35, Ballygunge Circular Road, Kolkata - 700 019,West Bengal,India
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Golden
Jubilee Celebration of Department
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International Symposium on Teaching,Research and Exploration in Biochemistry: 50 years of Journey Dated 6- 8 January 2006 |
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Symposium Abstracts Poster Abstracts
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Symposium
Abtracts
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Food and Agriculture Organization
of the United Nations (FAO) and Its Collaboration with Partners
Biplab K. Nandi Senior Food and Nutrition Officer
FAO Regional Office for Asia and the Pacific
Bangkok, Thailand
The Food and Agriculture Organization of the United Nations was founded in October 1945 and its membership comprised 42 nations that were committed to ensuring humanity’s freedom from hunger through the promotion of agricultural development and trade, improved nutrition, rural development and the pursuit of food security, whereby people could have access at all times to the food they need to lead active and healthy lives. Today, FAO serves 187 Member Nations and one Member Organization, the European Community.
FAO’s mandate is to raise levels of nutrition, improve agricultural productivity, better the lives of rural populations and contribute to the growth of the world economy.
FAO is a crucial source of expertise in agriculture, fisheries, forestry, economics, nutrition and sustainable development. The Organization assists its member countries by disseminating information, providing policy advice and technical assistance, setting standards and organizing fora to forge agreements aimed at promoting food security and the sustainable use of natural resources.
The World Food Summit (WFS) in 1996 adopted the goal of halving the number of hungry people in the world by 2015. While several countries have made some progress towards achieving this target at the national level, global progress has been disappointing. At the June 2002 World Food Summit: five years later (WFS: fyl), governments reaffirmed their determination to fulfill their earlier commitment. They acknowledged that the goal could only be attained by the combined efforts of all countries, rich and poor, and through partnerships between governments, international institutions, civil society and the private sector. They agreed, inter alia, to act as an International Alliance Against Hunger to achieve the WFS goal.
Regulation of mitochondrial glycerophosphate acyltransferase. Dipak Haldar, Kawalpreet K. Aneja and Ashim Malhotra. Biological Sciences, St. John’s University, Queens, New York 11439.
The importance of mitochondrial
glycerophosphate acyltransferase is to control the quality of membrane phospholipids
by selectively positioning fatty acids in cellular phospholipids and perhaps
also to divert fatty acid for the synthesis of triacylglycerol. Recently,
we have focused our attention on how the acyltransferase is regulated. In
one approach, we have determined in the rat genome two sequences that can
serve as promoter for the enzyme. Preliminary results suggest the distal
but not the proximal promoter is inducible by glucose/insulin in starved-refed
cells. In another approach, we have found evidence that phosphorylation
of the enzyme plays an important role in regulating its activity. The rat
enzyme possesses consensus sites for different kinases. The acyltransferase
activity of isolated mitochondria is stimulated by casein kinase II (J.
Biol. Chem. 280: 19527-34, 2005), PKC and is inhibited by PKA
and AMPK. PKC could directly phosphorylate immunoprecipitated acyltransferase.
When intact COS-1 cells were treated with a PKC activator, the mitochondrial
acyltransferase was stimulated and was S/T phosphorylated. (Supported by
NIH grant GM-57643 and a grant from Estee Lauder)
Life and Journey of an RNA Regulator Inside the Cell
Dr. Gourisankar Ghosh
Introns present in pre-mRNAs are
removed by splicing, which requires five small nuclear ribonucleo-protein
(snRNP) particles and several other protein factors. Among the other
proteins involved in splicing are the Ser-Arg (SR) proteins and SR protein
kinases (SRPK). SR proteins are modular RNA binding proteins comprising
of one or two N-terminal RNA recognition motifs (RRMs)
and a C-terminal domain rich in multiple RS dipeptides (RS domain). SRPKs
specifically phosphorylate the SR proteins and the phosphorylated SR proteins
promote the recruitment of snRNPs at the site of splicing, allowing the
formation of the spliceosome, a large multiprotein-RNA complex which catalyzes
the splicing reaction. I will discuss the mechanism how SRPK1
phosphorylates ASF/SF2, a member of the SR protein family and the implication
of phosphorylation on both constitutive and alternative splicing.
Dr. HaraPrasad Ghosh
The tumor suppressor in lung cancer
(TSLC1) gene encodes a membrane glycoprotein containing extensive homology
in the extracellular domain with Ig-superfamily cell adhesion molecules.
