Evelyne Walker-Nasir

Oct-2 DNA binding transcription factor: functional consequences of phosphorylation and glycosylation

Phosphorylation and O-GlcNAc modification often induce conformational changes and allow the protein to specifically interact with other proteins. Interplay of phosphorylation and O-GlcNAc modification at the same conserved site may result in the protein undergoing functional switches. We describe that at conserved Ser/Thr residues of human Oct-2, alternative phosphorylation and O-GlcNAc modification (Yin Yang sites) can be predicted by the YinOYang1.2 method. We propose here that alternative phosphorylation and O-GlcNAc modification at Ser191 in the N-terminal region, Ser271 and 274 in the linker region of two POU sub-domains and Thr301 and Ser323 in the POUh subdomain are involved in the differential binding behavior of Oct-2 to the octamer DNA motif. This implies that phosphorylation or O-GlcNAc modification of the same amino acid may result in a different binding capacity of the modified protein. In the C-terminal domain, Ser371, 389 and 394 are additional Yin Yang sites that could be involved in the modulation of Oct-2 binding properties.

Phosphorylation and glycosylation interplay: Protein modifications at hydroxy amino acids and prediction of signaling functions of the human β3 integrin family

Protein functions are determined by their three‐dimensional structures and the folded 3‐D structure is in turn governed by the primary structure and post‐translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post‐translational modifications, like phosphorylation and O‐linked N‐acetylglucosamine (O‐GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer‐assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi‐directional (outside‐in and inside‐out) signaling events. The β3 integrin family, including, αIIbβ3 and αvβ3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the β3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside‐out and outside‐in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin‐associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay. J. Cell. Biochem. 99: 706–718, 2006.

Immediate‐early gene regulation by interplay between different post‐translational modifications on human histone H3

In mammalian cells, induction of immediate‐early (IE) gene transcription occurs concomitantly with histone H3 phosphorylation on Ser 10 and is catalyzed by mitogen‐activated protein kinases (MAPKs). Histone H3 is an evolutionarily conserved protein located in the core of the nucleosome, along with histones H2A, H2B, and H4. The N‐terminal tails of histones protrude outside the chromatin structure and are accessible to various enzymes for post‐translational modifications (PTM). Phosphorylation, O‐GlcNAc modification, and their interplay often induce functional changes, but it is very difficult to monitor dynamic structural and functional changes in vivo. To get started in this complex task, computer‐assisted studies are useful to predict the range in which those dynamic structural and functional changes may occur. As an illustration, we propose blocking of phosphorylation by O‐GlcNAc modification on Ser 10, which may result in gene silencing in the presence of methylated Lys 9. Thus, alternate phosphorylation and O‐GlcNAc modification on Ser 10 in the histone H3 protein may provide an on/off switch to regulate expression of IE genes. J. Cell. Biochem. 103: 835–851, 2008.

Role of sialic acid and sulfate groups in cervical mucus physiological functions: study of Macaca radiata glycoproteins

The influence of charged groups in glycoproteins was investigated to assess their effect on the physiological functions of bonnet monkey cervical mucus. The macromolecular glycoproteins from peri-ovulatory, midcycle phase cervical mucus were treated with Pronase, trypsin and chymotrypsin and the enzyme-resistant glycoproteins purified by gel filtration on Sepharose 4B and a high molecular weight component containing carbohydrates, proteins and sulfate groups was recovered in high yield. This material still reacted with an antiserum directed against purified midcycle glycoprotein but not against another antiserum directed against luteal phase purified glycoproteins. Upon treatment with Pronase, trypsin and chymotrypsin, asialoglycoproteins and desulfated asialoglycoproteins released fragments of low molecular sizes, none of which reacted with the anti-midcycle glycoprotein antiserum. Cervical mucus collected from the estrogenic phase displayed a morphology supporting sperm migration, and this mucus retains the same morphology and reacts with the anti-midcycle glycoprotein antiserum following mild treatment with sialidase and subsequently with Pronase. These results imply that charged carbohydrate groups help maintain the structural and functional integrity of the mucus glycoprotein in its biological environment.

MAPRes: An Efficient Method to Analyze Protein Sequence Around Post-Translational Modification Sites

Functional switches are often regulated by dynamic protein modifications. Assessing protein functions, in vivo, and their functional switches remains still a great challenge in this age of development. An alternative methodology based on in silico procedures may facilitate assessing the multifunctionality of proteins and, in addition, allow predicting functions of those proteins that exhibit their functionality through transitory modifications. Extensive research is ongoing to predict the sequence of protein modification sites and analyze their dynamic nature. This study reports the analysis performed on phosphorylation, Phospho.ELM (version 3.0) and glycosylation, OGlycBase (version 6.0) data for mining association patterns utilizing a newly developed algorithm, MAPRes. This method, MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for post‐translational modifications), is based on mining association among significantly preferred amino acids of neighboring sequence environment and modification sites themselves. Association patterns arrived at by association pattern/rule mining were in significant conformity with the results of different approaches. However, attempts to analyze substrate sequence environment of phosphorylation sites catalyzed for Tyr kinases and the sequence data for O‐GlcNAc modification were not successful, due to the limited data available. Using the MAPRes algorithm for developing an association among PTM site with its vicinal amino acids is a valid method with many potential uses: this is indeed the first method ever to apply the association pattern mining technique to protein post‐translational modification data. J. Cell. Biochem. 104: 1220–1231, 2008.

