BIOmics Hands-On Workshop & Conference

POSTER ABSTRACTS

 

Singaram et al	Transgenic sugarcane resistant to shoot borer
Kovalevskiy et al	Papillomavirus E1 helicase hexamer maintains an asymmetric state in the absence of DNA and nucleotide cofactors
Pal	Molecular chaperones, protein mis-folding and human diseases
Balli et al	Factor V A4070G mutation in patients with homozygous Factor V Leiden
Ram et al	Biophysical characterisation and structural studies on signal transduction proteins DevR and DevS in Mycobacterium tuberculosis
Doğan et al	Identification, cloning and expression analysis of the ABA-induced HVA22-like protein from Triticum dicoccoides, wild emmer wheat under drought conditions
Yildirim et al	Effect of antioxidant treatment on the MAPK-pathway of the heart õn streptozotocin-induced diabetic rats
Prasanth et al	Biophysical, structural & functional studies on outer membrane protein OmpC and its variants from Salmonella typhi using protein engineering
Shittu et al	Derivatives as selective estrogen receptors α & β modulators (SERMS)
Roszczyk	Database of the nuclear proteome of Arabidopsis thaliana
Gladki et al	Drug targets prediction based on interactomics
Patana et al	The human UDP-glucuronosyltransferases: studies on substrate binding and catalytic mechanism
Odolczyk et al	Rescue of ÆF508-CFTR mutant by inhibition of protein-protein interaction
Siedlecki et al	A novel literature-based approach for detecting modules of functionally interacting genes
Zielinska et al	Meaning of alternatives mRNA sequences and synonymous mutations
Singh SK et al	MicroRNA profiling of rat brain oligodendroglial lineage cells
Osamor et al	Simultaneous and single gene expression computational analysis for malaria treatment discovery
Gandhi et al	dGas41: a genetic suppressor of RNAi, microRNA and heterochromatin silencing pathways in Drosophila melanogaste
Mishra et al	miRomics of development and stress in local indica rice varieties
Cohen et al	Understanding Endoplasmic Reticulum Inheritance
Breker et al	Dynamics of protein localization in yeast and their impact on endoplasmic reticulum (ER) functions
Lebenthal et al	Mounting computational evidence for functionality of Fantom non-coding RNA
Snir et al	De novo function prediction: identification of novel photosynthetic proteins
Levy et al	Prediction of 3D metal binding sites from translated gene sequences based on remote-homology templates
Kunik, Alon et al	Large scale analysis of antibodies, antigens and immunogenic interactions
Doniger et al	A Comparative Genome-wide Study of ncRNAs in Trypansomatids
Zhu et al	Phosphoproteomic analysis of ethylene-regulated protein phosphorylation in etiolated seedlings of Arabidopsis mutant ein2 using two-dimensional separations coupled with a hybrid quadrupole time-of-flight mass spectrometer
Saygi et al	Structural studies on mutant wheat metallothioneins

 

 

 

Poster board #1

 

Transgenic sugarcane resistant to shoot borer

 

Arvinth Singaram, MN Premachandran , N Subramonian

 

Sugarcane Breeding Institute (ICAR), Coimbatore-641 007, India.

 

Sugarcane (Saccharum spp. hybrid) is an important crop in the tropical and subtropical parts of the world, grown mainly for its use in sugar and ethanol production. Among the factors affecting sugarcane and sugar productivity insect pests play a major role and is estimated to cause a reduction of around 20 % in cane yield and 15 % in sugar recovery in general. The shoot borer, Chilo infuscatellus Snellen attacks the crop at young shoot stage and results in a very high amount of yield loss. A loss of 10 – 20 % of young shoots is very common during the infestation and during very serious infestation of shoot borer the loss may be much higher. Due to lack of shoot borer resistance in sugarcane germplasm it is difficult to develop resistant varieties through conventional breeding methods. The introduction of genes expressing insecticidal proteins was successful in many other crops.  In the present study sugarcane transgenics were developed with cry1Ab gene, which produces the insecticidal protein in the plant.

 

In vitro bioassay of Cry1Aa, Cry1Ab and Cry1Ac protoxins was conducted through surface diet contamination of shoot borer (Chilo infuscatellus) and variation in mortality percentage was observed. The LC₅₀ of Cry1Ab was the lowest indicating that it was more toxic than Cry1Aa and Cry1Ac. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene for shoot borer resistance through particle bombardment and Agrobacterium-mediated transformation systems. Gene integration was confirmed with PCR analysis in the regenerated plants. Southern analysis exhibited multiple integration sites in case of particle bombardment and a single site integration in Agrobacterium mediated transformants. Cry1Ab expression was confirmed and the amount of protein expressed in different events varied from 0.007% to 1.73% of the total soluble leaf protein.  In vivo bioassay of the transgenic sugarcane was carried out with neonate larvae of shoot borer. Lower percentage of dead hearts were seen in transgenics compared with the untransformed control plants. The potential of insect resistant sugarcane with cry1Ab was discussed in the light of the results obtained.

Back to top of page

 

 

Poster board #2

 

Papillomavirus E1 helicase hexamer maintains an asymmetric state in the absence of DNA and nucleotide cofactors

 

OV Kovalevskiy,¹ D Sizov,² AA Lebedev,¹ MN Isupov,³ CM Sanders,⁴ AA Antson¹

¹York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, UK; ²Taras Shevchenko Kiev State University, Biology Faculty, Virology Department, Kiev, Ukraine; ³Henry Wellcome Building for Biocatalysis, School of Biosciences, University of Exeter, Exeter, UK; ⁴Institute for Cancer Studies, University of Sheffield, Sheffield, UK

 

Concerted, stochastic and sequential mechanisms of action have been proposed for different hexameric AAA+ molecular motors. We determined the crystal structure of the E1 helicase from bovine papillomavirus, where asymmetric assembly is for the first time observed in the absence of nucleotide cofactors and DNA. Surprisingly, the ATP-binding sites adopt specific conformations linked to positional changes in the DNA-binding hairpins, which follow a wave-like trajectory, as observed previously in the E1/DNA/ADP complex. The protein's assembly thus maintains such an asymmetric state in the absence of DNA and nucleotide cofactors, allowing consideration of the E1 helicase action as the propagation of a conformational wave around the protein ring. The data imply that the wave's propagation within the AAA+ domains is not necessarily coupled with a strictly sequential hydrolysis of ATP. Since a single ATP hydrolysis event would affect the whole hexamer, such events may simply serve to rectify the direction of the wave's motion. Currently we are focused on investigating the interaction of E1 with forked DNA substrates.

Back to top of page

 

 

 

Poster board #3

 

Molecular chaperones, protein mis-folding and human diseases

 

Mohinder Pal

Protein Crystallography Group, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton

 

The function of a protein depends crucially upon it's attaining the correct ÒnativeÓ conformation often with the help of molecular chaperones. Mis-folded proteins are unable to perform their normal function and can cause disease in humans.

