subjects
- *
LIPIDS , *CELL membranes , *ENDOCYTOSIS , *CIRCULAR dichroism , *MICELLES , *NUCLEAR magnetic resonance , *PHOSPHOINOSITIDES , *SURFACE plasmon resonance - Abstract
Abstract: Disabled-2 (Dab2) is an adaptor protein involved in several biological processes ranging from endocytosis to platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the inner leaflet of the plasma membrane. As a result of platelet activation, Dab2 is released from α-granules and associates with both the αIIbβ3 integrin receptor and sulfatide lipids on the platelet surface through its N-terminal region including the PTB domain (N-PTB), thus, modulating platelet aggregation. Thrombin, a strong platelet agonist, prevents Dab2 function by cleaving N-PTB within the two basic motifs required for sulfatide association, a reaction that is prevented when Dab2 is bound to these sphingolipids. We have characterized the membrane binding properties of Dab2 N-PTB using micelles enriched with Dab2 lipid ligands, sulfatides and PtdIns(4,5)P2. Remarkably, NMR spectroscopy studies suggested differences in lipid-binding mechanisms. In addition, we experimentally demonstrated that sulfatide- and PtdIns(4,5)P2-binding sites overlap in Dab2 N-PTB and that both lipids stabilize the protein against temperature-induced unfolding. We found that whereas sulfatides induced conformational changes and facilitated Dab2 N-PTB penetration into micelles, Dab2 N-PTB bound to PtdIns(4,5)P2 lacked these properties. These results further support our model that platelet membrane sulfatides, but not PtdIns(4,5)P2, protect Dab2 N-PTB from thrombin cleavage. [Copyright &y& Elsevier]
- Published
- 2011
- Full Text
- View/download PDF
39. An internal linker and pH biosensing by phosphatidylinositol 5-phosphate regulate the function of the ESCRT-0 component TOM1.
- Author
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Xiong W, Roach TG, Ball N, Corluka M, Beyer J, Brown AM, and Capelluto DGS
- Abstract
Target of Myb1 (TOM1) facilitates the transport of endosomal ubiquitinated proteins destined for lysosomal degradation; however, the mechanisms regulating TOM1 during this process remain unknown. Here, we identified an adjacent DXXLL motif-containing region to the TOM1 VHS domain, which enhances its affinity for ubiquitin and can be modulated by phosphorylation. TOM1 is an endosomal phosphatidylinositol 5-phosphate (PtdIns5P) effector under Shigella flexneri infection. We pinpointed a consensus PtdIns5P-binding motif in the VHS domain. We show that PtdIns5P binding by TOM1 is pH-dependent, similarly observed in its binding partner TOLLIP. Under acidic conditions, TOM1 retained its complex formation with TOLLIP, but was unable to bind ubiquitin. S. flexneri infection inhibits pH-dependent endosomal maturation, leading to reduced protein degradation. We propose a model wherein pumping of H
+ to the cytosolic side of endosomes contributes to the accumulation of TOM1, and possibly TOLLIP, at these sites, thereby promoting PtdIns5P- and pH-dependent signaling, facilitating bacterial survival., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
- View/download PDF
40. The repertoire of protein-sulfatide interactions reveal distinct modes of sulfatide recognition.
- Author
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Capelluto DGS
- Abstract
Sulfatide is an abundant glycosphingolipid in the mammalian nervous system, kidney, trachea, gastrointestinal tract, spleen, and pancreas and is found in low levels in other tissues. Sulfatide is characterized by the presence of a sulfate group in the hydrophilic galactose moiety, with isoforms differing in their sphingosine base and the length, unsaturation, and hydroxylation of their acyl chain. Sulfatide has been associated with a variety of cellular processes including immune responses, cell survival, myelin organization, platelet aggregation, and host-pathogen interactions. Structural studies of protein-sulfatide interactions markedly advanced our understanding of their molecular contacts, key-interacting residues, orientation of the sulfatide in its binding site, and in some cases, sulfatide-mediated protein oligomerization. To date, all protein-sulfatide interactions are reported to display dissociation constants in the low micromolar range. At least three distinct modes of protein-sulfatide binding were identified: 1) protein binding to short consensus stretches of amino acids that adopt α-helical-loop-α-helical conformations; 2) sulfatide-bound proteins that present the sulfatide head group to another protein; and 3) proteins that cage sulfatides. The scope of this review is to present an up-to-date overview of these molecular mechanisms of sulfatide recognition to better understand the role of this glycosphingolipid in physiological and pathological states., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Capelluto.)