The intracellular cytoplasmic domain (CT) contains a protein 4.1 (FERM)
binding motif and a PDZ-interacting motif. TSLC1 is ubiquitously expressed
in normal tissues but its expression is silenced by promoter hypermethylation
in a number of primary tumors or tumor cells. Restoration of TSLC1 expression
suppressed, tumorigenicity of a number of cancer cells (Nature Genetics,
2001). In nontransformed cells, TSLC1 induce synapse formation in neuronal
cells and induce cell-cell adhesion in spermatogenesis and in epithelial
cells. Restoration of TSLC1 expression in a tumor cell resulted into (i)
enhancement of cell-cell adhesion, (ii) suppression of anchorage independent
growth transformation and (iii) suppression of in vivo tumorigenesis
(Cancer Res., 2003). Expression of TSLC1 from a recombinant adenovirus vector
(Ad-TSLC1) inhibited cell proliferation and induced apoptosis in a cancer
cell line. Intratumoral injection of Ad-TSLC1 suppressed growth of tumor
in mice. Both the FERM-binding and the PDZ-interacting motifs were critical
for the cell-cell adhesion, proapoptotic and oncosuppressive activities
of TSLC1 (Oncogene, 2004). Recent studies suggest that TSLC1 suppresses
epithelial-mesenchymal transition (EMT) that contributes to invasion and
metastasis of cancer cells (J. Biol. Chem., 2005).
Title: Omega 3 fatty acids present in fish oil inhibit breast cancer growth by inducing apoptotic pathway and by inhibiting cell growth signals.
Nandini Ghosh-Choudhury, Ph. D.
An association between dietary fats and the incidence of breast cancer has been implicated from epidemiological and experimental studies. High fat diet, rich in 6 polyunsaturated fatty acids (PUFA), may be associated with higher risk of breast cancer incidence in Western countries. Populations whose dietary intake is primarily 3 PUFAs, in contrast, have a lower incidence of breast cancer. The 3 PUFAs, which occur at high levels in some fish oils, exert protective effects against breast and other common cancers and also inhibit lung and liver metastasis of human breast cancer cells in animal models. Tumor burden induced by MDA-MB-231 human breast cancer cells was significantly reduced in 10% fish oil-fed mice as compared to that in mice fed with control diet. In order to determine the molecular mechanism of fish oil mediated reduction of tumor growth, we examined the role of PI 3 kinase/Akt signaling. Immunecomplex kinase assay showed decreased PI 3 kinase activity in the tumor lysates from fish oil fed mice. Similarly, Akt kinase activity, the downstream target of PI 3 kinase, was reduced in fish oil fed tumors. A mechanism by which PI 3 kinase signaling is regulated, involves the tumor suppressor protein PTEN (phosphatase and tensin homolog deleted from chromosome 10). PTEN dephosphorylates the PI 3 kinase product PI 3,4,5-tris-phosphate and is often mutated in breast cancer. Therefore, we examined the expression of PTEN in breast tumor lysates. Immunoblot analysis of the tumor lysates showed increased expression of PTEN in the fish oil fed mice. These data provide the first evidence of regulation of a tumor suppressor protein by dietary intervention to reduce breast tumor growth. Since PI 3 kinase-Akt-PTEN axis regulates the survival of cells, we investigated the role of proteins, which play important role in tumor cell apoptosis. Immunoblot analysis demonstrated reduced expression of the anti-apoptotic BclXL in the tumor of fish oil fed animals. In contrast, the expression of proapoptotic cytochrome c was increased in these tumor lysates. Furthermore, activating phosphorylation of the p65 subunit of NFB transcription factor, which regulates the expression of many genes that play important role in apoptosis, was significantly reduced in the tumor lysates of fish oil fed animals. This reduced phosphorylation indicates that NFB activity is inhibited in fish oil fed tumor samples.
Eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) are the two major long chain 3
PUFAs found in fish oil. To confirm the inhibition of NFB observed
in vivo, we performed electrophoretic mobility shift assay using
NFB DNA binding element and nuclear extracts of MDA-MB-231 cells
incubated with EPA and DHA. Both these 3 PUFAs significantly reduced
the DNA binding activity of NFB. Also both these PUFAs reduced the
transcription of a reporter gene driven by NFB DNA element. BclXL
is a target gene of NFB. Transcription of BclXL was significantly
reduced in the presence of DHA and EPA. Together these data present the
first mechanistic role of dietary fish oil and 3 PUFAs in
reducing breast tumor growth.
ROLE OF THYROID HORMONE IN GLUTATHIONE HOMEOSTASIS IN THE DEVELOPING RAT BRAIN.