 

The primary aim of this research is to characterize the interaction surfaces of molecular chaperones in order to elucidate their modus operandi and to stabilise the native protein with chemical ligands. Serum Amyloid P Component (SAP) is a homo-pentameric plasma protein of 25kDa subunits that binds to the pathological deposits of misfolded protein characteristic of a group of diseases called the Amyloidoses. SAP is thought not only to stabilise amyloid fibers but also to protect them from proteolytic and cell mediated degradation. SAP is also a putative molecular chaperone as it has been shown to assist in the correct folding of chemically denatured enzymes in vitro. In addition to binding mis-folded polypeptides SAP can also bind and unfold Ôoff-pathwayÕ folding intermediates.SAP was co-crystallized with three different aminoalkyl phosphonates that bind at the amyloid recognition site, and the X-ray crystal structures were determined.

 

A secondary aim of this work is to understand the enhanced amyloidogenic potential of L55P and V30M Transthyretin (TTR) protein. TTR misfolding has been implicated in number of human diseases such as senile systemic amyloidosis, familial amyloid polyneuropathy (FAP) and familial amyloid cardiopathy. TTR protein is a thyroxin binding protein (14kDa) existing as a tetramer in vivo. L55P and V30M mutant TTR are most aggressive and most common mutants respectively in causing FAP.L55P and V30M mutant TTR protein were expressed in E.coli and purified using anion-exchange chromatography and gel filtration. L55P and V30M TTR were co-crystallised with MDS84, a compound that has been demonstrated to stabilize the tetramer in vitro. The x-ray structures of L55P and V30M, TTR mutant protein has been determined.

 

Presently, I am refining these SAP-ligand complex structures to further elucidate the ligand interactions in order to correlate them with binding affinities determined by Isothermal Calorimetry (ITC). The L55P, V30M and WT TTR co-complexes with MDS84 will also be further refined and their binding affinities determined by ITC.

Back to top of page

 

 

 

Poster board #4

 

Factor V A4070G mutation in patients with homozygous Factor V Leiden

Sezen Balli, Aysenur Ozturk, Nejat Akar

 

Department of Pediatric Moleculer Pathology and Genetics, Faculty of Medicine, Ankara University, Ankara, Turkey.

 

A common polymorphism (FV Leiden) in the FV gene that causes a missense mutation, FV Arg506Gln, results with phenotype of APC resistance in the majority of effected individuals. The mutation is associated with a significant increase in thrombotic risk. The frequency of FVL is about 8 percent in our population.

 

A4070G (His1299Arg) polymorphism in exon 13 of FV gene can influence factor V levels and contribute to the activated protein C (APC) resistance phenotype ending with thrombosis risk. However, there exist several people over the age 70 with both mutations but not experienced thrombosis.

 

The aim of this study is to determine the role of combination of FVL and FV A4070G mutation in patients with and without thrombosis. DNA was isolated by conventional methods. Amplification  of exon 13 of the factor V gene was performed by polymerase chain reaction (PCR) and amplified fragments was digested with restriction endonuclease enzyme.

FVL was determined with Light Cycler (Roche) with commercial procedure.

 

First we screened  80 homozygous FV Leiden ; of these 7 were controls and 69 were thrombotic patients. One individual showed HR2 haplotype in heterozygosite state indicating a cis position of these two mutations. Then we screened patients (n:181) and controls(n:168) over the age of 70.  15 (%8.28)  of  181 individuals with thrombosis were found to have FV A4070G in heterozygous state.  15 (%8.92) heterozygous individuals were determined among 168 controls. There was no significant difference between the two groups. In these two groups FVL frequency is %35.80 and %8.33 respectively. Further, FV A4070G mutation did not have any effect on thrombosis with an odds ratio of 1.12 (0.50 -2.48 p=0.77)  in FV Leiden non carriers  and 0.21 (0.01-2.59 p=0.56) in FV Leiden heterozygous carriers.

 

In conclusion , the results of our study show that FV A4070G  mutation is not a risk factor in individuals over the age of 70. A4070G polymorphism might be located in cis position with FVL mutation which need further investigation.

 

(Supported by Ankara University Research Fund )

Back to top of page

 

 

 

Poster board #5

 

Biophysical characterisation and structural studies on signal transduction proteins DevR and DevS in Mycobacterium tuberculosis

T Jeethy Ram¹, S Ranjani¹, K Anujainthi¹, Jaya Tyagi², S Krishnaswamy¹

¹Centre of Excellence in Bioinformatics, School of Biotechnology, Madurai Kamaraj University, Madurai. India; ²Dept of Biotechnology, AIIMS, New Delhi, India

 

DevR–DevS is a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. DevR and DevS are regulated in response to oxygen stress. This signalling system is involved in the survival of the bacilli in dormant state and may be responsible for latent tuberculosis in one-third of the worldÕs population. Latent tuberculosis is a major public health concern because it acts as a reservoir of M. tuberculosis that can remain undetected for decades before re-emerging as active disease. As conventional drugs are less effective against persistent bacteria, these signalling systems can be utilized for the development of new antitubercular drugs.

 

Dev R and DevS₂₀₁ proteins of Mycobacterium tuberculosis have been overexpressed in E. coli. Purification and matrix-assisted refolding of both proteins were done using affinity chromatography. For final purification, gel filtration chromatography was done using Superdex G-75. DevS is a sensor kinase protein with a molecular weight of 26.5kDa. As DevS is a membrane associated protein only C-terminal portion is expressed. DevR is a response regulator protein with a molecular weight of 25.5kDa. Phosphorylation of the response regulator switches on its C-terminal domain to perform its associated function such as DNA-binding or enzymatic activity. CD spectrum of the phosphorylated sensor kinase, DevS shows conformational change when compared with unphosphorylated protein. Biophysical characterisation using CD and Fluorescence Spectroscopy were done for both proteins. Crystallization trials are on going for both phosphorylated DevR and DevS.

Back to top of page

 

 

 

Poster board #6

 

Identification, cloning and expression analysis of the ABA-induced HVA22-like protein from Triticum dicoccoides, wild emmer wheat under drought conditions

 

Esen Doğan and Hikmet Budak

Sabanci University, Biological Sciences and Bioengineering Program, Istanbul, Turkey

 

Drought is the major stress factor that has a negative effect on plants. With the availability of water resources getting more limited, usage of water for agricultural purposes is also getting more restricted. Consequently, it is critical that mechanisms responsible for drought resistance and molecules that are effective in these mechanisms be studied. It is well established that abscisic acid (ABA)-mediated signaling is involved in many of the stress conditions, including drought stress. HVA22, first isolated from barley (Hordeum vulgare L.), is one of the proteins whose expression is induced and regulated by ABA. To date, many homologues of HVA22 were identified in various organisms such as fungi, plants and animals, but not in prokaryotes, implying that HVA22 and HVA22-like proteins play a unique role in eukaryotes.

