GENETIC ANALYSIS OF \u3cem\u3eSERF\u3c/em\u3e GENE FUNCTION IN \u3cem\u3eDrosophila melanogaster\u3c/em\u3e AND ITS CONTRIBUTION TO A FLY MODEL OF SPINAL MUSCULAR ATROPHY

The Serf gene is evolutionarily highly conserved but its biological function is not known in any organism. In human, SERF1/H4F5 was first identified as a modifier of the disease Spinal Muscular Atrophy (SMA). SMA is caused by mutations in the Survival Motor Neuron 1(SMN1) gene leading to diminished levels of the Smn protein. More than 90% of patients with the most severe form of SMA have deletions that remove SERF1 as well as mutaions within SMN1. Hence, loss of Serf activity is hypothesized to exacerbate SMA disease progression. The primary motivation of this thesis was to test this intriguing but yet unverified hypothesis using a model organism, Drosophila melanogaster. To genetically manipulate Serf activity I created deletion, overexpression and knockdown alleles of Serf. I found that Serf is non-essential for viability in Drosophila and that null mutants have no obvious developmental defects. However, the loss of Serf gene activity results in diminished adult locomotion. In addition, Serf null mutants show lower Smn protein abundance. As Smn mRNA levels do not change with Serf manipulation, regulation likely occurs at the level of Smn protein translation or stability. I tested the impact of Serf in SMA by altering Serf expression in a fly SMA model harboring equivalent Smn point mutations as those that cause SMA in human patients. I found that diminished Serf levels exacerbate the observed mutant phenotype in growth, development and viability which correlates with decreased Smn protein abundance. Importantly, the simple overexpression of Serf in certain Smn mutant backgrounds increases the Smn protein abundance, which in some cases, correlates with a partial rescue of the associated phenotypic defects. In addition to being required for maximal Smn abundance, I found that Serf gene expression directly correlates with the abundance of toxic α-synuclein protein seen in a fly Parkinson’s disease model. These data support a role for Serf in protein homeostasis relevant to proteins active in at least two distinct neurodegenerative diseases. My study has also revealed that Serf influences lifespan in Drosophila. Loss of Serf reduces lifespan by 20-30% whereas ubiquitous overexpression of Serf results in an equivalent extension of the normal lifespan. Lifespan extension occurs even when Serf overexpression is restricted to muscles, neurons or only adult tissues. Change in lifespan with Serf manipulation inversely correlates with the accumulation of poly-ubiquitinated protein aggregates, a marker of tissue aging. These aggregates are marked with Ref(2)p/p62, a target of autophagy. Analysis of expression of several genes in the autophagy pathway suggests that Serf expression may promote longevity, at least in part, by upregulating the life-extending autophagy pathway. Serf gene expression also correlated with a modest resistance to oxidative stress and changes in the abundance of a mitochondrial marker protein, mitofusin, suggesting the possibility that Serf activity may impact mitochondrial function. Taken together, these studies establish Serf as a modifier of the Smn-limited SMA phenotype and reveal previously unknown roles for the Serf gene in Drosophila mobility and lifespan

Evidence of Conformational Changes in Adsorbed Lysozyme Molecule on Silver Colloids

In this article, we discuss metal-protein interactions in the Ag-lysozyme complex by spectroscopic measurements. The analysis of the variation in relative intensities of SERS bands reveal the orientation and the change in conformation of the protein molecules on the Ag surface with time. The interaction kinetics of metal-protein complexes has been analyzed over a period of three hours via both Raman and absorption measurements. Our analysis indicates that the Ag nanoparticles most likely interact with Trp-123 which is in close proximity to Phe-34 of the lysozyme molecule.Comment: 15 pages, 6 figure

A docking interaction study of the effect of critical mutations in ribonuclease a on protein-ligand binding

Enzymes with ribonucleolytic activity play a pivotal role in gene expression and cellular homeostasis by altering the levels of cellular RNA. Ribonuclease A has been the most well studied of such enzymes whose histidine residues (His12 and His119) play a crucial role in the catalytic mechanism of the protein. The ligands chosen for this study, 2′CMP and 3′CMP, act as competitive substrate analog inhibitors of this enzyme. Using molecular graphics software freely available for academic use, AutoDock and PyMol, we demonstrate that substitution of either histidine residue by alanine causes marked changes in the distances between these critical residues of the enzyme. The ligands in the docked conformation (particularly on mutation of His119 to Ala) compensate for the altered free energy and hydrogen bonding abilities in these new protein‐ligand complexes

