Oxidized GroEL can function as a chaperonin.

Here, we report on the facilitated reactivation (85%) of oxidatively inactivated rhodanese by an oxidized form of the molecular chaperone GroEL (ox-GroEL). Reactivation by ox-GroEL required a reductant, and the enzyme substrate, sodium thiosulfate. Also, we found that ox-GroEL formed a complex with oxidatively inactivated rhodanese as shown by differential centrifugation and fluorescence spectroscopy. Ox-GroEL was obtained upon incubation of native GroEL for 16 h with 5 mM hydrogen peroxide. Under these conditions, GroEL was shown to retain its quaternary and secondary structures, but it displayed an increased exposure of hydrophobic surfaces as detected with 1,1'-bis(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS) fluorescence. Additionally, ox-GroEL was significantly more sensitive towards proteolysis with trypsin compared to the native form of the protein. The oxidatively inactivated form of rhodanese, also had an increased exposure of hydrophobic surfaces, as previously reported. Thus, the proteins binding appeared to be mediated by hydrophobic interactions. Unlike in prior reactivation studies that involved native GroEL or alpha-crystallin, we have clearly shown that an oxidized form of GroEL can function as a molecular chaperone in the reactivation of oxidatively inactivated rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.

[1]  M. Lundrigan,et al.  Enhanced hydrogen peroxide sensitivity and altered stress protein expression in iron-starved Mycobacterium smegmatis , 1997, Biometals.

[2]  Enrique Herrero,et al.  Mitochondrial Hsp60, Resistance to Oxidative Stress, and the Labile Iron Pool Are Closely Connected in Saccharomyces cerevisiae * , 2002, The Journal of Biological Chemistry.

[3]  G. Melkani,et al.  GroEL interacts transiently with oxidatively inactivated rhodanese facilitating its reactivation. , 2002, Biochemical and biophysical research communications.

[4]  P. Voziyan,et al.  Polyols induce ATP-independent folding of GroEL-bound bacterial glutamine synthetase. , 2002, Archives of biochemistry and biophysics.

[5]  A. Horwich.  Protein folding in the cell , 2002 .

[6]  C. Wang,et al.  GroEL-assisted dehydrogenase folding mediated by coenzyme is ATP-independent. , 2001, Biochemical and biophysical research communications.

[7]  G. Vecchio,et al.  GroEL-assisted refolding of adrenodoxin during chemical cluster insertion. , 2001, European journal of biochemistry.

[8]  S. Li,et al.  Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase. , 2001, The international journal of biochemistry & cell biology.

[9]  S. Cuzzocrea,et al.  Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury. , 2001, Pharmacological reviews.

[10]  J. A. Mendoza,et al.  alpha-Crystallin facilitates the reactivation of hydrogen peroxide-inactivated rhodanese. , 2000, Biochemical and biophysical research communications.

[11]  M. Glocker,et al.  Mass Spectrometry Unravels Disulfide Bond Formation as the Mechanism That Activates a Molecular Chaperone* , 2000, The Journal of Biological Chemistry.

[12]  L. Gierasch,et al.  Basis of substrate binding by the chaperonin GroEL. , 1999, Biochemistry.

[13]  J. Smith,et al.  Thiolation of the γB-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide* , 1999, The Journal of Biological Chemistry.

[14]  U. Jakob,et al.  Chaperone Activity with a Redox Switch , 1999, Cell.

[15]  Carl Frieden,et al.  Purification of GroEL with low fluorescence background. , 1998, Methods in enzymology.

[16]  E. Stadtman,et al.  Modification of protein surface hydrophobicity and methionine oxidation by oxidative systems. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[17]  B. Friguet,et al.  Age-related decline of rat liver multicatalytic proteinase activity and protection from oxidative inactivation by heat-shock protein 90. , 1996, Archives of biochemistry and biophysics.

[18]  G. Campo,et al.  Ligand-induced Conformational Changes of GroEL Are Dependent on the Bound Substrate Polypeptide* , 1996, The Journal of Biological Chemistry.

[19]  Carl Frieden,et al.  Determination of regions in the dihydrofolate reductase structure that interact with the molecular chaperonin GroEL. , 1996, Biochemistry.

[20]  A. Spector,et al.  Alpha-crystallin can act as a chaperone under conditions of oxidative stress. , 1995, Investigative ophthalmology & visual science.

[21]  E. Eisenstein,et al.  The Hydrophobic Nature of GroEL-Substrate Binding (*) , 1995, The Journal of Biological Chemistry.

[22]  P. Horowitz,et al.  Bound substrate polypeptides can generally stabilize the tetradecameric structure of Cpn60 and induce its reassembly from monomers. , 1994, The Journal of biological chemistry.

[23]  F. Hartl,et al.  Molecular chaperone functions of heat-shock proteins. , 1993, Annual review of biochemistry.

[24]  P. Horowitz,et al.  Characterization of a stable, reactivatable complex between chaperonin 60 and mitochondrial rhodanese. , 1992, The Journal of biological chemistry.

[25]  G. Lorimer,et al.  Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures. , 1992, The Journal of biological chemistry.

[26]  M. Fisher.  Promotion of the in vitro renaturation of dodecameric glutamine synthetase from Escherichia coli in the presence of GroEL (chaperonin-60) and ATP. , 1992, Biochemistry.

[27]  J. Sambrook,et al.  Protein folding in the cell , 1992, Nature.

[28]  G. Kurzban,et al.  Purification of bovine liver rhodanese by low-pH column chromatography. , 1991, Protein expression and purification.

[29]  G. Lorimer,et al.  Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. , 1991, The Journal of biological chemistry.

[30]  F. Hartl,et al.  Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate , 1991, Nature.

[31]  G. Lorimer,et al.  Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis by chaperonin 60 (groEL) are K+ dependent. , 1990, Biochemistry.

[32]  F. Hartl,et al.  Protein-catalysed protein folding. , 1990, Trends in biotechnology.

[33]  P. Horowitz,et al.  Oxidative inactivation of rhodanese by hydrogen peroxide produces states that show differential reactivation. , 1989, The Journal of biological chemistry.

[34]  R. Siezen,et al.  Oxidative modifications to crystallins induced in calf lenses in vitro by hydrogen peroxide. , 1989, Experimental eye research.

[35]  Roger W. Hendrix,et al.  Homologous plant and bacterial proteins chaperone oligomeric protein assembly , 1988, Nature.

[36]  W. Hol,et al.  The covalent and tertiary structure of bovine liver rhodanese , 1978, Nature.

[37]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[38]  G. Weber,et al.  Dimer formation from 1-anilino-8-naphthalenesulfonate catalyzed by bovine serum albumin. Fluorescent molecule with exceptional binding properties , 1969 .

[39]  G. Weber,et al.  Dimer formation from 1-amino-8-naphthalenesulfonate catalyzed by bovine serum albumin. A new fluorescent molecule with exceptional binding properties. , 1969, Biochemistry.