 

A previous study (Ergen & Budak 2009) showed that HVA22 is differentially expressed in root tissue of a drought-tolerant genotype of wild emmer wheat [Triticum turgidum spp. diccocoides (Korn.) Thell.], which originated from southeastern Turkey.  In this study, for the first time, we cloned the full-length cDNA of HVA22-like protein, which is expressed differentially in root tissue of Triticum diccocoides under prolonged drought condition (9 days). Sequence analysis indicated that the protein product is a transmembrane protein with two predicted transmembrane regions. Presence of the protein was confirmed with expression studies using Escherichia coli and SDS-PAGE analysis. Further studies concerning sub-cellular localization of the protein and related function is also planned.

Back to top of page

 

 

 

Poster board #7

 

Effect of antioxidant treatment on the MAPK-pathway of the heart õn streptozotocin-induced diabetic rats

 

Samet S Yildirim, Esma N Zeydanli, Duygu Akman, Belma Turan

Departments of Biophysics, School of Medicine, Ankara University, Ankara, Turkey.

 

Oxidative stress contributes to the development of a wide range of diseases including diabetes and results from an imbalance between the production of reactive oxygen and the systemÕs ability to readily detoxify the reactive intermediates and repair the resulting damage. Cells are normally able to defend themselves against the oxidative stress induced damage by regulating the cellular redox status through the antioxidant systems. The present study was performed to examine the effect of antioxidant treatment (sodium selenate; 15 μmol/kg/day for 4 weeks) of diabetes on the actors of MAPK pathway such as ERK and NF-кB. We measured total and phosphorylated ERK and NF-кB levels in the heart tissue by using Western Blot technique. Total and Phospho - ERK levels were increased about 40% compared to those of the controls.  We also observed 30% decrease in the level of total NF-кB while 85% increase in the level of Phospho - NF-кB. Sodium selenate treatment of the diabetic rats preserved these altered levels of MAPK - actors, significantly. For comparison, we also treated the diabetic rats with another antioxidant, omega - 3E for the same period and obtained the similar benefical effects on these parameters. In conclusion, the results of the present study show that diabetes induced a significant increase in the levels of oxidative stress and/or caused a defect into the antioxidant defence system. Furthermore, our data demonstrated that antioxidant supplementation might be beneficial for diabetes therapy due to an improvement of the antioxidant defense system against the diabetes-induced altered cell defense state.

 

(Supported by TUBITAK SBAG-107S427&SBAG-107S304)

Back to top of page

 

 

 

Poster board #8

 

Biophysical, structural & functional studies on outer membrane protein OmpC and its variants from Salmonella typhi using protein engineering

 

P Prasanth¹, PD Kumar¹, MK Mathew² and S Krishnaswamy¹

¹Centre of Excellence in Bioinformatics, School of Biotechnology, Madurai Kamaraj University, Madurai;  ²National Centre for Biological Sciences, Bangalore

 

OmpC is the major b-barrel integral outer membrane protein of Salmonella enterica serovar Typhi. OmpC is known to illicit humoral and cell mediated Immune response in patients infected with S.typhi. It is also involved in inducing apoptosis. It interacts with phages and secretory proteins like Lactoferrin. OmpC belongs to the class of general porins. It is a homotrimer with molecular weight of 117 KDa. Recombinant OmpC has been over expressed in E.coli, purified and refolded in presence of nonionic detergents.

 

Two variants of Green Fluorescent Proteins GFPUV and GFPUV4 were cloned into loop 7 of OmpC to get OmpC-GFPUV and OmpC-GFPUV4 respectively. Flexible linkers were used to facilitate GFP folding. OmpC-GFPUV has been refolded and functionally characterized. However, the fluorescence has been lost possibly due improper folding of GFP in the loop. Expression and characterization of OmpC-GFPUV4 are underway. Loop deletion mutants were also made and purified.

 

Fluorescence and circular dichroism studies showed mutants possess similar characteristics as Native protein. Functional characterization and pore size determination were done using liposome swelling assays. Crystals were obtained for native, refolded as well as loop deletion mutants of OmpC. Crystals of loop deletion mutant diffracted to 3.5� at ESRF Synchrotron source. These results will be discussed.

Back to top of page

 

 

 

Poster board #9

 

Derivatives as selective estrogen receptors α & β modulators (SERMS)

 

LAJ Shittu ¹, RK Shittu ²

 

¹Reproductive endocrinology & bioinformatics unit, Department of Anatomical Sciences, College of Health Sciences, University of Abuja, Gwagwalada, Abuja, Nigeria; ²Microbiology units, Bolomedics laboratory, Egbeda, Lagos, Nigeria

 

Computational molecular docking modeling is now routinely used for the understanding of drug–receptor interaction in modern drug design and discovery due to the availability of bioinformatics veritable tools in recent times. Moreover, extensive studies have also shown that these computational generated models strongly support and help in the design of novel and more potent modulators by revealing the protein–ligands binding mechanisms involved in drug design.

 

Phytoestrogens are plant derived estrogenic compounds that mimic endogenous estrogens in their actions and they include lignans, stillbens, isoflavanoid and cumestans. The estrogen receptors (ERs) α and β are intracellular proteins responsible for controlling transcription of genes necessary for human development and reproduction. ER activity is usually modulated by the endogenous 17β-estradiol (E2) hormone that binds to nuclear ERs leading to recruitment of co-regulatory complexes, which control transcription of nuclear DNA necessary for human development and reproduction. However, irregularities in ER activity can lead to abnormal conditions such as breast, ovarian, prostate, endometrial and colonic cancers among others. Selective estrogen receptor modulators (SERMs) are synthetic compounds which are used to modulate ER activity. However, different concentration of SERMs usually produce varying combinations of actions such as agonistic, antagonistic and neutral when they are estrogen receptors bound depending upon the specific ER subtype and cell type in which the estrogen receptor is present.

 

Here, a virtual linux computer with bioinformatic triton software would be used for docking selective estrogen receptor modulators (SERMs) such as sesame lignans derivatives into estrogens receptors active sites. Sesame lignans derivatives protein structures would be generated and collected from the available literatures and we would study their specific interactions with the ER. It is hope that docked complexes would provide a better insight to design more of potent lignans modulators prior to their synthesis.

 

Since, some of these compounds have shown estrogenic activity in-vivo, we would carry out further study to optimize these structures to maximize activity and also be able to determine if there is a concentration range where such a compound might affect principally mitochondrial ERα.

Back to top of page

 

 

 

Poster board #10

 

Database of the nuclear proteome of Arabidopsis thaliana

 

Marlena Roszczyk

Bioinformatics Department, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

 

Despite constant increase of proteomic data, cellular localization of many proteins is still poorly characterized. Even for model organisms such as Arabidopsis thaliana, data inferred from direct assay is still insufficient, whereas localization predictions based on available bioinformatics tools are often elusive and contradict each other.

