scholarly journals Structural, in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides

2020 ◽  
Author(s):  
Wei Song ◽  
Carter J. Gottschalk ◽  
Tuo-Xian Tang ◽  
Andrew Biscardi ◽  
Jeffrey F. Ellena ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Surface Expression ◽  
Lipid Binding ◽  
Binding Assays ◽  
Binding Motifs ◽  
Cellular Processes ◽  
Amino Acid Region ◽  
Aggregation Processes ◽  
Acyl Chains ◽  
Hydrophilic Region

AbstractDisabled-2 (Dab2) is an adaptor protein that regulates numerous cellular processes. Among them, Dab2 modulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin αIIbβ3 receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with both the integrin αIIbβ3 receptor and sulfatides, both of which are known to be pro-aggregatory mediators, 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 a combined methodology including 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. A hydrophilic region, primarily mediated by R42, is responsible for the interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with the acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The interacting Dab2 SBP residues with sulfatide resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.

scholarly journals Direct Binding of Human NK Cell Natural Cytotoxicity Receptor NKp44 to the Surfaces of Mycobacteria and Other Bacteria

2008 ◽  
Vol 76 (4) ◽  
pp. 1719-1727 ◽  
Author(s):  
Semih Esin ◽  
Giovanna Batoni ◽  
Claudio Counoupas ◽  
Annarita Stringaro ◽  
Franca Lisa Brancatisano ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Surface Expression ◽  
Binding Assays ◽  
Igg Antibody ◽  
Direct Stimulation ◽  
Activating Receptors ◽  
Accessory Cells

ABSTRACT Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56bright. In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

scholarly journals Cooperative Interaction of Nck and Lck Orchestrates Optimal TCR Signaling

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 834
Author(s):  
Frederike A. Hartl ◽  
Jatuporn Ngoenkam ◽  
Esmeralda Beck-Garcia ◽  
Liz Cerqueira ◽  
Piyamaporn Wipa ◽  
...  
Keyword(s):  
T Cell ◽  
Ligand Binding ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Cooperative Interaction ◽  
Tcr Signaling ◽  
Binding Motifs ◽  
Adaptive Immune

The T cell antigen receptor (TCR) is expressed on T cells, which orchestrate adaptive immune responses. It is composed of the ligand-binding clonotypic TCRαβ heterodimer and the non-covalently bound invariant signal-transducing CD3 complex. Among the CD3 subunits, the CD3ε cytoplasmic tail contains binding motifs for the Src family kinase, Lck, and the adaptor protein, Nck. Lck binds to a receptor kinase (RK) motif and Nck binds to a proline-rich sequence (PRS). Both motifs only become accessible upon ligand binding to the TCR and facilitate the recruitment of Lck and Nck independently of phosphorylation of the TCR. Mutations in each of these motifs cause defects in TCR signaling and T cell activation. Here, we investigated the role of Nck in proximal TCR signaling by silencing both Nck isoforms, Nck1 and Nck2. In the absence of Nck, TCR phosphorylation, ZAP70 recruitment, and ZAP70 phosphorylation was impaired. Mechanistically, this is explained by loss of Lck recruitment to the stimulated TCR in cells lacking Nck. Hence, our data uncover a previously unknown cooperative interaction between Lck and Nck to promote optimal TCR signaling.

scholarly journals Elucidation of the anti-autophagy mechanism of the Legionella effector RavZ using semisynthetic LC3 proteins

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Aimin Yang ◽  
Supansa Pantoom ◽  
Yao-Wen Wu
Keyword(s):  
Legionella Pneumophila ◽  
Lipid Binding ◽  
Effector Protein ◽  
Intracellular Pathogen ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Acyl Chains ◽  
Pathogenic Microbes ◽  
Lir Motif ◽  
Lipid Binding Site

Autophagy is a conserved cellular process involved in the elimination of proteins and organelles. It is also used to combat infection with pathogenic microbes. The intracellular pathogen Legionella pneumophila manipulates autophagy by delivering the effector protein RavZ to deconjugate Atg8/LC3 proteins coupled to phosphatidylethanolamine (PE) on autophagosomal membranes. To understand how RavZ recognizes and deconjugates LC3-PE, we prepared semisynthetic LC3 proteins and elucidated the structures of the RavZ:LC3 interaction. Semisynthetic LC3 proteins allowed the analysis of structure-function relationships. RavZ extracts LC3-PE from the membrane before deconjugation. RavZ initially recognizes the LC3 molecule on membranes via its N-terminal LC3-interacting region (LIR) motif. The RavZ α3 helix is involved in extraction of the PE moiety and docking of the acyl chains into the lipid-binding site of RavZ that is related in structure to that of the phospholipid transfer protein Sec14. Thus, Legionella has evolved a novel mechanism to specifically evade host autophagy.

scholarly journals The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading

2019 ◽  
Vol 4 ◽  
pp. 117
Author(s):  
Maria Shamin ◽  
Tomasz H. Benedyk ◽  
Stephen C. Graham ◽  
Janet E. Deane
Keyword(s):  
Lipid Transfer Protein ◽  
Direct Interaction ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
Binding Assays ◽  
Lipid Transfer Proteins ◽  
Lipid Antigens ◽  
Saposin B ◽  
Protein Components ◽  
Lipid Loading

Background: Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. Methods: In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. We performed equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions. Results: We could not demonstrate a direct interaction between SapB and CD1d using any of these binding assays. Conclusions: This work establishes comprehensively that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process.

scholarly journals Increased fatty acyl saturation of phosphatidylinositol phosphates in prostate cancer progression