P.K.Sarkar Division of Neurobiology, Indian Institute of Chemical Biology
Jadavpur, Kolkata 7000064, India
Hypothyroidism in the developing rat brain is associated with enhanced oxidative stress, one of the earliest manifestations of which is a decline in the level of glutathione (GSH). To investigate the role of thyroid hormone (TH) on GSH homeostasis, the effect of TH on -glutamyl transpeptidase (GT) and -glutamyl–cysteine synthetase (GCS) – two key enzymes involved in the metabolism of GSH, was studied.
Contrary to expectation, hypothyroidism significantly declined the specific activity of cerebral GT in vivo as well as in primary cultures of astrocytes. Correspondingly, intraperitoneal injection of thyroid hormone (TH) to rat pups led to rapid stimulation of GT . The activation of the astrocyte ectoenzyme, GT, by TH do not explain the decline in intracellular GSH in the hypothyroid brain but may be conducive to GSH synthesis in neurons and their protection from oxidative stress.
Hypothyroidism in the developing
brain significantly declined the specific activity of GCS, the rate limiting
enzyme for the synthesis of GSH. In primary cultures of astrocytes, TH upregulated
the level of GSH with a maximal increase at 6 hour along with a parallel
increase in the activity of GCS. Inhibition of GCS activity by buthionine
sulfoximine totally abolished the TH-induced increase in the level of GSH
suggesting that the hormonal regulation of GSH is primarily controlled by
GCS . Mechanistic details of these investigations will be discussed.
Alternative Careers in Drug Development/Pharmaceutical Industry
Dr. Poushali Mukherjea
Holding a Ph.D. or other doctoral degree in biological sciences traditionally defines a career path in academia with strong emphasis on research and teaching. While that option certainly remains the dream for many students, various alternate career choices ranging from technical services, patent law, science journalism, and science policy are now available. The biotech/pharmaceutical industry offers a diverse array of career paths ranging from bench research in early discovery, clinical research, project management, medical writing, regulatory affairs and even sales and marketing. My presentation will focus on providing a brief snapshot of the drug development process in the US pharmaceutical industry.
One of FDA’s (Food and Drug Administration) most popular and enduring books called "From Test Tubes to Patients" describes how the promise of an investigational compound in the test tube may ultimately be used to treat a patient. Starting from 5,000-10,000 compounds typically tested in the early discovery laboratories, about 250 end up in preclinical testing in animals, about 10 enter early human trials and maybe 2 of these compounds are tested in large-scale clinical trials. Such a complicated process costs vast amounts of time and money; an estimated 11 years and 800 million dollars from bench to bedside.
However, the process of discovering,
developing and testing new drugs encompasses some of the most exciting areas
of scientific discovery. This opens up a vast range of careers to biologists,
organic chemists and even computer scientists and provides multiple avenues
for career transitions and mobility. With Western demand for Indian drug
ingredients and research services is surging many of these exciting opportunities
are right around the corner.
Mitochondrial import of tRNA: biochemical paths to discovery.
Samit Adhya. Indian Institute of Chemical biology
Biochemistry is a chemical view of biological processes. Biochemical experiments give insights into the molecular mechanics of cellular activities, and provide leads to be pursued by geneticists and cell biologists. This thesis will be postulated with reference to the author’s own research experiences over the last twenty years. The evolution of a scientific research project from a problem of translation control, to one of mitochondrial tRNA import, towards potential therapeutic applications, will be tracked using the biochemist’s looking glass. The relevance of such biochemical approaches in the post-genomic era will be discussed.
Structure and Dynamics of a
Gene-regulatory DNA Loop
Sankar Adhya Laboratory of Molecular Biology ,National Cancer Institute ,National Institutes of Health ,Bethesda, Maryland 20892 USA
Nucleoprotein complexes comprising short DNA loops (150 bp or less) are involved in a wide variety of DNA transactions (e.g. transcription regulation, replication and recombination) in both prokaryotes and eukaryotes. In these higher-order nucleoprotein complexes, proteins bound to spatially separated sites on DNA can interact with each other by looping out the relatively stiff intervening DNA. We investigated the structure, DNA trajectories, and dynamics of Gal repressosome, which is a higher-order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli. During repressosome assembly, a 113 bp DNA loop is formed by interaction of two GalR dimers, bound to spatially separated operators, OE and OI, flanking the gal promoters. Genetic analysis and complementing mathematical and physical studies indicated that GalR dimers interact directly and form a V-shaped stacked tetramer in repressosome, further stabilized by binding of a DNA-bending protein (HU) to an architecturally critical position on the DNA. The tetramerization interface was defined by isolating and characterizing looping-defective, suppressor of looping-defective, and gain-of-function mutations in GalR.
