scholarly journals Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nilmani Singh ◽  
Adriana Reyes-Ordoñez ◽  
Michael A. Compagnone ◽  
Jesus F. Moreno ◽  
Benjamin J. Leslie ◽  
...  
Keyword(s):  
Single Molecule ◽  
Learning Algorithm ◽  
Specific Interaction ◽  
Lipid Vesicles ◽  
Lipid Binding ◽  
Ph Domain ◽  
Structural Requirements ◽  
Pulldown Assay ◽  
Ph Domains ◽  
Functional Relevance

AbstractPleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.

scholarly journals Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

2020 ◽  
Author(s):  
Nilmani Singh ◽  
Adriana Reyes-Ordoñez ◽  
Michael A. Compagnone ◽  
Jesus F. Moreno Castillo ◽  
Benjamin J. Leslie ◽  
...  
Keyword(s):  
Single Molecule ◽  
Learning Algorithm ◽  
Specific Interaction ◽  
Lipid Vesicles ◽  
Lipid Binding ◽  
Ph Domain ◽  
Structural Requirements ◽  
Pulldown Assay ◽  
Ph Domains ◽  
Functional Relevance

ABSTRACTPleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employed a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domaincontaining proteins examined, 36 (54%) were found to have affinity for PIPs with various specificity, the majority of which had not been reported before. Further investigation of ARHGEF3 revealed structural requirements and functional relevance of its newly discovered PI(4,5)P2 binding. A recursive-learning algorithm based on the assay results of the 67 proteins was generated to analyze the sequences of 246 human PH domains, which predicted 48% of them to bind PIPs. A collection of the predicted PIP-binding proteins was assayed, with the vast majority found to bind PIPs. Collectively, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.

scholarly journals Pleckstrin homology (PH) domains and phosphoinositides

2007 ◽  
Vol 74 ◽  
pp. 81-93 ◽  
Author(s):  
Mark A. Lemmon
Keyword(s):  
Ph Domain ◽  
Pleckstrin Homology ◽  
High Affinity ◽  
Weak Binding ◽  
Ph Domains ◽  
Common Domain ◽  
Functional Relevance ◽  
Phox Homology ◽  
Adp Ribosylation Factor ◽  
Homology Domains

PH (pleckstrin homology) domains represent the 11th most common domain in the human proteome. They are best known for their ability to bind phosphoinositides with high affinity and specificity, although it is now clear that less than 10% of all PH domains share this property. Cases in which PH domains bind specific phosphoinositides with high affinity are restricted to those phosphoinositides that have a pair of adjacent phosphates in their inositol headgroup. Those that do not [PtdIns3P, PtdIns5P and PtdIns(3,5)P2] are instead recognized by distinct classes of domains including FYVE domains, PX (phox homology) domains, PHD (plant homeodomain) fingers and the recently identified PROPPINs (b-propellers that bind polyphosphoinositides). Of the 90% of PH domains that do not bind strongly and specifically to phosphoinositides, few are well understood. One group of PH domains appears to bind both phosphoinositides (with little specificity) and Arf (ADP-ribosylation factor) family small G-proteins, and are targeted to the Golgi apparatus where both phosphoinositides and the relevant Arfs are both present. Here, the PH domains may function as coincidence detectors. A central challenge in understanding the majority of PH domains is to establish whether the very low affinity phosphoinositide binding reported in many cases has any functional relevance. For PH domains from dynamin and from Dbl family proteins, this weak binding does appear to be functionally important, although its precise mechanistic role is unclear. In many other cases, it is quite likely that alternative binding partners are more relevant, and that the observed PH domain homology represents conservation of structural fold rather than function.

scholarly journals Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes

2020 ◽  
Vol 6 (8) ◽  
pp. eaay5736 ◽  
Author(s):  
Eiji Yamamoto ◽  
Jan Domański ◽  
Fiona B. Naughton ◽  
Robert B. Best ◽  
Antreas C. Kalli ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Bound States ◽  
Lipid Binding ◽  
Dependent Manner ◽  
Ph Domain ◽  
Concentration Dependent Manner ◽  
Phosphatidylinositol Phosphate ◽  
Ph Domains ◽  
Experimental Values ◽  
Dynamics Simulations

Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.

