Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry.

The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure 1). Usually molecular chaperones exhibit differential affinities with different conformations of the substrate. With the exception of the sHsp family of molecular chaperones, the shift from a high-affinity binding state to the low-affinity release state is triggered by ATP binding and hydrolysis (Haselback and Buchner, 2015). Aggregation prevention assay is a simple, yet definitive assay to determine the chaperone activity of heat labile proteins such as Maltodextrin glucosidase (MalZ), Citrate Synthase (CS) and NdeI. This is based on the premise that proteins with chaperone like activity should prevent protein substrates (MalZ, CS and NdeI) from thermal aggregation. Here, we describe a detailed protocol for aggregation prevention assay using two different chaperone proteins, resistin and MoxR1, identified from our lab. Resistin, a human protein (hRes) and MoxR1 a Mycobacterium tuberculosis protein were analysed for their effect on prevention of MalZ/Citrate Synthase (CS)/NdeI aggregation. Figure 1.Mechanism of action of molecular chaperones. Citrate synthase folds via increasingly structured intermediates (I1, I2) from the unfolded state (U) to the folded state (N). Under heat shock conditions, this process is reversed.

Functional characterization of PeIF5B as eIF5B homologue from Pisum sativum.

We earlier reported 'PeIF5B' as a novel factor from Pisum sativum that has sequence similarity to eIF5B (S. Rasheedi, S. Ghosh, M. Suragani et al., P. sativum contains a factor with strong homology to eIF5B, Gene 399 (2007) 144-151). The main aim of the present study was to perform functional characterization of PeIF5B as an eIF5B homologue from plant system. PeIF5B shows binding to Met - tRNA(f)(Met), hydrolyses GTP and interacts with ribosomes. In vivo growth complementation analysis shows that PeIF5B partially complements its yeast homologue. Interestingly, PeIF5B mainly localizes in the nucleus as confirmed by nuclear localization signal (NLS) prediction, confocal imaging and immunoblots of cellular fractions. Similar to the yeast eIF5B but unlike the human orthologue, PeIF5B is an intron-less gene. This study highlights PeIF5B's role as a functional eIF5B homologue possibly participating in nuclear translation in plant system.

Localization of resistin and its possible roles in the ovary of a vespertilionid bat, Scotophilus heathi.

The aim of the present study was to evaluate the expression and effect of resistin on ovarian activities of Scotophilus heathi. Immunohistochemical study showed marked variation in resistin immunostaining during different reproductive phases. Most intense immunostaining of resistin was noticed in thecal-interstitial cells in ovary during the period of delayed ovulation, the period of increased androgen synthesis and suppressed ovulation. The changes in ovarian resistin level also correlated positively with circulating leptin level and body white adipose tissue accumulation. The in vitro study showed that resistin alone preferentially stimulated progesterone synthesis, but with luteinizing hormone (LH) stimulated androgen secretion. Resistin alone dose-dependently increased expression of LH-receptor, steroidogenic acute regulatory protein and insulin receptor proteins in the ovary, whereas together with LH showed dose-dependent stimulatory effect on expression of androgen receptor and insulin receptor proteins in the ovary. In conclusion, during the period of fat accumulation increased ovarian resistin level may be responsible for increased androgen synthesis through insulin receptor mediated pathways in the ovary of S. heathi.

Effects of resistin on ovarian folliculogenesis and steroidogenesis in the vespertilionid bat, Scotophilus heathi.

The bat Scotophilus heathi exhibit prolonged anovulatory condition known as delayed ovulation coinciding with the period of extensive fat accumulation. The present study was undertaken to find out whether extensive accumulation of fat in S. heathi is responsible for suppression of ovarian activity by increasing production of adipokine resistin in the bat. This was achieved by (a) investigating variation in serum resistin level in relation to the changes in the body fat mass and (b) evaluating the effect of resistin treatment on ovarian activity with reference to steroid synthesis. An attempt was also made to determine whether resistin mediate its effects on ovary through signal transducer and activator of transcription 3 (STAT3) signaling mechanism. The results showed significant seasonal variation in serum resistin level with the peak level coinciding with the period of maximum fat accumulation, high circulating androgen level and period of anovulation. The treatment with resistin to the bat caused increase in androstenedione due to stimulatory effects on 3ß-hydroxysteroid dehydrogenase, but decrease in estradiol level due to inhibitory effect on aromatase. Resistin treatment increased androgen receptor protein together with increased insulin receptor but not through conventional luteinizing hormone receptor and steroidogenic acute regulatory protein mediated pathways. This study further showed that resistin treatment increases androstenedione synthesis and up-regulates insulin receptor in the ovary through STAT3 mediated pathways. These findings suggest that obese women through increased resistin synthesis may causes development of non-ovulatory antral follicles through insulin receptor signaling cascade.