In the repressosome, the alignment of the operators in the DNA loop could be either a parallel or an antiparallel mode. As each mode can have two alternative geometries differing in the mutual stacking of the OE- and OI-bound GalR dimers, it is possible to have four different trajectories in the repressosome. Modeling of the repressosome based on evaluation of DNA elastic energy, as well as its visualization by atomic force microscopy, suggested a mutual antiparallel, rather than parallel, orientation of the two gal operators in an under-twisted DNA loop. Feasibilities of the DNA trajectories were further tested by constructing four different potential loops with mutant GalR heterodimers bound to specifically designed hybrid operators in such a way as to give rise to only one of the four possible trajectories. The results confirmed that OE and OI adopt a mutual antiparallel orientation with OI on the top of OE in an under-twisted DNA loop.
By using single DNA molecules and magnetic tweezers, we detected DNA looping directly and characterized its kinetics, thermodynamics, and supercoiling dependence. Some of the physical parameters of the loop in single molecular states were also monitored by following the tethered particle motion, which distinguished between stable and dynamic state of the loop.
DNA REPAIR: A Recipe for Survival by Maintaining Genomic Integrity after Assaults from Within And Without
Sankar Mitra Sealy
Center for Molecular Science and Department of Biochemistry & Molecular
Biology
University of Texas Medical Branch,
Galveston, TX 77555
Contrary to a common notion, the cellular genomes (both nuclear and mitochondrial) are quite unstable, not only due to spontaneous chemical reactions, (e.g., base loss, C-deamination and O6G methylation by S-adenosylmethionine), but also due to reactions with reactive oxygen species (ROS). ROS are continuously generated as respiration by-products in the mitochondria and produce a variety of DNA lesions including a multitude of oxidized bases, abasic(AP) sites (APS ) and their oxidation products (OAS), and strand breaks in both nuclear (nu) and mitochondrial (mt) DNA. Similar types of damage are also induced by external genotoxic agents including radiation and anti-cancer drugs.
All organisms have developed defense mechanisms to handle such damage which, if left unrepaired, can kill cells via apoptosis or induce mutations. Repair of DNA damage is universal and involve multiple mechanisms. For common damage, e.g., uv-induced pyrimidine cyclobutane dimers or O6 methylG( m6G), repair involves direct reversal. We co-discovered the latter repair protein in 1980 and named it MGMT. We were also the first to clone human MGMT (in 1990).
The predominant endogenous genomic damage is caused by ROS, which are repaired mostly via the base excision repair (BER) process that was first delineated in E.coli, and was shown to involve 4-5 enzymes. The damaged bases are excised by glycosylases and the resulting APS and their cleavage products are repaired by AP-endonucleases (APE) to generate 3′-OH termini. A repair DNA polymerase (Pol I in E. coli or Pol β in mammalian cells) fills in the missing nucleotide (and also removes the 5’ blocking group). Genomic integrity is restored by sealing with a DNA ligase.
It is now clear that in vivo BER is far more complex because of the need to address many issues including: (1) repair of nu vs. mt DNA; (2) lesion recognition by glycosylases in the presence of a vast excess of undamaged chromatinized DNA; (3) how repair is affected by the cell cycle and aging; (4) intracellular trafficking of glycosylases and APE; (5) preferential repair of active sequences constituting a small fraction of the genome; (6) efficient repair affected by covalent modification and proteome network.
During last ten years, we have addressed some of these issues and our findings are summarized as follows. (1) ROS signals nu and mt accumulation of glycosylases and APE; (2) aging inhibits glycosylase entry into mitochondria; (3) mtAPE is derived from nuAPE after removal of the N-terminal nuclear localization signal by a novel, ROS-activated protease. (4) We discovered a new family of oxidized base-specific mammalian glycosylases that we named NEIL1 and 2, and (5) an APE-independent BER sub-pathway involving NEIL1/2, unlike APE-dependent BER initiated by the other two oxidized base-specific glycosylases OGG1 and NTH1.(6) Selective interactions of two NEILs with specific sets of proteins support our model that NEIL1’s primary role is in the repair of lesions in the replicating genome, while NEIL2 preferentially repairs damage in the transcribed sequences.
Finally,in an effort to explore the impact of endogenous genome damage, we generated a conditional mouse cell line lacking APE alleles but expressing human APE. We have shown that APE is essential in mammalian cells, unlike in yeast or E.coli, and that APS and OAS induce apoptosis. Our current model postulates that these persistent lesions in mt genomes cause a spiraling increase in mt ROS which then activate nuclear signaling for the mt-dependent apoptosis cascade.
