Intrarenal urothelium proliferation: an unexpected early event following ischemic injury.

Identification of renal cell progenitors and recognition of the events contributing to cell regeneration following ischemia-reperfusion injury (IRI) are a major challenge. In a mouse model of unilateral renal IRI, we demonstrated that the first cells to proliferate within injured kidneys were urothelial cells expressing the progenitor cell marker cytokeratin 14. A systematic cutting of the injured kidney revealed that these urothelial cells were located in the deep cortex at the corticomedullary junction in the vicinity of lobar vessels. Contrary to multilayered bladder urothelium, these intrarenal urothelial cells located in the upper part of the medulla constitute a monolayered barrier and express among uroplakins only uroplakin III. However, like bladder progenitors, intrarenal urothelial cells proliferated through a FGF receptor-2 (FGFR2)-mediated process. They strongly expressed FGFR2 and proliferated in vivo after recombinant FGF7 administration to control mice. In addition, IRI led to FGFR phosphorylation together with the selective upregulation of FGF7 and FGF2. Conversely, by day 2 following IRI, renal urothelial cell proliferation was significantly inhibited by FGFR2 antisense oligonucleotide administration into an intrarenal urinary space. Of notice, no significant migration of these early dividing urothelial cells was detected in the cortex within 7 days following IRI. Thus our data show that following IRI, proliferation of urothelial cells is mediated by the FGFR2 pathway and precedes tubular cell proliferation, indicating a particular sensitivity of this structure to changes caused by the ischemic process.

[The interaction of the GroEL chaperone with early kinetic intermediates of renaturing proteins inhibits the formation of their native structure]. / Vzaimodeistvie shaperona GroEL s rannimi kineticheskimi promezhutochnymi sostoianiiami renaturiruiushchikh belkov ingibiruet formirovanie ikh nativnoi struktury.

The main function of the chaperone GroEL is to prevent nonspecific association of nonnative protein chains and provide their correct folding. In the present work, the renaturation kinetics of three globular proteins (human alpha-lactalbumin, bovine carbonic anhydrase, and yeast phosphoglycerate kinase) in the presence of different molar excess of GroEL (up to 10-fold) was studied. It was shown that the formation of the native structure during the refolding of these proteins is retarded with an increase in GroEL molar excess due to the interaction of kinetic protein intermediates with the chaperone. Mg(2+)-ATP and Mg(2+)-ADP weaken this interaction and decrease the retarding effect of GroEL on the protein refolding kinetics. The theoretical modeling of protein folding in the presence of GroEL showed that the experimentally observed linear increase in the protein refolding half-time with increasing molar excess of GroEL must occur only when the protein adopts its native structure outside of GroEL (i.e. in the free state), while the refolding of the protein in the complex with GroEL is inhibited. The dissociation constants of GroEL complexed with the kinetic intermediates of the proteins studied were evaluated, and a simple mechanism of the functioning of GroEL as a molecular chaperone was proposed.

Interaction of 4,4'-dithiodipyridine with Cys(458) triggers disassembly of GroEL.

Chaperonin GroEL, consisting of two seven-subunit rings stacked back-to-back, is disassembled by interaction of 4, 4'-dithiodipyridine (DTP) with Cys(458) located close to the intersubunit contacts within and between the rings. The thiol group of Cys(458) is inaccessible to the probe being buried into the pocket locked by segment Asn(475)-Asn(487). Flexibility of this segment is proposed to induce the "open" state of the pocket and accommodate the bulky probe inside so that the consequential irreversible shifts in the pocket constituents disassemble GroEL. This scheme is supported by the finding that DTP-induced disassembly of GroEL is facilitated by ATP, which specifically stimulates a local shift of the segment Asn(475)-Asn(487) into solution.

Targeting of GroEL to SecA on the cytoplasmic membrane of Escherichia coli.

Chaperonin GroEL has been found to interact with isolated cytoplasmic membrane of Escherichia coli. Interaction requires Mg ions, whereas MgATP inhibits, and inhibition is stronger in the presence of co-chaperonin GroES. "Heat-shock" of the membrane at 45 degrees C destroys irreversibly its ability to bind GroEL. The binding of GroEL is characterized by saturation with a maximum of about 100 pmol GroEL bound per mg of total membrane protein, indicating a limited capacity and specificity of the membrane to bind GroEL. According to results of immunoblotting analysis and cleavable photoactivable cross-linking, a membrane target of GroEL is SecA, a protein known as a central component of the translocation machinery. Moreover, in some cases GroEL could modulate a cycle of association of SecA with the membrane by stimulating release of SecA from the membrane. A physiological role of targeting of GroEL in or close to the protein-conducting membrane apparatus is discussed.

Chaperonin-promoted post-translational membrane insertion of a multispanning membrane protein lactose permease.

Using an in vitro membrane-free translation system from Escherichia coli, it is shown that chaperonin GroEL added cotranslationally interacts with newly synthesized lactose permease (LacY), a polytopic membrane protein, thereby preventing aggregation. Subsequently, when the isolated GroEL-LacY complex is incubated with inverted membrane vesicles, the permease is inserted into the membrane in a MgATP-dependent manner. Post-translational membrane insertion is also observed when aggregation of newly synthesized LacY is prevented by addition of the nonionic detergent n-dodecyl-beta,D-maltoside during translation in place of GroEL. No membrane integration occurs with right-side-out vesicles, indicating that LacY interacts specifically only with the cytosolic face of the membrane. Ligand thiodigalactoside protection against alkylation of the Cys-148 residue in the permease shows proper post-translational insertion. Moreover, limited proteolysis of soluble LacY either complexed with GroEL or in detergent indicates that the newly synthesized protein assumes a conformation that is comparable to that of native, membrane-embedded permease prior to insertion into the membrane.