In silico determination of intracellular glycosylation and phosphorylation sites in human selectins: implications for biological function

Post‐translational modifications provide the proteins with the possibility to perform functions in addition to those determined by their primary sequence. However, analysis of multifunctional protein structures in the environment of cells and body fluids is made especially difficult by the presence of other interacting proteins. Bioinformatics tools are therefore helpful to predict protein multifunctionality through the identification of serine and threonine residues wherein the hydroxyl group is likely to become modified by phosphorylation or glycosylation. Moreover, serines and threonines where both modifications are likely to occur can also be predicted (YinYang sites), to suggest further functional versatility. Structural modifications of hydroxyl groups of P‐, E‐, and L‐selectins have been predicted and possible functions resulting from such modifications are proposed. Functional changes of the three selectins are based on the assumption that transitory and reversible protein modifications by phosphate and O‐GlcNAc cause specific conformational changes and generate binding sites for other proteins. The computer‐assisted prediction of glycosylation and phosphorylation sites in selectins should be helpful to assess the contribution of dynamic protein modifications in selectin‐mediated inflammatory responses and cell–cell adhesion processes that are difficult to determine experimentally. J. Cell. Biochem. 100: 1558–1572, 2007.

CREB in long‐term potentiation in hippocampus: Role of post‐translational modifications‐studies In silico

The multifunctionality of proteins is dictated by post‐translational modifications (PTMs) which involve the attachment of small functional groups such as phosphate and acetate, as well as carbohydrate moieties. These functional groups make the protein perform various functions in different environments. PTMs play a crucial role in memory and learning. Phosphorylation of synaptic proteins and transcription factors regulate the generation and storage of memory. Among these is the cAMP‐regulated element binding protein CREB that regulates CRE containing genes like c‐fos. Both phosphorylation and acetylation control the function of CREB as a transcription factor. CREB is also susceptible to O‐GlcNAc modification, which inhibits its activity. O‐GlcNAc modification occurs on the same or neighboring Ser/Thr residues akin to phosphorylation. An interplay between these modifications was shown to operate in nuclear and cytoplasmic proteins. In this study computational methods were utilized to predict different modification sites in CREB. These in silico results suggest that phosphorylation, O‐GlcNAc modification and acetylation modulate the transcriptional activity of CREB and thus dictate its contribution to synaptic plasticity. J. Cell. Biochem. 112: 138–146, 2011.

In Silico Modulation of Apoptotic Bcl-2 Proteins by Mistletoe Lectin-1: Functional Consequences of Protein Modifications

The mistletoe lectin‐1 (ML‐1) modulates tumor cell apoptosis by triggering signaling cascades through the complex interplay of phosphorylation and O‐linked N‐acetylglucosamine (O‐GlcNAc) modification in pro‐ and anti‐apoptotic proteins. In particular, ML‐1 is predicted to induce dephosphorylation of Bcl‐2‐family proteins and their alternative O‐GlcNAc modification at specific, conserved Ser/Thr residues. The sites for phosphorylation and glycosylation were predicted and analyzed using Netphos 2.0 and YinOYang 1.2. The involvement of modified Ser/Thr, and among them the potential Yin Yang sites that may undergo both types of posttranslational modification, is proposed to mediate apoptosis modulation by ML‐1. J. Cell. Biochem. 103: 479–491, 2008.

Galactose: A Specifically Recognized, Terminal Carbohydrate Moiety in Biological Processes

Glycoproteins and glycolipids carrying diverse oligosaccharide structures are involved in countless molecular interactions in physiologic and pathologic situations. Defining the specific carbohydrate moieties expressed in a particular set of molecules is a challenging task that could eventually explain how glycoproteins and glycolipids contribute to the physiology of normal cells and how their alterations could lead to pathologic states. A simple example is the ABO blood group system: in individuals with blood group B, the marker is defined by its terminal linked galactose, and substitution of its hydroxyl group at C2 by an N-acetyl group results in the formation of N-acetylgalactosamine, the blood group A marker. This review focuses on the importance of terminal linked galactose and its derivatives in different normal and pathological conditions. The involvement of various sugars residues sub-terminal to galactose and its derivatives was also evaluated on the basis of the galactosylation data taken from different publicly available carbohydrate databases. We conclude that those sugars penultimate to galactose, with their different types of linkages and anomery, contribute to the structure and functions of carbohydrate moieties with a terminal galactose.

In silico modulation of HMGN-1 binding to histones and gene expression by interplay of phosphorylation and O-GlcNAc modification

Utilizing different computational methods; phosphorylation, O-GlcNAc modification and Yin Yang sites are predicted in HMGN-1. Prediction results suggest that interplay of phosphorylation and O-GlcNAc modification regulates binding and removal of HMGN-1 with the nucleosome and its translocation from nucleus to cytoplasm and back to nucleus, consequently modulating gene expression.