Human resistin, a proinflammatory cytokine, shows chaperone-like activity.

Resistin, a cysteine-rich adipocytokine, proposed as a link between obesity and diabetes in mice, was shown as a proinflammatory molecule in humans. We earlier reported that human resistin (hRes), a trimer, was resistant to heat and urea denaturation, existed in an oligomeric polydispersed state, and showed a concentration-dependent conformational change. These properties and an intimate correlation of hRes expression with cellular stress prompted us to investigate hRes as a possible chaperone. Here, we show that recombinant human resistin was able to protect the heat-labile enzymes citrate synthase and Nde1 from thermal aggregation and inactivation and was able to refold and restore their enzymatic activities after heat/guanidinium chloride denaturation. Furthermore, recombinant human resistin could bind misfolded proteins only. Molecular dynamics-based association-dissociation kinetics of hRes subunits pointed to resistin being a molecular chaperone. Bis-ANS, which blocks surface hydrophobicity, abrogated the chaperone activity of hRes, establishing the importance of surface hydrophobicity for chaperone activity. Replacement of Phe49 with Tyr (F49YhRes), a critical residue within the hydrophobic patch of hRes, although it could prevent thermal aggregation of citrate synthase and Nde1, was unable to refold and restore their activities. Treatment of U937 cells with tunicamycin/thapsigargin resulted in reduced hRes secretion and concomitant localization in the endoplasmic reticulum. Escherichia coli transformants expressing hRes could be rescued from thermal stress, pointing to hRes's chaperone-like function in vivo. HeLa cells transfected with hRes showed protection from thapsigargin-induced apoptosis. In conclusion, hRes, an inflammatory protein, additionally exhibited chaperone-like properties, suggesting a possible link between inflammation and cellular stress.

The translation initiation factor, PeIF5B, from Pisum sativum displays chaperone activity.

We earlier documented the structural and functional characterization of PeIF5B factor from Pisum sativum that shows strong homology to the universal translation initiation factor eIF5B (Rasheedi et al., 2007, 2010 [12,13]). We now show that PeIF5B is an unusually thermo-stable protein resisting temperatures up to 95 °C. PeIF5B prevents thermal aggregation of heat labile proteins, such as citrate synthase (CS) and NdeI, under heat stress or chemical denaturation conditions and promotes their functional folding. It also prevents the aggregation of DTT induced insulin reduction. GTP appears to stimulate PeIF5B-mediated chaperone activity. In-vivo, PeIF5B over expression significantly enhances, the viability of Escherichia coli cells after heat stress (50 °C). These observations lead us to conclude that PeIF5B, in addition to its role in protein translation, has chaperone like activity and could be likely involved in protein folding and protection from stress.

Expression, purification and ligand binding properties of the recombinant translation initiation factor (PeIF5B) from Pisum sativum.

Gene encoding a novel translation initiation factor PeIF5B from Pisum sativum with sequence similarity to eIF5B from H. sapiens, D. melanogaster, S. cerevisiae as well as archaeal aIF5B from M. thermoautotrophicum was earlier reported by us. We now describe the expression and purification of 96 kDa recombinant PeIF5B (rPeIF5B) protein. Using fluorescence and circular dichroism spectra analyses, we show that Mg(2+) binding does not lead to any change in PeIF5B aromatic amino acid micro-environment, whereas GTP binding induces significant changes in the local environment of the aromatic amino acids. However, the protein undergoes changes in secondary structure upon metal ion and nucleotide binding. Charged initiator tRNA binding to PeIF5B is found to be cofactor dependent. PeIF5B binds to GTP in vitro as evident from autoradiography. Based on homology modeling of the catalytic domain of PeIF5B, we could confirm the conformational changes in PeIF5B following ligand binding.

Characterization of LEF4 ligand binding property and its role as part of baculoviral transcription machinery.