Our studies have so far raised more questions than we have answered, and we are currently pursuing them.
(Research supported by USPHS grants
CA53791, CA31721, CA81063, ES07572, ES08457, and ES06676)
Stimulus-responsive RelA dimer activation via p100, a bona fide IB protein
Soumen Basak Department of Chemistry and Biochemistry, University of California, San Diego
SUMMARY
Many different stimuli activate the transcription factor NF-k B/RelA, within diverse physiological contexts. Although three canonical stimulus-responsive NF-B inhibitors IB, -, and - have been identified, they do not appear to respond stimulus specifically; all three are degraded and resynthesized in response to a variety of stimuli to regulate the nuclear DNA binding activity of NF-B with characteristic temporal profiles. However, we find that the dynamics of NF-B/RelA activity downstream of the lymphotoxin- receptor (LTR) are not accounted for by degradation profiles of the three IB proteins. Instead biochemical analysis identifies the NF-B2 p100 protein as a bona fide IB inhibitor of NF-k B/RelA dimers that mediates stimulus-specific RelA dimer activation in response to LTR but not TNF signaling. Indeed, signaling genes nfkb2, ikk1, and nik, previously shown to be required for p100 processing and degradation, are also critical for LTR-mediated RelA dimer activation, whereas the three known IB proteins are dispensable. Our results suggest that cells in different physiological or pathological states may differentially regulate their relative responsiveness to specific stimuli by controlling the fraction of RelA dimers bound to so-called "canonicalversus "non-canonical" regulators.
Down Regulation of DNA Replicative and GSL Biosynthetic Gene-Products in Apoptotic Carcinoma Cells.
Subhash Basu1, Rui Ma1, Patrick J. Boyle1, Yoonie Cho1, Manju Basu1, and Sipra Banerjee2 . 1Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556; 2 Department of Cancer Biology, Cleveland Clinic, Cleveland, OH.
In normal cells the preeminent mechanism of cell death is by apoptosis. However, in cancer cells the components of the apoptotic pathway may exist but not always in the ativated forms. The human breast carcinoma MCF-7 and SKBR-3 are different in expressing pro-oncogene (HER2, ER &PgR) and pro-apoptotic (p53) proteins. In this study, the cells were treated with the apoptotic anti-cancer agents, cis-platin (Pol- inhibitor/DNA crosslinker; 10-80 m M) or L-PPMP (glycosyltransferase GlcT inhibitor; 1-8 m M). These drugs initiated apoptosis in a dose-dependent manner as measured by morphological changes and the phosphatidylserine flopping on the cell membrane by fluorescent dye PSS-380 binding assay (Basu et al. Glyco J. 20, 563-577, 2004). Both caspase-8 and -3 were activated in cells treated with cis-platin as detected by Western Blot. Caspase-3 and -9 activation is being studied in the cells treated with L-PPMP. The extrinsic and intrinsic pathways were suggested for treatment with cis-platin and L-PPMP, respectively. Little is known about the stability of the replisome during apoptosis. DNA helicases are motor proteins that catalyze the melting of dsDNA during DNA replication, recombination, and repair. Previously, DNA helicase-III was characterized as a part of the replisome complex isolated from carcino-embryonic cells. Helicase activity was quantitated using a novel (ROME) assay system that measures the release of oligonucleotides (20-60 nt) from radiolabeled DNA. A dose-dependent decrease in DNA pol- (80%) and DNA helicase (60%) activities wasobserved upon treatment with cis-platin. A sharp decrease of activities of the glycolipid: sialyltransferases [SAT-2 (CMP-NeuAc; GM3 2-8 sialyltransferase and SAT-4 (CMP-NeuAc: GM1a 2-3 sialyltransferase)] was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin. Further studies on the expression of ER- short and long forms on the apoptotic MCF cell surfaces during the initiation process is of interest at present. An ovarian carcinoma cell line (SKOV-3) has also been treated with several anti-cancer drugs (cis-platin and L-PPMP) and the process of apoptosis was initated in as early as 2 hours after treatment with concomitant activation of Caspases 3, as well as 9.
Activation of the apoptotic pathways in different carcinoma cells by two different routes ( i) extrinsic receptor mediated pathway and
ii) intrinsic mitochondrial membrane
permiabilization pathway) in the presence of cis-platin and L-PPMP
have been investigated in the present studies.