Pleckstrin homology domains: not just for phosphoinositides

2004 ◽  
Vol 32 (5) ◽  
pp. 707-711 ◽  
Author(s):  
M.A. Lemmon
Keyword(s):  
Subcellular Localization ◽  
Human Genome ◽  
Small Gtpases ◽  
Membrane Targeting ◽  
Ph Domain ◽  
Cellular Membranes ◽  
Pleckstrin Homology ◽  
Ph Domains ◽  
Common Domain ◽  
Homology Domains

PH domains (pleckstrin homology domains) are the 11th most common domain in the human genome and are best known for their ability to target cellular membranes by binding specifically to phosphoinositides. Recent studies in yeast have shown that, in fact, this is a property of only a small fraction of the known PH domains. Most PH domains are not capable of independent membrane targeting, and those capable of doing so (approx. 33%) appear, most often, to require both phosphoinositide and non-phosphoinositide determinants for their subcellular localization. Several recent studies have suggested that small GTPases such as ARF family proteins play a role in defining PH domain localization. Some others have described a signalling role for PH domains in regulating small GTPases, although phosphoinositides may also play a role. These findings herald a change in our perspective of PH domain function, which will be significantly more diverse than previously supposed.

scholarly journals The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function

1998 ◽  
Vol 9 (8) ◽  
pp. 1981-1994 ◽  
Author(s):  
Wolfgang Nagel ◽  
Pierre Schilcher ◽  
Lutz Zeitlmann ◽  
Waldemar Kolanus
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Biological Function ◽  
Common Mechanism ◽  
Membrane Association ◽  
Bruton’S Tyrosine Kinase ◽  
Ph Domain ◽  
Bruton's Tyrosine Kinase ◽  
Ph Domains

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ∼100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.

scholarly journals Investigating Serum and Tissue Expression Identified a Cytokine/Chemokine Signature as a Highly Effective Melanoma Marker

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3680
Author(s):  
Marco Cesati ◽  
Francesca Scatozza ◽  
Daniela D’Arcangelo ◽  
Gian Carlo Antonini-Cappellini ◽  
Stefania Rossi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Learning Algorithm ◽  
Profile Analysis ◽  
Pearson Correlation ◽  
Tissue Expression ◽  
Support Vector ◽  
Expression Data ◽  
Serum Samples ◽  
Tissue Samples ◽  
Mortality And Morbidity

The identification of reliable and quantitative melanoma biomarkers may help an early diagnosis and may directly affect melanoma mortality and morbidity. The aim of the present study was to identify effective biomarkers by investigating the expression of 27 cytokines/chemokines in melanoma compared to healthy controls, both in serum and in tissue samples. Serum samples were from 232 patients recruited at the IDI-IRCCS hospital. Expression was quantified by xMAP technology, on 27 cytokines/chemokines, compared to the control sera. RNA expression data of the same 27 molecules were obtained from 511 melanoma- and healthy-tissue samples, from the GENT2 database. Statistical analysis involved a 3-step approach: analysis of the single-molecules by Mann–Whitney analysis; analysis of paired-molecules by Pearson correlation; and profile analysis by the machine learning algorithm Support Vector Machine (SVM). Single-molecule analysis of serum expression identified IL-1b, IL-6, IP-10, PDGF-BB, and RANTES differently expressed in melanoma (p < 0.05). Expression of IL-8, GM-CSF, MCP-1, and TNF-α was found to be significantly correlated with Breslow thickness. Eotaxin and MCP-1 were found differentially expressed in male vs. female patients. Tissue expression analysis identified very effective marker/predictor genes, namely, IL-1Ra, IL-7, MIP-1a, and MIP-1b, with individual AUC values of 0.88, 0.86, 0.93, 0.87, respectively. SVM analysis of the tissue expression data identified the combination of these four molecules as the most effective signature to discriminate melanoma patients (AUC = 0.98). Validation, using the GEPIA2 database on an additional 1019 independent samples, fully confirmed these observations. The present study demonstrates, for the first time, that the IL-1Ra, IL-7, MIP-1a, and MIP-1b gene signature discriminates melanoma from control tissues with extremely high efficacy. We therefore propose this 4-molecule combination as an effective melanoma marker.