- Published
- 2022
- Full Text
- View/download PDF
41. Editorial: Signaling Proteins for Endosomal and Lysosomal Function.
- Author
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Capelluto DGS, Conde CB, Tumbarello DA, and van den Bogaart G
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Published
- 2021
- Full Text
- View/download PDF
42. Protein Trafficking or Cell Signaling: A Dilemma for the Adaptor Protein TOM1.
- Author
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Roach TG, Lång HKM, Xiong W, Ryhänen SJ, and Capelluto DGS
- Abstract
Lysosomal degradation of ubiquitinated transmembrane protein receptors (cargo) relies on the function of Endosomal Sorting Complex Required for Transport (ESCRT) protein complexes. The ESCRT machinery is comprised of five unique oligomeric complexes with distinct functions. Target of Myb1 (TOM1) is an ESCRT protein involved in the initial steps of endosomal cargo sorting. To exert its function, TOM1 associates with ubiquitin moieties on the cargo via its VHS and GAT domains. Several ESCRT proteins, including TOLLIP, Endofin, and Hrs, have been reported to form a complex with TOM1 at early endosomal membrane surfaces, which may potentiate the role of TOM1 in cargo sorting. More recently, it was found that TOM1 is involved in other physiological processes, including autophagy, immune responses, and neuroinflammation, which crosstalk with its endosomal cargo sorting function. Alteration of TOM1 function has emerged as a phosphoinositide-dependent survival mechanism for bacterial infections and cancer progression. Based on current knowledge of TOM1-dependent cellular processes, this review illustrates how TOM1 functions in coordination with an array of protein partners under physiological and pathological scenarios., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roach, Lång, Xiong, Ryhänen and Capelluto.)
- Published
- 2021
- Full Text
- View/download PDF
43. Structural, thermodynamic, and phosphatidylinositol 3-phosphate binding properties of Phafin2.
- Author
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Tang TX, Jo A, Deng J, Ellena JF, Lazar IM, Davis RM, and Capelluto DG
- Subjects
- Phosphatidylinositol Phosphates metabolism, Protein Binding, Protein Domains, Structure-Activity Relationship, Thermodynamics, Vesicular Transport Proteins metabolism, Phosphatidylinositol Phosphates chemistry, Vesicular Transport Proteins chemistry
- Abstract
Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments. Here, we show that human Phafin2 is a moderately elongated monomer of ∼28 kDa with an intensity-average hydrodynamic diameter of ∼7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an α/β structure and predicts ∼40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.4°C. The folding-unfolding curves, obtained using urea- and guanidine hydrochloride-mediated denaturation, indicate that Phafin2 undergoes a two-state native-to-denatured transition. Analysis of these transitions shows that the free energy change for urea- and guanidine hydrochloride-induced Phafin2 denaturation in water is ∼4 kcal mol
-1 . PtdIns(3)P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns(3)P-binding domains of the protein in endomembrane compartments., (© 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)- Published
- 2017
- Full Text
- View/download PDF
44. Identification of Lipid Binding Modulators Using the Protein-Lipid Overlay Assay.
- Author
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Tang TX, Xiong W, Finkielstein CV, and Capelluto DGS
- Subjects
- Binding Sites, Collodion chemistry, Drug Evaluation, Preclinical methods, Glutathione Transferase chemistry, Humans, Hydrogen-Ion Concentration, Intracellular Signaling Peptides and Proteins chemistry, Ligands, Protein Binding, Vesicular Transport Proteins chemistry, Lipid Metabolism, Phospholipids chemistry, Proteins chemistry
- Abstract
The protein-lipid overlay assay is an inexpensive, easy-to-implement, and high-throughput methodology that employs nitrocellulose membranes to immobilize lipids in order to rapid screen and identify protein-lipid interactions. In this chapter, we show how this methodology can identify potential modulators of protein-lipid interactions by screening water-soluble lipid competitors or even the introduction of pH changes during the binding assay to identify pH-dependent lipid binding events.
