nan Nasir-ud-Din

Signaling through sphingolipid microdomains of the plasma membrane: the concept of signaling platform

Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners [1] and temporary elaboration of supramolecular structures [2], to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptors cytoplasmic domains and intracellular signaling and adaptor proteins [3]. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptors cytoplasmic domains, an event that initiates the signaling cascade [4]. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular non-receptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor [5]. The concept of the signaling platform [6] has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling.The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane [7,8]. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact [9]. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.

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.

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.

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.

Carbohydrate moeity of Plasmodium falciparum glycoproteins: The nature of the carbohydrate‐peptide linkage in the MSP‐2 glycoprotein

Metabolic labelling of Plasmodium falciparum parasites with [3H]GIcN, [3H]Man, [3H]Gal and [3H]ethanolamine, and subsequent purification by SDS‐PAGE of the labelled material provided effective labelling of the MSP‐1, 195 kDa, and MSP‐2, 42‐53 kDa, glycoproteins. Reductive β‐elimination of the MSP‐2 released from the gel consisted of glycopeptides containing labelled sugars. Processing of the eliminated components and identification of the sugar residues demonstrated the presence of N‐acetylglucosaminitol and N‐acetylgalactosaminitol amongst other labelled sugars. Reductive β‐elimination with sodium hydroxide‐sodium borotritide‐borohydride showed the presence of glucosaminitol and alanine in the hydrolysis products. The MSP‐2 was retained on solid phase wheat‐germ agglutinin and was released from the lectin by treatment with GIcNAc. Upon treatment with O‐glycanase the MSP‐2 glycoprotein released lablled amino sugar, and derived oligosaccharides on treatment with exoglycosidases released labelled components corresponding to the metabolically incorporated sugars. Labelled Gal was incorporated into the MSP‐2 glycoprotein using [3H]UDP‐Gal and galactosyltransferase. The galactosylated glycoprotein released labelled Gal upon treatment with β‐galactosidase. The results of the present study suggest that the carbohydrate chains of the MSP‐2 glycoprotein are attached to the protein backbone via GlcNAc‐ and GalNAc‐sedne/threonine in O‐glycosyl linkage and the glycoprotein has terminal GlcNAc and Gal residues. The carbohydrate moieties of MSP‐2, glycoprotein consist mainly of short chains linked to the protein core.