Abstract and title of the presentation in Golden jubilee celebration symposium on "Teaching, Research and Exploring Biochemistry: Fifty years of journey" at the Dept. of Biochemistry, CU
Title:Fine tuning of gene expression: one RNA regulates another
Dr. Suvendra Bhattacharyya
Abstract:
Over the last decade, post transcriptional
regulation of gene expression has emerged as one the most active fields
of modern molecular biology. Before being translated into proteins, mRNA
under goes several surveillance check points that ensure specific temporal
and spatial expression pattern of each gene in metazoan. The emerging idea
of post transcriptional gene silencing by tiny regulatory RNA molecules
such as microRNA and siRNAs has provided us with both opportunities and
new challenges. Impairment of small RNA-mediated fine tuning of gene expression
is evidenced in different human diseases including cancer. Recent advancement
towards understanding principles of post transcriptional gene repression
and its possible implications will be discussed.
PHOSPHORYLATION OF YEAST 60S RIBOSOME BIOGENESIS FACTOR Tif6p, THE YEAST HOMOLOGUE OF MAMMALIAN TRANSLATION INITIATION FACTOR eIF6, IS REQUIRED FOR REGULATION OF 60S RIBOSOME BIOGENESIS AND CONSEQUENTLY TRANSLATION IN YEAST CELLS
Uttiya Basu, Partha Ray, Romit Majumdar, and Umadas Maitra
Albert Einstein College of Medicine, Bronx, NY 10461, USA
Eukaryotic translation initiation
factor 6 (eIF6) can bind to the 60S ribosomal subunit and prevent its association
with the 40S ribosomal subunit. In yeast cells, Tif6p, the yeast homologue
of mammalian eIF6, is essential for the biogenesis of 60S ribosomal subunits
because the protein is necessary for the processing of 35S pre-rRNA in the
nucleolus to form the mature 25S and 5.8S rRNAs, constituents of the 60S
ribosomal particle. In the present work, we have isolated a protein kinase
from rabbit reticulocyte lysates that phosphorylates recombinant human eIF6.
Mass spectrometric analysis identified it as casein kinase I. The site of
phosphorylation, which is highly conserved from yeast to mammals, has been
identified to be the serine residues at positions 174 (major site) and 175
(minor site). The same serine residues are also phosphorylated by the nuclear
isoform of yeast CK I (Hrr25p) in yeast cells. Failure to phosphorylate
at these sites caused a loss of cell growth and viability. Additional experiments
showed that while both the phosphorylated and unphosphorylated forms of
Tif6p bound well to mature 60S and pre-60S ribosomal particles, failure
to phosphorylate Tif6p resulted in inhibition of 35S pre-rRNA processing
to mature 25S rRNA. Furthermore, while wild-type Tif6p was distributed both
in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala
and Ser175Ala) became a constitutively nuclear protein. Taken together,
these results suggest that phosphorylation of Ser-174 and Ser-175 by Hrr25p
in nucleolus plays a critical role in the nuclear export of Tif6p and regulates
60S ribosome biogenesis and consequently translation in yeast cells.
PHOSPHORYLATION REGULATION OF MANNOSYLPHOSPHO DOLICHOL SYNTHASE : UNITY IN DIVERSITY
Dipak K. Banerjee. Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00936-5067. USA
Mannosylphospho dolichol (Dol-P-Man),
a mannosyl donor in the assembly of Glc3Man9GlcNAc2-PP-Dol
(LLO) for asparagine-linked glycoproteins is synthesized by the transfer
reaction GDP-mannose + Dol-P Û
Dol-P-Man + GDP, and catalyzed by mannosylpho dolichol synthase (DPMS).
DPMS activity has been detected in higher eukaryotes, yeasts, protozoan
parasites, plant fungi, nematodes as well as in archaebacteria. In S.
cerevisiae, DPM1 is a structural gene and its disruption is lethal to
the organism. Partial deficiency of DPMS has also been reported in congenital
disorder of glycosylation (CDG). DPMS is distributed in two groups: Group
I, a single-component enzyme represented by S. cerevisiae DPM1p;
and Group II is a multi-component enzyme represented by human DPMS, containing
a catalytic subunit DPM1 and two accessory proteins DPM2 and DPM3. Group
I DPMS shares 50-60% amino acid identity, has a transmembrane domain near
the C-terminus, and can be expressed in E.coli as a functional enzyme.