Late expression factor 4 (LEF4) is one of the four identified subunits of Autographa californica nucleopolyhedrosis virus (AcNPV) encoded RNA polymerase that carries out transcription from viral late and very late promoters. This 464-amino acid baculovirus-encoded protein also harbors 5' mRNA capping activity that includes RNA 5' triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities. Hydrolysis of 5' triphosphate RNA and free NTPs is metal ion dependent property of the protein. In the present communication, we describe the structural changes in the recombinant LEF4 protein following ligand binding. Metal ion binding causes some alteration in the conformation around aromatic amino acids whereas there is no effect on tryptophan fluorescence on GTP binding in absence and presence of metal ion. It is found that GTP and divalent cation cofactor produce some prominent changes in the secondary structure of the protein. Electrophoretic mobility shift assay (EMSA) shows that LEF4 is the probable factor that acts as anchor to dock the viral RNA polymerase on the very late polyhedrin promoter (Ppolh) facilitated by other factors.

Single-nucleotide polymorphisms in peroxisome proliferator-activated receptor gamma and their association with plasma levels of resistin and the metabolic syndrome in a South Indian population.

Studies on the association of the Pro12Ala and C1431T polymorphisms of PPAR? with diabetes and obesity have revealed extensive population-dependent variations. However, association of these polymorphisms with the metabolic syndrome and its individual components has not been well investigated in the Indian population. The Indian population harbours the maximum number of diabetics in the world who are thus more susceptible to metabolic disorders. We screened a South Indian population (N=699) for a possible association of these polymorphisms with the metabolic syndrome (MS) and type 2 diabetes. We also investigated the correlation of these two single-nucleotide polymorphisms (SNPs) with plasma resistin levels. The C1431T SNP was associated with higher levels of plasma resistin (P=0.017). Furthermore, C1431T was associated with resistin in different tertiles. Prevalence of the 'Pro-C' haplotype decreased with increasing tertiles of resistin (84.1% to 75.4%, P=0.037). Plasma resistin levels were not found to be associated with MS and type 2 diabetes. These results point to a likely association of plasma resistin levels with PPAR? polymorphisms in the Indian population.

Biophysical characterization and unfolding of LEF4 factor of RNA polymerase from AcNPV.

Late expression factor 4 (LEF4) is one of the four subunits of Autographa californica nuclear polyhedrosis virus (AcNPV) RNA polymerase. LEF4 was overexpressed in Escherichia coli and recombinant protein was subjected to structural characterization. Chemical induced unfolding of LEF4 was investigated using intrinsic fluorescence, hydrophobic dye binding, fluorescence quenching, and circular dichroism (CD) techniques. The unfolding of LEF4 was found to be a non-two state, biphasic transition. Intermediate states of LEF4 at 2M GnHCl and 4M urea shared some common structural features and hence may lie on the same pathway of protein folding. Steady-state fluorescence and far-UV CD showed that while there was considerable shift in the wavelength of emission maximum (lambda(max)), the secondary structure of LEF4 intermediates at 2M GnHCl and 4M urea remained intact. Further, temperature induced denaturation of LEF4 was monitored using far-UV CD. This study points to the structural stability of LEF4 under the influence of denaturants like urea and temperature. Although LEF4 is an interesting model protein to study protein folding intermediates, in terms of functional significance the robust nature of this protein might reflect one of the several strategies adapted by the virus to survive under very adverse environmental and physiological conditions.

Pisum sativum contains a factor with strong homology to eIF5B.

Immunoscreening of a cDNA expression library, prepared from 7 days old young shoots of pea (Pisum sativum), identified a novel gene comprising of 2586 bp open reading frame (ORF) with 381 bp and 532 bp 5' and 3'untranslated regions (UTRs), respectively. Sequence analysis of this gene, termed as PeIF5B, revealed striking homology to eukaryotic translation initiation factor eIF5B - a sequence homologue of prokaryotic translation initiation factor IF2. Southern blot analyses indicated that PeIF5B exists as a single copy gene in P. sativum genome. Northern blot hybridization revealed the presence of a 7 kb transcript in pea plant. In vitro translation using rabbit reticulocyte lysate system yielded a protein corresponding to 116 kDa which was higher than the calculated value of 96 kDa. Phylogenetic analyses of PeIF5B placed it closer to eIF5B from yeast, human and Drosophila. Pfam domain search analysis pointed to its likely role as a translation initiation factor. The presence of an eIF5B-like factor in a plant system will aid in better understanding of the mechanism of translation initiation in plants.