 

The current study is an attempt to create a plausible database of A. thaliana nuclear proteome, which may be used as a reference in a mass-spectometry analysis of the nucleus. The obtained database of 5662 proteins consists of two parts, overlapping for 401 proteins:

             951 A. thaliana proteins which have Gene Ontology [1] annotation GO:0005634 (cellular component: nucleus) of evidence code IC, IDA, IEP, IGI, IMP, IPI, RCA or TAS. The chosen evidence codes guarantee that the localization was inferred from direct or high-throughput experiments or - in case of computational analysis - was reviewed by a curator.

             5112 A. thaliana proteins, which were successfully mapped on the set of Saccharomyces cerevisiae proteins of known nuclear localization. The localization of each yeast protein was determined by a direct analysis of the fluorescense signal of its fusion with GFP [2]. The names of 21 cellular components for yeast proteins taken from [2] were generalized to 6 main localizations: CYT (cytoplasm), DIV (proteins associated with cell division components and cytoskeleton), MIT (mitochondrion), NUC (nucleus), SEC (proteins associated with secretion or exported), VAC (vacuole and its membrane). The localizations were mapped onto A. thaliana proteins on the basis of sequence homology with S.cerevisiae proteins computed by the BLASTP program. As A. thaliana sequences often have many S. cerevisiae homologues due to genes duplications, the attained localizations of A. thaliana proteins are sometimes the result of merging data from few different S. cerevisiae proteins.

 

The presented approach may be used to prepare similar databases for other organisms of known genome.

 

 [1]            Ashburner M et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet.  25(1), 25-29 (2000).

[2]             Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O'Shea EK, and JS Weissman. Global analysis of protein expression in yeast. Nature, 425, 737-741 (2003).

Back to top of page

 

 

 

Poster board #11

 

Drug targets prediction based on interactomics

 

Arek Gladki¹, Piotr Zielenkiewicz^1,2

¹Bioinformatics Department, Institute of Biochemistry & Biophysics, Polish Academy of Sciences; ²Plant Molecular Biology Dept, Warsaw University, Warszawa, Poland

 

BACKGROUND: Misuse and overuse of antibiotics led to multi drug resistance and increased lethality in some bacterial infections. There is a need for novel antibiotics nowadays.  Finding new potential drug targets in bacteria can be considered as one of the solutions. Discoveries of new targets potentially lead to new small drug particles with novel antimicrobial mechanisms.

 

Bacterial essential proteins are in the scope of interest. There is a problem with theoretical protein essentiality evaluation. It was shown that topological parameters of the interactome can be good predictors, with node degree being the most popular one.

 

METHODS: Two experimental interactomes (of E. Coli and C. pylori) were used to predict the network of interactions for three other species (M. tuberculosis, Y. pestis, S. aureus).  Interaction transfer was based on sequence similarity. Psi-BLAST and COG families were used.

 

Orthology assignment between proteins from different species (with known experimental interactions and with interactions being predicted) comprised the key action in the methodology. Therefore, a few approaches were proposed. At first, the commonly accepted Reciprocal Best Hit (RBH) method, which is known to have a high specificity, was proposed. Two other types of methods were proposed to extend and/or change the scope of the orthology assignment (increasing the sensitivity, with none or acceptable specificity loss). In the first case (OHMQ – One Hit Multiple Query methods), some protein duplication events were taken into account by enabling assignment (with or without COG families-based filtering) of a few proteins from species with known experimental interactions to one protein for which predictions were being made. In the second type of approach (extended RBH methods), assignments (with COG families-based filtering) for proteins being second best hits were also made possible.

 

In each case, predicted interaction networks were analyzed and 5% of the most connected proteins were treated as hubs. They were filtered out to the set of proteins not having a human homolog. Found potential drug targets were ranked based on absence/presence/position in all approaches. Finally, found drug targets were evaluated manually using Pubmed and Drugbank.

 

RESULTS: Potential drug targets found in S. aureus can serve as a promisingexample. Lumazine synthase (DMRL synthase), indicated as a drug target in each method, belongs to the group of enzymes involved in the biosynthesis of riboflavin, which are considered as attractive drug targets  (Pubmed, PMID 14690539). Para-aminobenzoate synthase found using the extended RBH-based methods, is necessary for the synthesis of folic acid. Sulfanilamide is an approved drug against this protein acting as its competitive inhibitor (DrugBank, DB00259). Anthranilate synthase, found in the OHMQ method case, was also confirmed to be an attractive drug target (Pubmed, PMID 18952181).

Back to top of page

 

 

 

Poster board #12

 

The human UDP-glucuronosyltransferases: studies on substrate binding and catalytic mechanism

 

Anne-Sisko Patana¹, Mika Kurkela², Moshe Finel², Adrian Goldman^{1, 3}

¹Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Finland; ²DDTC, Faculty of Pharmacy, University of Helsinki, Finland;  ³Neuroscience Center, University of Helsinki, Finland

 

UDP-glucuronosyltransferases (UGTs, EC 2.4.1.17) are membrane bound glycosyltransferases located in the endoplasmic reticulum (ER). They play an important role in the metabolism and subsequent elimination of many xeno- and endobiotics from our body. They catalyse the transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) to endogenic or exogenic compounds, usually highly lipophilic molecules. As a consequence the aqueous solubility of the substrate (aglycone) is increased and it is more easily excreted from the body. UGTs consist of two domains located on the lumenal side of the ER membrane, a single-pass transmembrane helix and a short cytosolic tail. Currently, 19 different UGTs are known in man and, based on sequence and gene organisation, they are divided into three subfamilies: UGT1A, UGT2A, and UGT2B. UGTs belong to the GT1 family of glycosyltransferases. The reaction mechanism in several members of the GT1s family is a serine protease-like catalysis in which a His-Asp pair in the N-terminal domain deprotonates a hydroxyl on the aglycone for subsequent nucleophilic attack on the sugar donor.

 

We have examined several residues to understand UDPGA binding and to identify the most likely catalytic dyad in human UGTs. In the case of UGT1A9 the catalytic pair may be H37 and either D143 or D148. H37 is not totally conserved, however, and in UGTs 1A4 and 2B10 it is replaced by either Pro or Leu, respectively. Interestingly, both UGT1A4 and UGT2B10 mainly catalyse N-glucuronidation reactions, while most other UGTs catalyse O-glucuronidation. Thus we have investigated the roles of H37, D143 and D148 in UGT1A9 by site-directed mutagenesis, activity and kinetic analyses using both O- and N-substrates. The results suggest that H37 may not be essential for N-glucuronidation by UGT1A9. We suggest that, as N-nucleophiles are usually stronger than O-nucleophiles, they may not require general base activation by H37. Co-substrate binding was studied in UGT1A6, UGT1A9, UGT1A10 and UGT2B7 isoforms by site-directed mutagenesis, modelling, single-point activity and kinetic measurements with several substrates. The results indicated that residues H371, E379, D395 and Q396 (UGT1A6 numbering) are involved in UDPGA binding.