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Atsushi Koizumi ◽  
Shintaro Narita ◽  
Hiroki Nakanishi ◽  
Masaki Ishikawa ◽  
Satoshi Eguchi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Progression ◽  
Fatty Acyl ◽  
Human Prostate ◽  
Pathological Stage ◽  
Double Bonds ◽  
Prostate Hyperplasia ◽  
Cellular Processes ◽  
Acyl Chains ◽  
Phosphatidylinositol Phosphates

Abstract Phosphoinositides (PIPs) participate in many cellular processes, including cancer progression; however, the metabolic features of PIPs associated with prostate cancer (PCa) are unknown. We investigated PIPs profiles in PTEN-deficient prostate cancer cell lines, human prostate tissues obtained from patients with PCa and benign prostate hyperplasia (BPH) specimens using mass spectrometry. In immortalized normal human prostate PNT1B cells, PTEN deficiency increased phosphatidylinositol tris-phosphate (PIP3) and decreased phosphatidylinositol mono- and bis-phosphate (PIP1 and PIP2), consistent with PTEN’s functional role as a PI(3,4,5)P3 3-phosphatase. In human prostate tissues, levels of total (sum of all acyl variants) phosphatidylinositol (PI) and PIP1 in PCa were significantly higher than in BPH, whereas PIP2 and PIP3 contents were significantly lower than in BPH. PCa patients had significantly higher proportion of PI, PIP1, and PIP2 with 0–2 double bonds in acyl chains than BPH patients. In subgroup analyses based on PCa aggressiveness, mean total levels of PI with 0–2 double bonds in acyl chains were significantly higher in patients with pathological stage T3 than in those with pathological stage T2. These data indicate that alteration of PIPs level and the saturation of acyl chains may be associated with the development and aggressiveness of prostate cancer, although it is unknown whether this alteration is causative.

scholarly journals Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function

Blood ◽  
2009 ◽  
Vol 113 (2) ◽  
pp. 338-346 ◽  
Author(s):  
Ramon Urrea Moreno ◽  
Juana Gil ◽  
Carmen Rodriguez-Sainz ◽  
Elena Cela ◽  
Victor LaFay ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Critical Role ◽  
Lipid Binding ◽  
Inflammatory Disorder ◽  
C2 Domain ◽  
Biophysical Modeling ◽  
Binding Assays ◽  
Calcium Dependent ◽  
Pathogen Elimination ◽  
Cytotoxic Function

Abstract Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.

scholarly journals GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

2016 ◽  
Vol 36 (7) ◽  
pp. 1152-1163 ◽  
Author(s):  
Maoxiang Zhang ◽  
Jason E. Davis ◽  
Chunman Li ◽  
Jie Gao ◽  
Wei Huang ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Loop ◽  
Interaction Sites ◽  
G Protein Coupled

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at thetrans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

Interaction of Grb2 SH3 domain with UVRAG in an Alzheimer’s disease–like scenario

2014 ◽  
Vol 92 (3) ◽  
pp. 219-225 ◽  
Author(s):  
Kasturi Roy ◽  
Oishee Chakrabarti ◽  
Debashis Mukhopadhyay
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Binding Motifs ◽  
Src Homology 3 ◽  
Src Homology ◽  
Cellular Compartmentalization

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein which participates in trafficking pathways alongside its role in signaling. Proteins important for actin remodeling and cellular compartmentalization contain SRC Homology 3 (SH3) binding motifs that interact with Grb2. While studying the Grb2–amyloid precursor protein (APP) intracellular domain (AICD) interaction in Alzheimer’s disease cell line models, it was seen that Grb2 colocalized to compartments that mature into autophagosomes. The entrapping of AICD in the Grb2 vesicles and its clearance via autophagosomes was a survival contrivance on the part of the cell. Here, we report that Grb2, when in excess, interacts with ultraviolet radiation resistance-associated gene protein (UVRAG) under excess conditions of AICD–Grb2 or Grb2. The N-terminal SH3 domain of Grb2 specifically interacts with UVRAG, unlike the C-terminal SH3 domain. This interaction helps to understand the role of Grb2 in the autophagic maturation of vesicles.

scholarly journals C-terminal Recognition by 14-3-3 Proteins for Surface Expression of Membrane Receptors

2005 ◽  
Vol 280 (43) ◽  
pp. 36263-36272 ◽  
Author(s):  
Brian Coblitz ◽  
Sojin Shikano ◽  
Meng Wu ◽  
Sandra B. Gabelli ◽  
Lisa M. Cockrell ◽  
...  
Keyword(s):  
Genetic Selection ◽  
Surface Expression ◽  
Membrane Receptors ◽  
Binding Modes ◽  
Mode Iii ◽  
Binding Motifs ◽  
Random Peptide Library ◽  
Random Peptide ◽  
Native Proteins ◽  
Localization Signal

Diverse functions of 14-3-3 proteins are directly coupled to their ability to interact with targeted peptide substrates. RSX(pS/pT)XP and RXΦX(pS/pT)XP are two canonical consensus binding motifs for 14-3-3 proteins representing the two common binding modes, modes I and II, between 14-3-3 and internal peptides. Using a genetic selection, we have screened a random peptide library and identified a group of C-terminal motifs, termed SWTY, capable of overriding an endoplasmic reticulum localization signal and redirecting membrane proteins to cell surface. Here we report that the C-terminal SWTY motif, although different from mode I and II consensus, binds tightly to 14-3-3 proteins with a dissociation constant (KD) of 0.17 μm, comparable with that of internal canonical binding peptides. We show that all residues but proline in -SWTX-COOH are compatible for the interaction and surface expression. Because SWTY-like sequences have been found in native proteins, these results support a broad significance of 14-3-3 interaction with protein C termini. The C-terminal binding consensus, mode III, represents an expansion of the repertoire of 14-3-3-targeted sequences.

Sign in / Sign up

Export Citation Format

Share Document