31P NMR studies of the ATP/α-crystallin complex: Functional implications

Reddy, M. Chandra Sekhar; Palmisano, David V.; Groth-Vasselli, Barbara; Farnsworth, Patricia N.

doi:10.1016/0006-291x(92)90256-k

Cited by 35 publications

(20 citation statements)

References 12 publications

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“…chaperone function was enhanced by ATP [23]. In separate reports, ATP increased the refolding of xylose reductase by total a-crystallin [34], increased the binding of a-crystallin to lens membranes, and inhibited the chaperone activity of a plant sHsp [35,36]. Consistent with previous studies using aB-crystallin, the present results demonstrated that ATP enhanced the chaperone effect of MTB HSP 16.3 by twofold in the CS aggregation assay.…”

Section: Discussionsupporting

confidence: 92%

“…However, the specific role of ATP in the chaperone function of sHsps has been controversial [18,23,28,29]. Previous reports indicated that the chaperone activity of MTB HSP 16.3 may be ATP independent [13,19] while structural studies demonstrated an interaction between ATP and total bovine a-crystallin using equilibrium binding studies, intrinsic tryptophan fluorescence and 31 P NMR [23,25,34,35,37]. aB-Crystallin has also been reported to display an autokinase activity [38][39][40].…”

Section: Discussionmentioning

confidence: 99%

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Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human alphaB-crystallin

Valdez¹,

Clark²,

Wu³

et al. 2002

Eur J Biochem

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Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection. MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein aggregation in vitro, and it contains the core Ôa-crystallinÕ domain found in all sHsps. In this study we characterized the chaperone activity of recombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human aB-crystallin, a well characterized member of the sHsp family. Recombinant MTB HSP 16.3 was expressed in Escherichia coli and purified to apparent homogeneity. Similar to aB-crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 mM ATP the chaperone activity was enhanced by twofold. ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was observed with ATPcS, a nonhydrolyzable analog of ATP. Increased expression of MTB HSP 16.3 resulted in protection against thermal killing in E. coli at 48°C. While the sequence similarity between human aB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between these proteins containing the core Ôa-crystallinÕ domain are much closer.Keywords: ATP; human aB-crystallin; molecular chaperone; Mycobacterium tuberculosis HSP 16.3; small heat shock proteins.One-third of the world's population is infected with latent inactive tuberculosis and active tuberculosis is the leading cause of death due to an infectious disease [1]. Each year, new infections occur in 54 million people; 6.8 million people develop clinical disease, and 2.4 million cases result in death [2]. There is still limited knowledge of the molecular pathogenesis of the latent stage of this organism [3]. Individuals who have been infected with Mycobacterium tuberculosis can harbor stable dormant bacilli for decades before developing an active infection later in life [4]. Recent reports indicate an important role for M. tuberculosis (MTB) heat shock protein (HSP) 16.3 in the survival of MTB during prolonged periods of infection [5][6][7]. It was shown that MTB HSP 16.3, initially described as the immunodominant 14-or 16-kDa antigen [8][9][10][11], was a major component in tuberculosis infection in humans and played an important role in enhancing protein stability and survival [5]. Eighty-five percent of patients with active tuberculosis showed a positive reaction to this antigen, suggesting that this protein expressed in vivo had a key role in MTB infection [11,12]. The 14K antigen was later renamed MTB HSP 16.3 [13]. MTB HSP 16.3 accumulates to become the dominant protein in the latent stationary phase of M. tuberculosis infection [7]. Over-expression of HSP 16.3 in log phase growth of M. tuberculosis slowed the growth rate and protected against stationary phase autolysis in vitro [7]. MTB HSP 16.3 has been characterized as a membrane asso...

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human alphaB-crystallin

Valdez¹,

Clark²,

Wu³

et al. 2002

Eur J Biochem

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“…It is the additional exposure of bis-ANS-binding sites on ␣-crystallin by ATP that led to association. It may be pointed out here that the presence of ATP has already been reported to increase the binding of ␣-crystallin to lens membranes by 35% (46). This additional exposure is not because of unfolding or a molten globule-type swelling of ␣-crystallin but is due to reorganization of the hydrophobic residues.…”

