PubMed Health⌕ Search

Biomedical subjects

V Thulasiraman

Publications and source records attributed to V Thulasiraman.

12 recordsLinked to original sources
PubMed2001

Protein biochips for differential profiling.

Progress has been made in utilizing ProteinChip technology to profile and compare protein expression in normal and diseased states, particularly in the areas of cancer, infectious disease and toxicology. The past year has also seen the development of several novel chip types designed to analyze proteins in a fashion analogous to the array-based format of DNA microarrays. Some of these platforms may be used for differential profiling.

Bacterial Proteins↗
PubMed2001

Detection and identification of virulence factors in Yersinia pestis using SELDI ProteinChip system.

A rapid method for the detection, purification, and identification of proteins in bacterial extracts was developed using surface enhanced laser desorption/ionization (SELDI) ProteinChip technology. The effectiveness of this technique for monitoring the expression and identification of temperature- and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mammalian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degrees C (the temperature of the flea vector) to 37 degrees C (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degrees C and 37 degrees C in a Ca(2+)-deficient medium. Through an initial protein profiling of the crude bacterial extract on strong anion exchange and copper affinity, ProteinChip arrays detected five proteins that were up-regulated and three proteins that were down-regulated at 37 degrees C. Two of the proteins predominately expressed at 37 degrees C were semi-purified in less than two days. The two proteins were identified as catalase-peroxidase and Antigen 4. Aside from its speed, a salient feature of the SELDI technique is the microgram amounts of crude sample required for analysis.

Bacterial Proteins↗
PubMed1999

Molybdate inhibits hsp90, induces structural changes in its C-terminal domain, and alters its interactions with substrates.

To examine the biochemical mechanism by which hsp90 exerts its essential positive function on certain signal transduction proteins, we characterized the effects of molybdate and geldanamycin on hsp90 function and structure. Molybdate inhibited hsp90-mediated p56lck biogenesis and luciferase renaturation while enforcing salt-stable interactions with these substrates. Molybdate also reduced the amount of free hsp90 present in cell lysates, inhibited hsp90's ability to bind geldanamycin, and induced resistance to proteolysis at a specific region within the C-terminal domain of hsp90. In contrast, the hsp90 inhibitor geldanamycin prevented hsp90 from assuming natural or molybdate-induced conformations that allow salt-stable interactions with substrates. When these compounds were applied sequentially, the order of addition determined the effects observed, indicating that these agents had opposing effects on hsp90. We conclude that a specific region within the C-terminal domain of hsp90 (near residue 600) determines the mode by which hsp90 interacts with substrates and that the ability of hsp90 to cycle between alternative modes of interaction is obligatory for hsp90 function.

Amino Acid Sequence↗
PubMed1999

In vivo newly translated polypeptides are sequestered in a protected folding environment.

Molecular chaperones play a fundamental role in cellular protein folding. Using intact mammalian cells we examined the contribution of cytosolic chaperones to de novo folding. A large fraction of newly translated polypeptides associate transiently with Hsc70 and the chaperonin TRiC/CCT during their biogenesis. The substrate repertoire observed for Hsc70 and TRiC is not identical: Hsc70 interacts with a wide spectrum of polypeptides larger than 20 kDa, while TRiC associates with a diverse set of proteins between 30 and 60 kDa. Overexpression of a bacterial chaperonin 'trap' that irreversibly captures unfolded polypeptides did not interrupt the productive folding pathway. The trap was unable to bind newly translated polypeptides, indicating that folding in mammalian cells occurs without the release of non-native folding intermediates into the bulk cytosol. We conclude that de novo protein folding occurs in a protected environment created by a highly processive chaperone machinery and is directly coupled to translation.

Animals↗
PubMed1999

Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC.

von Hippel-Lindau (VHL) disease is caused by loss of function of the VHL tumor suppressor protein. Here, we demonstrate that the folding and assembly of VHL into a complex with its partner proteins, elongin B and elongin C (herein, elongin BC), is directly mediated by the chaperonin TRiC/CCT. Association of VHL with TRiC is required for formation of the VHL-elongin BC complex. A 55-amino acid domain of VHL is both necessary and sufficient for binding to TRiC. Importantly, mutation or deletion of this domain is associated with VHL disease. We identified two mutations that disrupt the normal interaction with TRiC and impair VHL folding. Our results define a novel role for TRiC in mediating oligomerization and suggest that inactivating mutations can impair polypeptide function by interfering with chaperone-mediated folding.

Chaperonins↗
PubMed1999

Dual role for Hsc70 in the biogenesis and regulation of the heme-regulated kinase of the alpha subunit of eukaryotic translation initiation factor 2.

The heme-regulated kinase of the alpha subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI.

Animals↗
PubMed1998

Evidence that Hsc70 negatively modulates the activation of the heme-regulated eIF-2alpha kinase in rabbit reticulocyte lysate.