Human DPM1 has only 30% amino acid identity to the Group I members, lacks
the transmembrane domain and cannot be expressed in E.coli. DPMS
from all sources however, has a consensus sequence to be phosphorylated
by cAMP-dependent protein kinase (PKA). We, therefore, hypothesized that
phosphorylation will not only upregulate the DPMS activity but will also
accelerate the angiogenic process. Our results suggest that (i) DPMS activity
from the PKA-deficient CHO mutants is low; (ii) recombinant S. cerevisiae
DPMS responds less to phosphorylation when serine-141 is replaced with
alanine by site-directed mutagenesis; and (iii) 8Br-cAMP upregulates the
DPMS activity, LLO biosynthesis, and the rate of angiogenesis. Supported
in part by grants from the Department of Defense DAMD17-03-1-0754, and NIH
U54-CA096297.
Title: Regulation of canonical and non-canonical NF-kappaB activationpathways: From chemistry to biology
Amanda Fusco, Don Vu, De-Bin Huang, Olga Savinova, Anu Moorthy, Rashmi
Talwar and Gourisankar Ghosh
Department of Chemistry & Biochemistry
Abstract: NF-kappaB is a group of closely related dimeric transcription factors that profoundly affects a large array of physiological activities such as immune and inflammatory responses, cell proliferation, growth and apoptosis. The combinatorial homo- and heterodimers are formed from five subunits; p50, p52, RelA, cRel and RelB. The most predominant of these dimers is p50/RelA heterodimer. A class of inhibitor proteins known as IkappaB tightly control transcriptional activity of the p50/RelA heterdimer. In resting cells these inhibitors form stable ternary complexes with the p50/RelA heterodimer and are degraded by the proteasome in response to extracellular stimuli. The activation pathways of p50/RelA is referred to as canonical pathways. The p52/RelB heterodimer is also a potent NF-kappaB dimer, which activates slowly by a different class of stimuli and the
activation pathways are known as non-canonical pathways. p52 is the processed form of p100 and the C-terminus of p100 is structurally similar to IkappaB. In resting cells RelB remains inhibited as the p100/RelB complex. Stimuli of non-canonical pathways activate p52/Rel through the degradation
of the C-terminal inhibitory domain of p100. It is unclear as to how RelA
and RelB act as targets for two distinct classes of inhibitors. I will describe structural and biochemical mechanisms of how RelA and RelB specifies two distinct pathways.
ROLE OF THYROID HORMONE IN GLUTATHIONE HOMEOSTASIS IN THE DEVELOPING RAT BRAIN.
P.K.Sarkar
Division of Neurobiology, Indian Institute of Chemical Biology
Jadavpur, Kolkata 7000064, India
Hypothyroidism in the developing rat brain is associated with enhanced oxidative stress, one of the earliest manifestations of which is a decline in the level of glutathione (GSH). To investigate the role of thyroid hormone (TH) on GSH homeostasis, the effect of TH on -glutamyl transpeptidase (GT) and -glutamyl–cysteine synthetase (GCS) – two key enzymes involved in the metabolism of GSH, was studied.
Contrary to expectation, hypothyroidism significantly declined the specific activity of cerebral GT in vivo as well as in primary cultures of astrocytes. Correspondingly, intraperitoneal injection of thyroid hormone (TH) to rat pups led to rapid stimulation of GT . The activation of the astrocyte ectoenzyme, GT, by TH do not explain the decline in intracellular GSH in the hypothyroid brain but may be conducive to GSH synthesis in neurons and their protection from oxidative stress.
Hypothyroidism in the developing brain significantly declined the specific activity of GCS, the rate limiting enzyme for the synthesis of GSH. In primary cultures of astrocytes, TH upregulated the level of GSH with a maximal increase at 6 hour along with a parallel increase in the activity of GCS. Inhibition of GCS activity by buthionine sulfoximine totally abolished the TH-induced increase in the level of GSH suggesting that the hormonal regulation of GSH is primarily controlled by GCS . Mechanistic details of these investigations will be discussed.
ABSTRACT
Expression of Superoxide Dismutase Gene Isoforms in Mustard Gas Induced Lung Injury. Sutapa Mukhopadhyay, Veera Rajaratnam, Shyamali Mukherjee, and Salil K. Das, Division of Cancer Biology, Department of Biomedical Sciences, Meharry Medical College, Nashville, TN.