Back to top of page

 

 

 

Poster board #13

 

Rescue of ÆF508-CFTR mutant by inhibition of protein-protein interaction

 

Norbert Odolczyk¹, Grzegorz Wieczorek¹, Danielle Tondelier³, Janine Fritsch³, Sandra Moriceau³, Patricia Melin Heschel⁴, FrŽdrique Becq⁴, Aleksander Edelman³,  Piotr Zielenkiewicz^1,2

 

¹Inst Biochemistry & Biophysics, Polish Acad Sci, Warsaw, Poland; ²Plant Molecular Biology Dept, Warsaw University, Warsaw, Poland; ³ INSERM, UniversitŽ Paris Descartes, FacultŽ de MŽdecine, Paris, France; ⁴ Inst Physiologie et Biologie Cellulaires, Univ Poitiers, CNRS, Poitiers, France

 

Background: Cystic Fibrosis conductance Transmembrane Regulator (CFTR) is a chloride channel and important ion and fluid homeostasis regulator. Mutation in the CFTR gene, causing a deletion of Phe508 in the first Nucleotide Binding Domain (NBD1) of CFTR protein, is the most common mutation responsible for Cystic Fibrosis. Analysis of molecular dynamics results led to a conclusion that the ÆF508 NBD1 domain has much more conformational freedom and tendency to expose significantly more hydrophobic surface than the wild type protein. This substantial exposure of hydrophobic surface could be the reason for the recognition of ÆF508-CFTR by the ER quality control system as misfolded protein and its degradation before reaching the epithelial cell membrane. As a consequence, we observe a strong phenotypic effect in patients with ÆF508 mutation.

 

Aim: Find small chemical compounds which may act as inhibitors of protein-protein interaction between mutated CFTR, and protein(s) responsible for its ER retention and/or premature degradation.

 

Methodology: Based on a Molecular Dynamics trajectory, we identified two pockets around the exposed hydrophobic surface of ÆF508-CFTR and treated them as two independent receptors for the Virtual Screening procedure. We have been screening structures of small molecules retrieved from the National Cancer Institute Database using the DOCK 6.0 program. Initially obtained complexes were gradually minimized and evaluated by seven scoring functions (D_Score, ChemScore, PMF_score, G_score, HPScore, HMScore and HSScore). Instead of using a consensus scoring protocol (because of lack of a possibility to construct a reliable test subset for newly identified receptors), we focused our attention on the top 2-5% results from each scoring function. A critical visual assessment resulted in selecting twelve chemical compounds for experimental tests. 

 

Results: Four of the tested small molecules efficiently allow the CFTR mutant to escape from the ER quality control system and result in the appearance of a functional CFTR channel in the epithelial cell membrane. This is the first known example for a successful, computer aided design of a protein-protein interaction inhibitor targeting a ÒsickÓ conformation of one of the participating proteins.

Back to top of page

 

 

 

Poster board #14

 

A novel literature-based approach for detecting modules of functionally interacting genes

 

P Siedlecki¹, S Kaczanowski¹ and P Zielenkiewicz^1,2

¹Bioinformatics Department, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawinskiego 5a, 02-106 Warsaw, Poland; ²Plant Molecular Biology Department, Warsaw University, Warsaw, Poland

 

One of the major challenges that face today's biotechnology is how to deal with the increasing knowledge about genes and proteins. The ability to discover modules of functionally interacting proteins (i.e. physical interactions, common regulation, etc.) has become a crucial factor not only for our understanding of the living organism, but also for our ability to more directly and specifically interact with certain pathways of cells bio-machinery.

 

The main obstacle here is the huge diversity of interaction types; it is hard to design a single high-throughput experiment to cover all of them. By contrast, the majority of potential interactions are already well described in the literature by various groups working on smaller parts of the cells bio-machinery. The possibility to use this knowledge and extract data from literature is tempting but there are few serious problems with such an approach. One of them is the fact that data is not unified; genes have different names and/or are often poorly described. Even GO annotations do not use the full richness of the biological language. Another major problem is with statistical significance of the extracted data; different keywords could occur in biological papers due to Òrandom chanceÓ - with sentences like Ònot similar to...Ó - or due to Òerrors in results/interpretationÓ.

 

To deal with some of theses problems we developed a novel methodology based on literature mining which is capable of unifying protein annotations based on extracted data. These annotations are then used for detecting functional interactions between proteins using highly specific keywords.

Back to top of page

 

 

 

Poster board #15

 

Meaning of alternatives mRNA sequences and synonymous mutations

 

Joanna Zielinska¹, Maria Sromek², Piotr Zielenkiewicz^1,3

¹Bioinformatics Department, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; ²The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland; ³Plant Molecular Biology Department, Warsaw University, Warsaw, Poland

 

The genetic code is degenerated and it is obvious that one protein sequence can be encoded by a lot of different mRNA's. Simultaneously it is not known if all alternatives can be really used by all living organisms. We can observe that different organisms often have different codon usage bias. Each RNA sequence has different properties, such as structure, length and stability. It is not clear how those differences influence translation. In some cases we can correlate translation rate with structure minimum free energy or codon usage. One synonymous mutation in non-coding region of RNA can change splicing and cause synthesis of different protein. It is supposed that one synonymous mutation in coding regions can change some protein properties or translation rate.

 

As an example RET proto-oncogene, one of the receptor tyrosine kinases, was used. We know that germline pathogenic mutation Y791F causes medullary thyroid carcinoma (MTC) and additional homozygotic single-nucleotide polymorphism in exon 13 T2307G (L769L) lower the age of patients. Similar correlation is observed in case of another pathogenic mutation C634R and homozygotic mutations in exon 15 C2712G (S904S) and exon 14 C2508T (S836S). mRNA's of those polymorphisms were compared at the level of structure, its free energy and codon usage. It is concluded that differences in patients age caused by synonymous mutations in RET proto-oncogene might be explained by changes of mRNA properties.

Back to top of page

 

 

 

Poster board #16

 

MicroRNA profiling of rat brain oligodendroglial lineage cells

 

Sunit Kumar Singh^1,2, Maryam Urooj¹, Salini Krishnan¹, Ritu Mishra¹, Chintan Chhatbar¹, Makoto Horiuchi², David Pleasure²

 

¹Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India;  ²Shriners Hospital for Children Northern California, Sacramento, CA-95817, USA

 

MicroRNAs (miRNAs) are endogenous small RNAs, which can regulate target mRNAs by binding to their 3Õ untranslated regions. miRNAs play important role in variety of functions, including developmental transitions, neuronal patterning, apoptosis, and haematopoiesis etc. miRNAs control the gene regulation by translational repression; decapping, deadenylation and/or cleavage of target mRNA. Determining spatial and temporal patterns of miRNA expression provides insight into their biological functions. Many miRNAs have been isolated from mammalian embryonic neurons and mature brain. In this preliminary study, we studied the miRNA expression pattern in three different stages of rat brain oligodendroglial lineage cells (OLCs), such as oligodendroglial progenitor (OP), immature (IM) and mature cell (MO) stage.