- Published
- 2017
- Full Text
- View/download PDF
45. Structure of the GAT domain of the endosomal adapter protein Tom1.
- Author
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Xiao S, Ellena JF, Armstrong GS, and Capelluto DG
- Abstract
Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain's association to Tollip's Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.
- Published
- 2016
- Full Text
- View/download PDF
46. Biophysical and molecular-dynamics studies of phosphatidic acid binding by the Dvl-2 DEP domain.
- Author
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Capelluto DG, Zhao X, Lucas A, Lemkul JA, Xiao S, Fu X, Sun F, Bevan DR, and Finkielstein CV
- Subjects
- Cell Membrane metabolism, Frizzled Receptors metabolism, Hydrogen-Ion Concentration, Protein Binding, Protein Structure, Tertiary, Signal Transduction, Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing metabolism, Biophysical Phenomena, Molecular Dynamics Simulation, Phosphatidic Acids metabolism
- Abstract
The Wnt-dependent, β-catenin-independent pathway modulates cell movement and behavior. A downstream regulator of this signaling pathway is Dishevelled (Dvl), which, among other multiple interactions, binds to the Frizzled receptor and the plasma membrane via phosphatidic acid (PA) in a mechanism proposed to be pH-dependent. While the Dvl DEP domain is central to the β-catenin-independent Wnt signaling function, the mechanism underlying its physical interaction with the membrane remains elusive. In this report, we elucidate the structural and functional basis of PA association to the Dvl2 DEP domain. Nuclear magnetic resonance, molecular-dynamics simulations, and mutagenesis data indicated that the domain interacted with the phospholipid through the basic helix 3 and a contiguous loop with moderate affinity. The association suggested that PA binding promoted local conformational changes in helix 2 and β-strand 4, both of which are compromised to maintain a stable hydrophobic core in the DEP domain. We also show that the Dvl2 DEP domain bound PA in a pH-dependent manner in a mechanism that resembles deprotonation of PA. Collectively, our results structurally define the PA-binding properties of the Dvl2 DEP domain, which can be exploited for the investigation of binding mechanisms of other DEP domain-interacting proteins., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
47. Structural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry.
- Author
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Sun F, Kale SD, Azurmendi HF, Li D, Tyler BM, and Capelluto DG
- Subjects
- Amino Acid Motifs, Binding Sites, Cell Line, Cell Membrane metabolism, Circular Dichroism, Host-Parasite Interactions, Humans, Kinetics, Magnetic Resonance Spectroscopy, Models, Biological, Mutation, Phytophthora cytology, Phytophthora genetics, Phytophthora pathogenicity, Plant Roots parasitology, Protein Binding, Protein Stability, Protein Unfolding, Proteins genetics, Proteins metabolism, Recombinant Fusion Proteins, Surface Plasmon Resonance, Temperature, Phosphatidylinositol Phosphates metabolism, Phytophthora metabolism, Plant Diseases parasitology, Proteins chemistry, Glycine max parasitology
- Abstract
Oomycetes such as Phytophthora sojae employ effector proteins that enter plant cells to facilitate infection. Entry of some effector proteins is mediated by RxLR motifs in the effectors and phosphoinositides (PIP) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effectors involved in PIP recognition. We have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that Avh5 is helical in nature, with a long N-terminal disordered region. NMR titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate, directly identified the ligand-binding residues. A C-terminal lysine-rich helical region (helix 2) was the principal lipid-binding site, with the N-terminal RxLR (RFLR) motif playing a more minor role. Mutations in the RFLR motif affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Mutations in the RFLR motif or in the basic region both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. Based on these findings, we propose a model where Avh5 interacts with PtdIns(3)P through its C terminus, and by binding of the RFLR motif, which promotes host cell entry.