Mustard gas exposure causes inflammatory lung diseases. Many inflammatory lung diseases are associated with reactive oxygen species (ROS). ROS are involved in the maintenance of physiological functions. In tissues, it is therefore essential to maintain a steady-state level of antioxidant activity to allow both for the physiological functions of ROS to proceed and at the same time preventing tissue damage. We have recently reported that mustard gas exposure decreases the overall activity of superoxide dismutase (SOD). However, in this study we did not identify whether the activity of each or a particular isoform of SOD was decreased. In the present study, we investigated the effects on each of the three isozymes [cytosolic Cu/Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2) and extracellular-SOD (SOD3)]. We were particularly interested in SOD3 since it is the only extracellular scavenger of the superoxide radical and the major SOD in lung. Adult guinea pigs were intratracheally injected single doses of CEES (4 mg/kg body weight) in ethanol. Control animals were injected with ethanol in the same way. The animals were sacrificed after 7 days and lungs were removed after perfusion with physiological saline. Mustard gas exposure caused lung injury with evidence of fibrosis. The lung injury was associated with a significant increase in the activity of SOD1, a significant decrease in the activity of SOD3 and no change in the activity of SOD2. Thus the decrease in the total activity of SOD was primarily due to the SOD3 isozyme. Mustard gas exposure caused a significant increase in the SOD1 gene and protein expression. However, it did not have any significant effect on the expression of gene and protein of either SOD2 or SOD3. Our results indicate that the overall decrease in the activity of SOD by mustard gas exposure is mediated by direct inactivation of the SOD3 gene. Existence of active and inactive forms of SOD3 has been described in human due to change in cysteine-cysteine disulfide bonding. We are currently investigating whether mustard gas-induced inactivation of SOD3 in lung is similarly mediated by a change in cysteine-cysteine disulfide bonding. [Supported by grant from the Army (DAMD 17-03-2-0054).
Phosphorylation-dependent regulation of nuclear NF- B activity
Sankar Ghosh Section of Immunobiology and Department of Molecular Biophysics & Biochemistry ,Yale University School of Medicine,New Haven, CT 06520, USA
The transcription factor NF-B plays a critical role in immune and inflammatory responses by augmenting the expression of many inducible, immediate response genes, including cytokines such as IL-1 and TNF , pro-inflammatory mediators such as COX-2, adhesion molecules such as E-selectin, co-stimulatory molecules such as B7, and chemokines such as MCP-1. NF- B acts as a critical co-ordinating element in the body's response to infection and injury, and thus is an important mediator of immune responses. The activation of NF- B involves the signal-induced degradation of the I B protein in cytosolic NF- B:l B complexes leading to the subsequent nuclear translocation of the released NF- B. It has been assumed that nuclear translocation of NF- B would be sufficient for activation of NF- B-dependent genes. However, studies carried out in our laboratory has demonstrated that the p65 subunit of NF- B has to be phosphorylated to become competent for transcription. In particular we discovered that the catalytic subunit of cyclic-AMP-dependent protein kinase was present in cytosolic NF- B:l B complexes and that degradation of I B in respoinse to inducers such as LPS led to phosphorylation of p65 at serine 276, a canaonical site for PKA-phosphorylation. Recent studies have also implicated acetylation and deacetylation of both p65 and histones as crucial regulatory steps. To establish the importance of post-translational events in regulating the activity of nuclear NF- B, we have created a series of knock-in mice expressing mutant forms of p65. The mice display remarkably interesting phenotypes, and our efforts in understanding these phenotypes will be discussed.
Human La protein functions as an RNA chaperone at the Ribosome assembly during internal initiation
Dr. Saumitra Das
Department of Microbiology and Cell Biology,Indian Institute of Science,Bangalore 560012, India
Human La protein has been implicated to play a role as a molecular chaperone to facilitate IRES structure and translational competence. Earlier, we have demonstrated that La protein interacts with the HCV-IRES element both in vitro and in vivo and also shown that this interaction enhances the efficiency of viral RNA translation. Among the three RNA recognition motif (RRM) of La protein, RRM2 was found to bind with high affinity with the SLIV, around the GCAC sequence near the initiator AUG. This interaction was shown to induce a conformational alteration in the IRES to facilitate the interaction with the ribosomal protein S5 and help in 48S ribosomal assembly. Recently, we have observed, that a synthetic peptide LaR2C, derived from the C-terminus of La-RRM2, retained the RNA binding ability, compete with the binding of cellular La protein to HCV IRES and acts as dominant negative inhibitor of internal initiation of translation. It appears LaR2C peptide interferes with the assembly of 48S complexes resulting in the accumulation of pre-initiation complexes that are incompetent for the 60S ribosomal subunit joining. This observation constitute the "proof of concept" that small molecule, which prevents binding of ribosome to the HCV IRES, may act as potent inhibitor of viral RNA translation and can be developed to an effective anti-HCV therapy. Further analyses, using series of internal deletion and point mutations within La protein, defined critical determinants for the assembly of ribosomal complexes on HCV IRES. The study reveals valuable insights and reinforces the role of host cell proteins in the formation of functional initiation complex on the hepatitis C virus RNA during internal initiation.
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