Section: Discussionmentioning

confidence: 54%

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

Biswas

Das

2004

Journal of Biological Chemistry

110

160

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ATP plays a significant role in the function of molecular chaperones of the large heat shock protein families. However, its role in the functions of chaperones of the small heat shock protein families is not understood very well. We report here a study on the role of ATP on the structure and function of the major eye lens chaperone ␣-crystallin. Our in vitro study shows that at physiological temperature, ATP induces the association of ␣-crystallin with substrate proteins. The association process is reversible and low affinity in nature with unit binding stoichiometry. 4,4 -Dianilino-1,1 -binaphthyl-5,5-disulfonic acid, dipotassium salt, binding studies show that ATP induces the exposure of additional hydrophobic sites on ␣-crystallin, but no appreciable enhancement of the same was observed for the substrate protein ␥-crystallin or carbonic anhydrase. An equilibrium unfolding study reveals that ATP at 3 mM concentration stabilizes the ␣-crystallin structure by 4.5 kJ/mol. The compactness induced by ATP makes it more resistant to tryptic cleavage. ATP-induced association of chaperone ␣-crystallin with substrate enhanced its aggregation prevention ability and also enhanced the refolding yield of lactate dehydrogenase from the unfolded state. Our results suggest that the binding of ATP to ␣-crystallin and not its hydrolysis is required for all these effects, as replacement of ATP by its nonhydrolyzable analogue adenosine-5 -O-(3-thiotriphosphate), tetralithium salt, reproduced all the results faithfully. The implication of the ATP-induced reversible protein-protein association at physiological temperatures on the functional role of ␣-crystallin in vivo is discussed.␣-Crystallin is the major protein component of the vertebrate eye lens and is composed of two subunits, ␣A and ␣B, 20 kDa each having 57% sequence homology between them. Both subunits associate to form a large oligomer with an average molecular mass of 800 kDa. It is a key member of the small heat shock protein (sHSP) 1 superfamily having a conserved core

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“…A functional relationship between ATP and chaperone-like activity for ␣-crystallin has been suggested by equilibrium binding studies, intrinsic tryptophan fluorescence, and 31 P nuclear magnetic resonance spectroscopy that demonstrated an interaction between ATP and total bovine ␣-crystallin (26,28). In the presence of 300 M ATP, a decrease of 36% in the ϭ 340 nm peak fluorescence intensity demonstrated that ATP binding induced conformational changes in total bovine ␣-crystallin (26).…”

Section: Discussionmentioning

confidence: 99%

ATP-enhanced molecular chaperone functions of the small heat shock protein human αB crystallin

Muchowski

Clark

1998

Proc. Natl. Acad. Sci. U.S.A.

141

111

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We report direct experimental evidence that human ␣B-crystallin, a member of the small heat shock protein family, actively participates in the refolding of citrate synthase (CS) in vitro. In the presence of 3.5 mM ATP, CS reactivation by ␣B-crystallin was enhanced approximately twofold. Similarly, 3.5 mM ATP enhanced the chaperone activity of ␣B-crystallin on the unfolding and aggregation of CS at 45°C. Consistent with these findings, cell viability at 50°C was improved nearly five orders of magnitude in Escherichia coli expressing ␣B-crystallin. SDS͞PAGE analysis of cell lysates suggested that ␣B-crystallin protects cells against physiological stress in vivo by maintaining cytosolic proteins in their native and functional conformations. This report confirms the action of ␣B-crystallin as a molecular chaperone both in vitro and in vivo and describes the enhancement of ␣B-crystallin chaperone functions by ATP.Molecular chaperones of the large heat shock protein families not only suppress protein unfolding and aggregation in response to stress but also actively participate in the refolding of denatured proteins in vitro, often in an ATP-dependent manner (1, 2). ␣B-crystallin is a protein that shares sequence and functional similarities with small heat shock proteins (sHsps) from numerous species (3, 4). ␣B-crystallin is expressed constitutively at high levels in the lens of the eye and at lower levels in many nonlens cells and tissues and is up-regulated dramatically in response to stress and pathological conditions in vivo (3, 5). Chaperone-like activity has been described for ␣B-crystallin from humans and various other species in suppressing protein unfolding and aggregation in response to thermal or chemical stress (4,(6)(7)(8)(9). Although the participation of ␣-crystallin and other sHsps in the refolding of proteins has been reported (4, 6, 10), the enhancement by ATP on the reactivation and refolding of proteins by ␣B-crystallin has not. To functionally characterize ␣B-crystallin as a molecular chaperone, its effects on the unfolding and refolding of citrate synthase (CS) in the presence and absence of ATP were studied in vitro. CS was chosen as a model protein for the characterization of unfolding and refolding reactions because it has been used extensively with molecular chaperones that include GroEL, Hsp90, and the sHsps (6,(11)(12)(13)(14).ATP is an abundant phosphorous metabolite in lenses from many species (15) that is present in lens cells at concentrations as high as 6.7 mM (16,17), which is among the highest levels of any cell in the body (18). High concentrations of ATP (4-8 mM) are found in skeletal muscle (19), in which high levels of ␣B-crystallin are expressed (20). The activity of GroEL and other chaperones on unfolding and refolding of proteins has been characterized at high concentrations of ATP (6,11,(21)(22)(23)(24), which may reflect a functional relationship between high concentrations of ATP and chaperone activity in all cells. The functional experiments with ␣B-crystallin in th...

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31P NMR studies of the ATP/α-crystallin complex: Functional implications

Cited by 35 publications

References 12 publications

Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human alphaB-crystallin

Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human alphaB-crystallin

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

ATP-enhanced molecular chaperone functions of the small heat shock protein human αB crystallin

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