 

miRNA array data provided us the information about the highly expressed miRNAs in three stages of OLCs. We have also found one miRNA (miR-138), which is differentially, expressed during the transition of cells from one stage to another stage. We identified few important target genes of array expressed miRNAs including the miR-138 by using bioinformatics prediction tools. However, these predictions based target genes should be considered tentative until they are validated through experimentation. This preliminary data generated in this study is expected to provide the platform for further studies regarding role of miRNAs in differentiation oligodendroglial lineage cells.

Back to top of page

 

 

 

Poster board #17

 

Simultaneous and Single Gene Expression Computational Analysis for Malaria Treatment Discovery

 

Victor Chukwudi Osamor¹, Ezekiel Femi Adebiyi ¹,  Seydou Doumbia²

 

¹Department of Computer and Information Sciences (Bioinformatics Unit), College of Science and Technology, Covenant University, Ota, Nigeria; ²Malaria Research Training Center (MRTC), University of Bamako, Mali

 

 

The major aim of this work is to develop an efficient and effective k-means algorithm to cluster malaria microarray data to enable the extraction of a functional relationship of genes for malaria treatment discovery. However, traditional k-means and most k-means variants are still computationally expensive for large datasets such as microarray data, which have large datasets with a large dimension size d. Huge data is generated and biologists have the challenge of extracting useful information from volumes of microarray data.  Firstly, in this work, we develop a novel k-means algorithm, which is simple but more efficient than the traditional k-means and the recent enhanced k-means. Using our method, the new k-means algorithm is able to save significant computation time at each iteration and thus arrive at an O(nk²) expected run time. Our new algorithm is based on the recently established relationship between principal component analysis (PCA) and the k-means clustering. We further prove that our algorithm is correct theoretically. Results obtained from testing the algorithm on three biological data and three non-biological data also indicate that our algorithm is empirically faster than other known k-means algorithms. We assessed the quality of our algorithm clusters against the clusters of known structure using the Hubert-Arabie Adjusted Rand index (ARI_HA), we found that when k is close to d, the quality is good (ARI_HA > 0.8) and when k is not close to d, the quality of our new k-means algorithm is excellent (ARI_HA > 0.9).  We compare three different k-means algorithms including our novel Metric Matrics k-means (MMk-means), results from an in vitro microarray data with the classification from an in vivo microarray data in order to perform a comparative functional classification of P. falciparum genes and further validate the effectiveness of our MMk-means algorithm. Lastly using clustering, R programming (with Wilcoxon statistical test) and the new microarray data of P. yoelli at the liver stage and the P. falciparum microarray data at the blood stages, we extracted twenty nine (29) viable P. falciparum and P. yoelli genes that can be used for designing a Polymerase Chain Reaction (PCR) primer for the detection of malaria at the liver stage.

Back to top of page

 

 

 

Poster board #18

 

dGas41: a genetic suppressor of RNAi, microRNA and heterochromatin silencing pathways in Drosophila melanogaster

 

Sumit Gandhi^1,2 and Utpal Bhadra¹

 

¹Functional Genomics and Gene Silencing Lab, Centre for Cellular and Molecular Biology, Hyderabad, India; ²Plant Biotechnology Division, Indian Institute of Integrative Medicine, Jammu, India

 

Recent studies have pointed out the role of RNA as a central factor in various transcriptional and post-transcriptional gene-silencing pathways. These silencing pathways depend on the components of RNA interference machinery in Drosophila. The variety in silencing responses, which can be elicited by RNA molecules, suggest a complex control that determines the choice in funneling. Biochemical approaches have identified some of the components of the core RNAi pathway in different organisms. However there is a need to systematically identify all the effectors involved. Genetic screening carried out in the lab has implicated roles of several previously characterized and still uncharacterized genes in RNA interference. Here we functionally characterize a novel gene dGas41 with respect to its role in RNA silencing pathway. Mutations in dGas41 result in loss of RNAi of white (eye color) gene in the GMR-wIR transgenic files. microRNA 277 mediated silencing is also lost in dGas41 mutations. Reduction in amount of several other mature miRNAs indicates that dGas41 mutants affect not only miR277 silencing, but the complete miRNA pathway. Previous studies from our lab and several others have shown the requirement of RNAi silencing components in heterochromatic silencing. Mutants of dGas41 also exhibit a loss of heterochromatic silencing of white gene in chromosomal inversion w[m4h] and loss of normal bristles in Sb[v]. This is further corroborated with loss of Histone 3 Lysine 9 (H3K9) methylation and Heterochromatin Protein 1 (HP1) delocalization in developing Drosophila embryos. Thus we show that dGas41 is a novel gene, which interconnects these RNA silencing pathways.

Back to top of page

 

 

 

Poster board #19

 

miRomics of development and stress in local indica rice varieties

 

Neeti Sanan Mishra, Rashmi R Sahoo, Deepti Mittal, Sudhir K Sopory and Sunil K Mukherjee

 

International Center for genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, INDIA

 

It is imperative to understand the mechanisms of growth and development in higher plants for improving plant adaptation towards abiotic and biotic stresses. Recently the miRNAs have emerged as important regulators of gene modulation and hence plant physiologies. To follow the miRNA networks involved in fine-tuning the plant development in normal and stressed environments we adopted the approach of cloning and comparative miRNA profiling.

 

We have cloned and identified ~40 new putative miRNAs from salt-stressed and Tungro virus infected local basmati rice variety. A few of these putative miRNAs were common to both suggesting a converging functional role of miRNAs in managing diverse stresses. The spatial and temporal accumulation of these miRNAs and their predicted targets is being validated by northern blotting and q-PCR studies. We also pyrosequenced (Illumina, GA) several cDNA libraries representing the developmental and salt-stressed physiologies of endogenous (indica) rice varieties. As a first step we identified and compared the digital expression status of known rice miRNAs within each data-set. We could identify 210, 169 and 172 known miRNAs from three main tissues viz. leaf, root and flower of local basmati variety; with 27, 10 and 8 miRNAs being uniquely represented in the respective tissues. On comparing the digital data of known rice miRNAs obtained from leaves of salt-stressed basmati seedlings and the unstressed seedlings of a salt-tolerant variety, Pokkali and identified 6 and 5 miRNAs, respectively, specific to each physiology. The validation of these findings by miRNA-array and q-PCR is in progress.

 

Our findings suggest that a thorough understanding of the miRNA expression patterns and their function will help unravel the mysteries of rice stress biology.