- Published
- 2013
- Full Text
- View/download PDF
48. The enigmatic role of sulfatides: new insights into cellular functions and mechanisms of protein recognition.
- Author
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Xiao S, Finkielstein CV, and Capelluto DG
- Subjects
- Animals, Autoimmunity, Cardiovascular Diseases etiology, Host-Pathogen Interactions, Humans, Immunity, Innate, Neoplasms etiology, Platelet Adhesiveness, Platelet Aggregation, Carrier Proteins metabolism, Sulfoglycosphingolipids metabolism
- Abstract
Sulfatides are sphingolipids commonly found at the surface of most of eukaryotic cells. Sulfatides are not just structural components of the plasma membrane but also participate in a wide range of cellular processes including protein trafficking, cell adhesion and aggregation, axon-myelin interactions, neural plasticity, and immune responses, among others. The intriguing question is how can sulfatides trigger such cellular processes? Their dynamic presence and specific localization at plasma membrane sites may explain their multitasking role. Crystal and NMR structural studies have provided the basis for understanding the mechanism of binding by sulfatide-interacting proteins. These proteins generally exhibit a hydrophobic cavity that is responsible for the interaction with the sulfatide acyl chain, whereas the hydrophilic, negatively charged moiety can be found either buried in the hydrophobic cavity of the protein or exposed for additional intermolecular associations. Since sulfatides vary in their acyl chain composition, which are tissue-dependent, more emphasis on understanding acyl chain specificity by sulfatide-binding proteins is warranted. Importantly, changes in cellular sulfatide levels as well as circulating sulfatides in serum directly impact cardiovascular and cancer disease development and progress. Therefore, sulfatides might prove useful as novel biomarkers. The scope of this review is to overview cell functions and mechanisms of sulfatide recognition to better understand the role of these lipids in health and disease.
- Published
- 2013
- Full Text
- View/download PDF
49. Structure, sulfatide binding properties, and inhibition of platelet aggregation by a disabled-2 protein-derived peptide.
- Author
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Xiao S, Charonko JJ, Fu X, Salmanzadeh A, Davalos RV, Vlachos PP, Finkielstein CV, and Capelluto DG
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Amino Acid Motifs, Amino Acid Sequence, Apoptosis Regulatory Proteins, Binding Sites, Circular Dichroism, Humans, Micelles, Models, Molecular, Molecular Sequence Data, Peptides metabolism, Phosphorylcholine analogs & derivatives, Phosphorylcholine chemistry, Platelet Adhesiveness drug effects, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Sulfoglycosphingolipids metabolism, Surface Plasmon Resonance, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins metabolism, Adaptor Proteins, Signal Transducing chemistry, Peptides chemistry, Peptides pharmacology, Platelet Aggregation drug effects, Sulfoglycosphingolipids chemistry, Tumor Suppressor Proteins chemistry
- Abstract
Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for α(IIb)β(3) integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes.
- Published
- 2012
- Full Text
- View/download PDF
50. The C2 domain of Tollip, a Toll-like receptor signalling regulator, exhibits broad preference for phosphoinositides.
- Author
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Ankem G, Mitra S, Sun F, Moreno AC, Chutvirasakul B, Azurmendi HF, Li L, and Capelluto DG
- Subjects
- Calcium metabolism, Humans, Intracellular Signaling Peptides and Proteins genetics, Kinetics, Mutation, Phosphatidylinositols chemistry, Protein Binding, Protein Structure, Tertiary, Protein Transport, Saccharomyces cerevisiae metabolism, Gene Expression Regulation physiology, Intracellular Signaling Peptides and Proteins metabolism, Phosphatidylinositols metabolism
- Abstract
TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid-protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.
- Published
- 2011
- Full Text
- View/download PDF
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