N-acetylglucosamine (GlcNAc-inducible gene GIG2 is a novel component of GlcNAc metabolism in Candida albicans

Candida albicans is an opportunistic fungal pathogen that resides in the human body as a commensal and can turn pathogenic when the host is immunocompromised. Adaptation of C. albicans to host niche-specific conditions is important for the establishment of pathogenicity, where the ability of C. albicans to utilize multiple carbon sources provides additional flexibility. One alternative sugar is N-acetylglucosamine (GlcNAc), which is now established as an important carbon source for many pathogens and can also act as a signaling molecule. Although GlcNAc catabolism has been well studied in many pathogens, the importance of several enzymes involved in the formation of metabolic intermediates still remains elusive. In this context, microarray analysis was carried out to investigate the transcriptional responses induced by GlcNAc under different conditions. A novel gene that was highly upregulated immediately following the GlcNAc catabolic genes was identified and was named GIG2 (GlcNAc-induced gene 2). This gene is regulated in a manner distinct from that of the GlcNAc-induced genes described previously in that GlcNAc metabolism is essential for its induction. Furthermore, this gene is involved in the metabolism of N-acetylneuraminate (sialic acid), a molecule equally important for initial host-pathogen recognition. Mutant cells showed a considerable decrease in fungal burden in mouse kidneys and were hypersensitive to oxidative stress conditions. Since GIG2 is also present in many other fungal and enterobacterial genomes, targeted inhibition of its activity would offer insight into the treatment of candidiasis and other fungal or enterobacterial infections

A comparative study of interaction of tetracycline with several proteins using time resolved anisotropy, phosphorescence, docking and FRET

A comparative study of the interaction of an antibiotic Tetracycline hydrochloride (TC) with two albumins, Human serum albumin (HSA) and Bovine serum albumin (BSA) along with Escherichia Coli Alkaline Phosphatase (AP) has been presented exploiting the enhanced emission and anisotropy of the bound drug. The association constant at 298 K is found to be two orders of magnitude lower in BSA/HSA compared to that in AP with number of binding site being one in each case. Fluorescence resonance energy transfer (FRET) and molecular docking studies have been employed for the systems containing HSA and BSA to find out the particular tryptophan (Trp) residue and the other residues in the proteins involved in the binding process. Rotational correlation time (θc) of the bound TC obtained from time resolved anisotropy of TC in all the protein-TC complexes has been compared to understand the binding mechanism. Low temperature (77 K) phosphorescence (LTP) spectra of Trp residues in the free proteins (HSA/BSA) and in the complexes of HSA/BSA have been used to specify the role of Trp residues in FRET and in the binding process. The results have been compared with those obtained for the complex of AP with TC. The photophysical behaviour (viz., emission maximum, quantum yield, lifetime and θc) of TC in various protic and aprotic polar solvents has been determined to address the nature of the microenvironment of TC in the protein-drug complexes

Prediction-based protein engineering of domain I of Cry2A entomocidal toxin of Bacillus thuringiensis for the enhancement of toxicity against lepidopteran insects

Issues relating to sustenance of the usefulness of genetically modified first generation Bt crop plants in the farmer’s field are of great concern for crop scientists. Additional biotechnological strategies need to be in place to safeguard the possibility for yield loss of Bt crop by other lepidopteran insects that are insensitive to the Cry1A toxin, and also against the possibility for emergence of resistant insects. In this respect, Cry2A toxin has figured as a prospective candidate to be the second toxin to offer the required protection along with Cry1A. In the present study, the entomocidal potency of Cry2A toxin was enhanced through knowledge-based protein engineering of the toxin molecule. Deletion of 42 amino acid residues from the N-terminal end of the peptide followed by the replacement of Lys residues by nonpolar amino acids in the putative transmembrane region including the introduction of Pro resulted in a 4.1–6.6-fold increase in the toxicity of the peptide against three major lepidopteran insect pests of crop plants