Back to top of page

 

 

 

Poster board #20

 

Understanding Endoplasmic Reticulum Inheritance

 

Yifat Cohen and Maya Schuldiner

 

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

 

The presence of various membrane-bound compartments (organelles) in Eukaryotic cells permits the coexistence of a diverse range of chemical microenvironments. Some organelles, like the endoplasmic reticulum (ER), mitochondria and chloroplasts cannot be made de novo, and must therefore be inherited from mother to daughter cell upon cell division. Despite the importance of this process for cells function and viability we still do not know the entire spectrum of genes involved in the inheritance of the ER. Here, we attempt to uncover the molecular mechanism of ER inheritance in the budding yeast Saccharomyces cerevisiae by performing comprehensive full genome screens for mutants defective in inheritance of the ER. Thus far we have established a screening method using a high throughput fluorescent microscopy system. The findings from the screen will be followed up by more focused tools to reach a mechanistic understanding of the process. Uncovering ER inheritance mechanisms in yeast and finding the similarities to mammals should allow rapid progress in our understanding of this process in all eukaryotes, whereas any differences in such a basic cellular mechanism, are important for anti-fungal drug therapies.

Back to top of page

 

 

 

Poster board #21

 

Dynamics of protein localization in yeast and their impact on endoplasmic reticulum (ER) functions

 

Michal Breker and Maya Schuldiner

 

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

 

To understand the complex molecular machinery of the cell we must study all the components and their dynamic interplay. High throughput tools allow gathering information about cells in a systematic manner and have generated extensive, albeit often static, characterizations of cellular components. One such dataset uses a library of all yeast proteins tagged with Green Fluorescent Protein (GFP) to describe their sub-cellular localization. Although many examples of this library has thus far only been used to look at localization under a single condition. My work therefore aims to comprehensively determine the localization of all yeast proteins under a large variety of different growth conditions. This will be performed utilizing the existing yeast GFP library combined with high throughput microscopy. The data acquired will be used to create an atlas of sub-cellular dynamics and to reach a mechanistic understanding of the molecular machinery required to regulate protein re-localizations. As proof of principal, we will focus on conditions that impact proteins of the endoplasmic reticulum (ER) and use them to elucidate regulatory mechanisms that coordinate the different ER functions.

Back to top of page

 

 

 

Poster board #22

 

Mounting computational evidence for functionality of Fantom non-coding RNA

Ilana Lebenthal and Ron Unger

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

A surprising observation in large scale studies of mammalian genome transcription is the large percent of the genomic DNA that is transcribed to RNA. The meaning of the transcription of so many genomic regions is still under debate, and it remains an open question whether most of the identified transcripts are in fact functional. Here we look for computational indirect evidence that can support the functionality of a 34,030 non-coding RNA (ncRNA) transcripts that were found in the Fantom3 project. We show that as a group this set of sequences is more conserved with human and rat than control sets of sequences taken randomly from the mouse genome. In particular, there are some Fantom sequences that show very high sequence conservation with the other species. We demonstrate that homologs of the Fantom ncRNA sequences in human and rat have more matches to ESTs in these organisms than homologs of the control sets. We show that the conserved subgroup of sequences is differentially expressed, and exhibits elevated expression levels in brain tissues. In addition, we were able to show that on average the Fantom ncRNA sequences have lower minimal free energy of folding than the control sets, partially because of statistically distinct dinucleotide composition. Taken these observations together, it is clear that as a group the Fantom ncRNA set is distinct from random sets from the genome. Therefore we conclude that many of these transcripts may indeed have biological function.

Back to top of page

 

 

 

Poster board #23

 

De novo function prediction: identification of novel photosynthetic proteins

 

Rotem Snir and Yanay Ofran

 

Lab of systems biology and functional genomics, Faculty of Life Sciences, Bar Ilan university, Ramat Gan, Israel

 

Photosynthesis (PS) is the main source of energy for nearly all forms of life. A better understanding of the molecular machinery that underlies PS would not only improve our understanding of bioenergetics, but may also lead to technologies that could imitate PS to the benefit of mankind. A thorough study of the mechanism of PS requires a comprehensive analysis of PS proteins. However, only a small fraction of PS proteins is annotated as such. Arguably, the vast majority of PS organisms are marine bacteria whose sequences may be known to us only from genomic and metagenomic projects that provide no functional annotation. Therefore, there is a need to develop high-throughput tools for the identification of such proteins from sequence. Here we present a sequence based approach for the identification of novel PS proteins. Using data mining approaches we built a dataset of PS proteins and generated a dictionary of PS sequence motifs which describe all PS functions. Using this dictionary we are able to identify unannotated PS proteins with high precision and recall.

Back to top of page

 

 

 

Poster board #24

 

Prediction of 3D metal binding sites from translated gene sequences based on remote-homology templates

 

Ronen Levy, Vladimir Sobolev and Marvin Edelman

 

Department of Plant Sciences, Weizmann Institute of Science, Rehovot Israel

 

We recently demonstrated an ability to effectively predict metal binding-sites from apo structures resolved by X-ray crystallography using the CHED algorithm (Babor et al, 2008). We now show that structures obtained by modeling translated gene sequences are sufficient for the same task (Levy et al, 2009). The basis for this is: the major overlap already achieved between structural and linear database space (> 40%), and the minor extent by which side chain modeling reduces predictive accuracy (~5%). The SeqCHED procedure (Levy et al, 2009) involves: inputting a translated gene sequence ("Target"); seeking a homologous PDB sequence ("Template"); structurally modeling Target side-chains using the Template backbone; outputting the predicted metal binding site using the CHED algorithm. Analysis of the approach finds selectivity to be uniformly high (~85-90%) irrespective of the level of sequence homology between Template and Target in the range of 18-100% identity. Below approximately 18% identity, the number of analyzable target-template pairs and predictability of metal binding sites falls off sharply. A full third of structural templates were found to have target partners only in the remote homology range of 18-30%. In this range, nonmetal-binding templates are calculated to be the majority and serve to predict with 50% sensitivity and ~85-90% selectivity at the geometric level.

 

 

Babor M, Gerzon S, Raveh B, Sobolev V, Edelman M (2008) Prediction of transition metal binding sites from apo protein structures. Proteins 70, 208-217.

 

Levy R, Edelman M, Sobolev V (2009) Prediction of 3D metal binding sites from translated gene sequences based on remote-homology templates.  Proteins 76, 365-374.