The role of the heat-shock cognate protein, Hsc70, in regulating the activity of the heme-regulated eIF-2alpha kinase (HRI) in hemin-supplemented rabbit reticulocyte lysate (RRL) in response to heat and oxidative stress was examined and compared with the effect of Hsc70 on HRI activation in response to heme deficiency. Hsc70 suppressed eIF-2alpha phosphorylation and maintained the guanine nucleotide exchange activity of eIF-2B in heme-deficient RRL and in hemin-supplemented RRL exposed to elevated temperatures (42 degrees C), denatured protein (reduced carboxymethylated bovine serum albumin, RCM-BSA), oxidized glutathione or Hg2+. The ability of Hsc70 to inhibit HRI activation was mediated through its ability to inhibit the hyper-autophosphorylation of transformed HRI, which causes the hyperactivation of HRI. Maintenance of protein-synthesis rate was observed to be an unreliable indicator of the ability of Hsc70 to suppress HRI activation in response to stress. While Hsc70 completely reversed protein synthesis inhibition caused by Hg2+. Hsc70 only partially reversed translational inhibition caused by oxidized glutathione (GSSG) or heat shock. The inability of Hsc70 to fully protect protein synthesis from inhibition induced by heat shock or GSSG was due to inability of Hsc70 to protect eIF-4 E from heat-induced dephosphorylation, and its inability to protect translational elongation from GSSG-induced inhibition, respectively. Activation of HRI in heat-shocked hemin-supplemented lysate correlated with a marked decrease in the pool of Hsc70 that was available to bind RCM-BSA and the loss of the interaction of Hsc70 with HRI. These observations indicate that heat shock induced the accumulation of a sufficient quantity of Hsc70 binding substrates (e.g., denatured protein) to sequester Hsc70 and inhibit the ability of Hsc70 to suppress HRI activation. Our results indicate that Hsc70 not only negatively modulates the activation of HRI in heme-deficienct RRL, but also in hemin-supplemented RRL in response to heat and oxidative stress.

Animals↗
PubMed1997

The role of the 90-kDa heat-shock protein and its associated cohorts in stabilizing the heme-regulated eIF-2alpha kinase in reticulocyte lysates during heat stress.

The heme-regulated eIF-2alpha kinase (HRI) is activated not only in heme-deficient rabbit reticulocyte lysates (RRL), but also in hemin-supplemented RRL treated with heat-shock, N-ethylmaleimide (MalNEt) or heavy metal ions. We have demonstrated previously that heat-shock proteins, Hsp90, Hsp70 and FKBP52, are associated with HRI in RRL; the association of HRI with Hsp90 and FKBP52, but not Hsp70, is enhanced by hemin. To study the role of Hsp90 and its associated cohorts in the regulation of HRI, we examined the interaction of these proteins with HRI in hemin-supplemented RRLs during heat or oxidative stress. The association of HRI with Hsp90, FKBP52 and p23 was maintained in heat-, MalNEt- or Hg2(+)-treated hemin-supplemented RRL. Glycerol gradient centrifugation and gel filtration on Sephacryl S-300 indicated that neither heat shock nor MalNEt-treatment affected the apparent molecular mass of HRI in hemin supplemented RRL. In addition, active HRI was coimmunoprecipitated with 8D3 anti-Hsp90 from both heme-deficient and MalNEt-treated hemin-supplemented RRL. These results demonstrate that activation of HRI in response to heat stress and oxidative stress does not require dissociation of Hsp90 from HRI. Furthermore, HRI activity was inhibited upon addition of hemin to Hsp90-depleted heme-deficient RRL, indicating that inhibition of HRI activity by hemin is not mediated by the reassociation of Hsp90 with HRI. We also examined the dynamics of the interaction of Hsp90 with HRI. Reconstitution of the interaction of Hsp90 with HRI was stimulated by elevated temperature and required both Mg2+ and ATP. Addition of purified Hsp90 to hemin-supplemented RRL which had been treated with MalNEt to inactivate its capacity to chaperone protein renaturation, protected HRI from irreversible denaturation and aggregation upon incubation at 41 degrees C. Our results suggest that Hsp90 interacts with HRI primarily in its capacity as a molecular chaperone, stabilizing HRI from denaturation under conditions of heat stress and oxidative stress.

Animals↗
PubMed1996

Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.

Renaturation of thermally denatured firefly luciferase in rabbit reticulocyte lysate (RRL) requires hsp90, hsc70, and other as yet unidentified RRL components [Schumacher, R.J., et al. (1994) J. Biol. Chem. 269, 9493-9499]. Benzoquinonoid ansamycins (BAs) have recently been shown to specifically bind hsp90 and inhibit its function. In this report, we present data that indicate BAs are specific inhibitors of hsp90 function. The effects of the BA geldanamycin (GA) on the kinetics of the luciferase renaturation in RRL were examined to gain insight into the mechanism by which GA inhibits the function of the hsp90 chaperone machinery. Chaperone-mediated renaturation of luciferase obeyed Michaelis-Menten kinetics. The GA inhibited luciferase renaturation uncompetitively with respect to ATP concentration and noncompetitively with respect to luciferase concentration, indicating that GA binds after the binding of ATP and that it binds to both the hsp90 chaperone machine/ATP complex and the hsp90 chaperone machine/ATP/luciferase complex. GA markedly decreased the Kapp of the hsp90 chaperone machine for ATP, suggesting that GA increases the binding affinity of the hsp90 chaperone machinery for ATP or it slows the rate of ATP hydrolysis. Consistent with the notion that GA specifically binds hsp90 and inhibits its function, addition of hsp90, but not hsc70, p60, or p23, reversed GA-induced inhibition of luciferase renaturation in RRL. Hsp90, hsc70, and the hsp cohorts p60, p48, and p23 were coimmunoprecipitated with luciferase from RRL. GA increased the steady-state levels of luciferase associated with hsp90/hsp70 chaperone machine complexes that contain p60 and blocked the association of the hsp90 cohort p23 with chaperone-bound luciferase. The data suggest that the function of the hsp90 chaperone machinery is not specific to its previously described interaction with steroid hormone receptors, and that it carries out some more generalized function in vivo.

Animals↗