scholarly journals How to control single-molecule rotation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Grant J. Simpson ◽  
Víctor García-López ◽  
A. Daniel Boese ◽  
James M. Tour ◽  
Leonhard Grill
Keyword(s):  
Electric Field ◽  
Single Molecule ◽  
Scanning Tunneling Microscope ◽  
Specific Interaction ◽  
Scanning Tunneling ◽  
Individual Molecule ◽  
Pivot Point ◽  
Dipolar Molecules ◽  
At Will ◽  
Orientation Of Molecules

Abstract The orientation of molecules is crucial in many chemical processes. Here, we report how single dipolar molecules can be oriented with maximum precision using the electric field of a scanning tunneling microscope. Rotation is found to occur around a fixed pivot point that is caused by the specific interaction of an oxygen atom in the molecule with the Ag(111) surface. Both directions of rotation are realized at will with 100% directionality. Consequently, the internal dipole moment of an individual molecule can be spatially mapped via its behavior in an applied electric field. The importance of the oxygen-surface interaction is demonstrated by the addition of a silver atom between a single molecule and the surface and the consequent loss of the pivot point.

scholarly journals Identification of an 11-residue portion of CTP–phosphocholine cytidylyltransferase that is required for enzyme–membrane interactions

1997 ◽  
Vol 325 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Jilin YANG ◽  
Jinxia WANG ◽  
Irene TSEU ◽  
Maciej KULISZEWSKI ◽  
Wensu LEE ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Fusion Proteins ◽  
Membrane Binding ◽  
Lipid Vesicles ◽  
Lipid Binding ◽  
Binding Domain ◽  
Membrane Interactions ◽  
Phosphocholine Cytidylyltransferase ◽  
N Terminus ◽  
L2 Cells

CTP–phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in the biosynthesis of phosphatidylcholine (PC) in many cells. Enzyme–membrane interactions appear to play an important role in CT activation. A putative membrane-binding domain appears to be located between residues 236 and 293 from the N-terminus. To map the membrane-binding domain more precisely, glutathione S-transferase fusion proteins were prepared that contained deletions of various domains in this putative lipid-binding region. The fusion proteins were assessed for their binding of [3H]PC/oleic acid vesicles. Fusion proteins encompassing residues 267–277 bound to PC/oleic acid vesicles, whereas fragments lacking this region exhibited no specific binding to the lipid vesicles. The membrane-binding characteristics of the CT fusion proteins were also examined using intact lung microsomes. Only fragments encompassing residues 267–277 competed with full-length 125I-labelled CT, expressed in recombinant Sf9 insect cells, for microsomal membrane binding. To investigate the role of this region in PC biosynthesis, A549 and L2 cells were transfected with cDNA for CT mutants under the control of a glucocorticoid-inducible long terminal repeat (LTR) promoter. Induction of CT mutants containing residues 267–277 in transfectants resulted in reduced PC synthesis. The decrease in PC synthesis was accompanied by a shift in endogenous CT activity from the particulate to the soluble fraction. Expression of CT mutants lacking this region in A549 and L2 cells did not affect PC formation and subcellular distribution of CT activity. These results suggest that the CT region located between residues 267 and 277 from the N-terminus is required for the interaction of CT with membranes.

scholarly journals Activation of the Lbc Rho Exchange Factor Proto-Oncogene by Truncation of an Extended C Terminus That Regulates Transformation and Targeting

1999 ◽  
Vol 19 (2) ◽  
pp. 1334-1345 ◽  
Author(s):  
Paola Sterpetti ◽  
Andrew A. Hack ◽  
Mariam P. Bashar ◽  
Brian Park ◽  
Sou-De Cheng ◽  
...  
Keyword(s):  
Biologically Active ◽  
Chromosome 7 ◽  
Chromosome 15 ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Exchange Factor ◽  
C Terminus ◽  
Ph Domains ◽  
Transforming Activity

ABSTRACT The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbloncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted α-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the α-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential.

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