Back to top of page

 

 

 

Poster board #25

 

 

Large scale analysis of antibodies, antigens and immunogenic interactions

 

Vered Kunik, Shahar Alon & Yanay Ofran

 

Lab of systems biology and functional genomics, Faculty of Life Sciences, Bar Ilan university, Ramat Gan, Israel

 

Characterizing the antigenic contacts between an antibody and an antigen is a key for understanding antigenic recognition and interaction and may promote the prediction of B-cell epitopes and rational antibody design. We have created a comprehensive non-redundant list of antigenic contacts, using a novel structure based method for distinguishing antigenic contacts from all antibody-antigen contacts. The residue-residue preferences of the derived antigenic contacts show a marked difference from that of other types of protein-protein interactions. Moreover, the different CDRs display large differences, both in residue-residue preferences and in amino-acid composition. Finally, the amino-acid composition of the epitopes differs from that of other protein-protein interfaces, a finding which may lead to an improved detection of B-cell epitopes.

Back to top of page

 

 

 

Poster board #26

 

A Comparative Genome-wide Study of ncRNAs in Trypansomatids

 

T Doniger, C Wachtel, R Katz, S Michaeli, R Unger

 

Faculty of Life Science, Bar-Ilan University, Ramat-Gan, ISRAEL

 

Recent studies have provided extensive evidence for multitudes of non-coding RNA (ncRNA) transcripts in a wide range of eukaryotic genomes. ncRNAs are emerging as key players in multiple layers of cellular regulation. With the availability of many whole genome sequences, comparative analysis has become a powerful tool to identify ncRNA. We undertake a systematic genome-wide in silico screen to search for novel ncRNAs in the genome of Trypanosoma brucei by comparative genomics. T. brucei was compared to 6 other sequenced Trypansomatids. A total of 8877 and 15,141 sequences were found to be conserved in six genomes and at least four genomes, respectively. Almost one third of the known ncRNA was found in six genomes, and about half were found in four genomes. Annotated sequences were then filtered out. Thus, yielding at total of 57 conserved unannotated sequences in six genomes and 126 in at least four genomes. Among this collection we identified tRNA-sec, previously annotated incorrectly in the T. brucei genome annotation as well as snoRNAs, and several novel ncRNAs of unknown function. Many of the predicted ncRNAs were validated experimentally and categorized to their families.

Back to top of page

 

 

 

 

Poster board #27

 

Phosphoproteomic analysis of ethylene-regulated protein phosphorylation in etiolated seedlings of Arabidopsis mutant ein2 using two-dimensional separations coupled with a hybrid quadrupole time-of-flight mass spectrometer

 

Hao Li, Wai Shing Wong, Lin Zhu, Hong Wei Guo, Joseph Ecker and Ning Li

 

Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong

 

Ethylene regulates a variety of stress responses and developmental adaptation in plants. In the present study, the phosphoproteomics is adopted to investigate the differential protein phosphorylation by ethylene in Arabidopsis ethylene-insensitive 2 (ein2) mutant. A total of 224 phosphopeptides were identified, of which 64 phosphopeptides were detected three or more times. Ethylene induces a general reduction in phosphorylated proteins in ein2. Totally, three ethyleneenhanced and three ethylene-repressible unique phosphopeptides were identified, respectively. Classification of the cellular functions of these phosphoproteins revealed that 55.5% of them are related to signaling and gene expression. Peptide sequence alignment reveals two highly conserved phosphorylation motifs, PRVD/GSx and SPDYxx. Alignment of these phosphopeptides with Arabidopsis proteins reveals five phosphorylation motifs. Both ethylene-enhanced and -repressible phosphopeptides present in these motifs. EIL-1, ERF110 transcription factors and Hua enhancer 4 (HEN4) are predicted to contain one of the phosphorylation motifs. The phosphorylation of the motif-containing peptides has been validated by the in vitro kinase assays coupled with MS analysis. The differential regulation of phosphorylation by ethylene is substantiated by Western dot blot analysis. Taken together, these results suggest that ethylene signals may be transduced by a phosphor-relay from receptors to transcriptional events via both ein2-dependent and - independent pathways.

Back to top of page

 

 

 

Poster board #28

 

Structural studies on mutant wheat metallothioneins

 

Ceren Saygi, Anil Akturk, Mert Aydin, Filiz Yeşilõrmak and Zehra Sayers

 

Sabanci University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey

 

Metallothioneins (MTs) are classified as low molecular weight, cysteine-rich, metal binding proteins. The large number of cysteine (Cys) residues in MTs bind a variety of metals by mercaptide bonds [1]. Most protein-metal binding studies are carried out on mammalian proteins and little is known about the structural features the plant MTs. A novel MT gene (dmt) in Triticum durum was identified and cloned for overexpression in E. coli [2]. T. durum metallothionein (dMT) displays three sequence domains: metal binding N terminus (β domain, 1-19^th residues) and C terminus (α domain, 61-75^th residues) and a long hinge region (20-60^th residues). Cysteines are clustered equally in N and C termini with a ÒCys-X-CysÓ motif (Cys-motif) and the hinge region possess no Cys residues. dMT was overexpressed in E.coli as a GST (glutathione-S-transferase) fusion protein (GSTdMT). Both GSTdMT and dMT cleaved from GST were purified and characterized by biochemical and biophysical methods. It was shown that GSTdMT binds 4±1 moles of Cd per one mole of protein and has a high tendency to form stable oligomeric structures [3]. The aims of the present work are investigation of the effect of removal of the hinge region connecting the two metal binding domains on the stability of the protein structure, and determination of the effect of Cys-motif modifications on the metal binding capacity and affinity of dMT. Furthermore removal of the hinge region will allow comparison with the structure of mammalian MTs which tend to possess short connecting hinge region. Structural features of all mutants will be investigated using biophysical methods such as gel filtration chromatography, SDS- and native PAGE, dynamic light scattering, atomic absorption spectroscopy and circular dichroism spectrometry. Removal and linking procedure of hinge region are executed by PCR techniques. The ÒchimericÓ dMT is inserted to the vector pEGX4T-2 and BL21 strain E.coli has been transformed with this construct. Cys motifs are modified to produce mutant proteins with CCSCG, GCSCC or CCSCC motifs.  Mutations are accomplished by site-directed mutagenesis and the mutant constructs are introduced into the pGEX4T-2 vector for expression in E. coli. Results of mutations on the expression of recombinant proteins and their metal-binding properties will be presented.

 [1] C. Cobbett, P. Goldsbrough, ÒPhytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis,Ó Annual Review of Plant Biology, vol. 53, no. 3, pp. 159-182, 2002.

[2] K. Bilecen, †.H. …ztŸrk, A.D. Duru, T. SŸtlŸ, M. Petoukhov, D.I. Svergun, M.H.J. Koch, U. Sezerman, I. Cakmak and Z. Sayers, ÒTriticum durum metallothionein: isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling,Ó The Journal of Biological Chemistry, vol. 280, no. 14, pp. 13701-13711, 2005.

[3] F. Dede, G. Dinler, and Z. Sayers, Ò3D Macromolecular Structure Analyses: Applications in Plant Proteins,Ó Proc. of the NATO Advanced Research Workshop, Published by Springer Verlag, Heidelberg, pg 135-146, 2006.

Back to top of page