Afshan Kaleem

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.

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.

Bioconversion potential of Trichoderma viride HN1 cellulase for a lignocellulosic biomass Saccharum spontaneum

The industrialisation of lignocellulose conversion is impeded by expensive cellulase enzymes required for saccharification in bioethanol production. Current research undertakes cellulase production from pretreated Saccharum spontaneum through Trichoderma viride HN1 under submerged fermentation conditions. Pretreatment of substrate with 2% NaOH resulted in 88% delignification. Maximum cellulase production (2603 ± 16.39 U/mL/min carboxymethyl cellulase and 1393 ± 25.55 U/mL/min FPase) was achieved at 6% substrate at pH 5.0, with 5% inoculum, incubated at 35°C for 120 h of fermentation period. Addition of surfactant, Tween 80 and metal ion Mn+2, significantly enhanced cellulase yield. This study accounts proficient cellulase yield through process optimisation by exploiting cheaper substrate to escalate their commercial endeavour.

Influence of the sequence environment and properties of neighboring amino acids on amino-acetylation: relevance for structure-function analysis.

Proteins function is regulated by co‐translational modifications and post‐translational modifications (PTMs) such as phosphorylation, glycosylation, and acetylation, which induce proteins to perform multiple tasks in a specified environment. Acetylation takes place post‐translationally on the ε‐amino group of Lys in histone proteins, allowing regulation of gene expression. Furthermore, amino group acetylation also occurs co‐translationally on Ser, Thr, Gly, Met, and Ala, possibly contributing to the stability of proteins. In this work, the influence of amino acids next to acetylated sites has been investigated by using MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for PTMs). MAPRes was utilized to examine the sequence patterns vicinal to modified and non‐modified residues, taking into account their charge and polarity. The PTMs data were further sub‐divided according to their sub‐cellular location (nuclear, mitochondrial, and cytoplasmic), and their association patterns were mined. The association patterns mined by MAPRes for acetylated and non‐acetylated residues are consistent with the existing literature but also revealed novel patterns. These rules have been utilized to describe the acetylation and its effects on the protein structure‐function relationship. J. Cell. Biochem. 114: 874–887, 2013.

Effect on the Ras/Raf signaling pathway of post-translational modifications of neurofibromin: in silico study of protein modification responsible for regulatory pathways

Mapping and chemical characterization of post‐translational modifications (PTMs) in proteins are critical to understand the regulatory mechanisms involving modified proteins and their role in disease. Neurofibromatosis type 1 (NF‐1) is an autosomal dominantly inherited disorder, where NF1 mutations usually result in a reduced level of the tumor suppressor protein, neurofibromin (NF). NF is a multifunctional cytoplasmic protein that regulates microtubule dynamics and participates in several signaling pathways, particularly the RAS signaling pathway. NF is a Ras GTPase‐activating protein (GAP) that prevents oncogenesis by converting GTP‐Ras to GDP‐Ras. This function of NF is regulated by phosphorylation. Interplay of phosphorylation with O‐GlcNAc modification on the same or vicinal Ser/Thr residues, the Yin Yang sites, is well known in cytoplasmic and nuclear proteins. The dynamic aspects of PTMs and their interplay being difficult to follow in vivo, we undertook this in silico work to predict and define the possible role of Yin Yang sites in NF‐1. Interplay of phosphorylation and O‐GlcNAc modification is proposed as a mechanism controlling the Ras signaling pathway. J. Cell. Biochem. 108: 816–824, 2009.

Process optimisation for the biosynthesis of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3 using submerged fermentation

This study deals with optimisation of cultural conditions for enhanced production of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3. Influence of different variables including incubation time, temperature, inoculum size, pH, nitrogen sources and metal ions has been studied. The optimum conditions for cellulase production were incubation period of 72 h, inoculum size 4% incubation temperature 37°C, pH 7, 0.25% ammonium sulphate, 0.2% peptone as inorganic and organic nitrogen source in case of Bacillus PC-BC6. In case of mutant Bacillus N3, optimal conditions were incubation period of 48 h, incubation temperature 37°C, inoculum size 3%, pH 7, 0.2% ammonium chloride and 0.15% yeast extract. Presence of MnSO4 and CaCl2 enhances the enzyme production by Bacillus PC-BC6 and mutant Bacillus N3, respectively. This study was innovative and successful in producing cellulase economically by using cheap indigenous substrate Saccharum spontaneum.

O-GlcNAc modification of the anti-malarial vaccine candidate PfAMA1: in silico-defined structural changes and potential to generate a better vaccine.

The complex life cycle of plasmodial parasites makes the selection of a single subunit protein a less than optimal strategy to generate an efficient vaccinal protection against malaria. Moreover, the full protection afforded by malarial proteins carried by intact parasites implies that immune responses against different antigens expressed in different phases of the cycle are required, but also suggests that native malarial antigens are presented to the host immune system in a manner that recombinant proteins do not achieve. The malarial apical membrane antigen 1 (AMA1) represents a suitable vaccine candidate because AMA1 is expressed on sporozoites and merozoites and allows them to invade hepatocytes and erythrocytes, respectively. Anti-AMA1 antibodies and cytotoxic T-cells are therefore expected to interfere both with the primary invasion of hepatocytes by sporozoites and with the later propagation of merozoites in erythrocytes, and thus efficiently counteract parasite development in its human host. AMA1 bears potential glycosylation sites and the human erythrocytic O-linked N-acetylglucosamine transferase (OGT) could glycosylate AMA1 through combinatorial metabolism. This hypothesis was tested in silico by developing binding models of AMA1 with human OGT complexed with UDP-GlcNc, and followed by the binding of O-GlcNAc with the hydroxyl group of AMA1 serine and threonine residues. Our results suggests that AMA1 shows potential for glycosylation at Thr517 and Ser498 and that O-GlcNAc AMA1 may constitute a conformationally more appropriate antigen for developing a protective anti-malarial immune response.