On the distribution of ligands within the asymmetric chaperonin complex, GroEL14.ADP7.GroES7.

In the presence of MgATP or MgADP the E. coli chaperonin proteins, GroEL and GroES, form a stable asymmetric complex with a stoichiometry of two GroEL7:one GroES7: seven MgADP. The distribution of the ligands between the two heptameric GroEL rings is crucial to our understanding of the mechanism of chaperonin-assisted folding, being either cis (i.e. [GroEL7.MgADP7.GroES7]-[GroEL7]) or trans (i.e. [GroEL7.MgADP7]-[GroEL7.GroES7]. On the basis of cross-linking experiments with 8-azido-ATP and the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), it was suggested that GroES and MgADP are bound to the same GroEL ring which resists proteinase K digestion [Nature 366 (1993) 228-233]. However, we find that the SPDP-promoted cross linking of GroES and GroEL occurs in the absence of Mg2+, ADP or ATP, which are required for the formation of the asymmetric complex. Cross-linking is shown to occur only when the SPDP-modified GroES is co-precipitated with GroEL by trichloracetic acid. Furthermore, there are structural grounds for questioning whether SPDP can crosslink, in a physiologically relevant manner, an amino group of GroES with any of the cysteinyl groups of GroEL.

ATP induces non-identity of two rings in chaperonin GroEL.

For its function, the Escherichia coli chaperonin GroEL requires the presence of ATP and co-chaperonin GroES. We have observed that ADP displays a two-step inhibition of GroEL-dependent ATP hydrolysis, wherein one-half of the GroEL ATPase sites is strongly inhibited by ADP while the other half is affected very mildly. It is suggested that interaction with ATP induces structural and functional differences between two initially identical rings in GroEL (inter-ring negative cooperativity) and that the subsequent binding of GroES occurs to the ring that is occupied first by ATP in a positively cooperative manner.

Chaperonins as potential gene regulatory factors. In vitro interaction and solubilization of NifA, the nif transcriptional activator, with GroEL.

A previous study (Govezensky, D., Greener, T., and Zamir, A. (1991) J. Bacteriol. 20, 6339-6346) indicated that the chaperonin GroEL was required for maximal expression from nif promoters in Klebsiella pneumoniae and nif-transformed Escherichia coli. That this requirement stemmed from the ability of GroEL to properly fold NifA, the nif transcriptional activator, was first supported by co-immunoprecipitation of NifA in K. pneumoniae extracts with anti-GroEL antibodies. In the present in vitro study, NifA, partially purified from E. coli overexpressing the protein, was diluted from a 6 M urea solution into a refolding buffer in the presence or absence of GroEL. Dilution in the absence of GroEL caused the complete precipitation of NifA. When present in the dilution buffer, GroEL bound NifA and maintained it in a soluble state. GroEL was also found to bind NifA newly synthesized in an in vitro translation system. For both NifA preparations, cochaperonin GroES and ATP promoted release of NifA from GroEL. These results provide evidence for the association of NifA with GroEL and for the role of both GroEL and GroES in the solubilization and thereby folding of the nif transcriptional activator.

Prediction of an inter-residue interaction in the chaperonin GroEL from multiple sequence alignment is confirmed by double-mutant cycle analysis.

A search for co-ordinated amino acid changes in the hsp60 family of chaperonins suggested that cysteine residues at positions 137 and 518 in the Escherichia coli chaperonin GroEL may interact with each other. In order to determine whether this interaction indeed exists we constructed a double-mutant cycle comprising wild-type GroEL, the single mutants Cys137-->Ser and Cys518-->Ser and the corresponding double mutant. The effects of the two mutations on the function of GroEL, in assisting the refolding of a non-folded protein substrate (rhodanese), are shown to be non-additive. It is also shown that ADP by itself specifically destabilizes the Cys518-->Ser mutant GroEL particle with this effect being suppressed in the double mutant. The observed pattern of co-ordinated mutations in the hsp60 family of chaperonins is thus shown to reflect a real interaction, though most likely indirect, between Cys137 and Cys518 in GroEL. Our study demonstrates that patterns of co-ordinated mutations combined with double-mutant cycle analysis can provide structural information on interactions in a protein without an available three-dimensional structure at atomic resolution.

Direct demonstration that ATP is in contact with Cys-137 in chaperonin GroEL.

The nonhydrolyzable ATP analogue ATP gamma S (adenosine 5'-3-O-(thio)triphosphate) is affinity cross-linked to GroEL by formation of a disulfide bridge in a peroxide-promoted reaction. By replacing with serine each of 3 cysteine residues in GroEL, it is shown that ATP gamma S specifically cross-links to Cys-137. It is thus demonstrated that the ATP bound to GroEL is in direct contact with Cys-137.

Mutation Ala2-->Ser destabilizes intersubunit interactions in the molecular chaperone GroEL.

The mutation Ala2-->Ser in the molecular chaperone GroEL increases positive co-operativity in ATP hydrolysis, as reflected by a change in the Hill coefficient from 2.36(+/- 0.23) for wild-type to 3.19(+/- 0.17) for the mutant. This amino acid replacement destabilizes the oligomeric structure of GroEL. It is shown that adenine nucleotides also have a specific destabilizing effect which is more pronounced in the case of the Ala2-->Ser mutant. Addition of GroES or the non-folded protein ligand rhodanese blocks the destabilizing effect of adenine nucleotides for both wild-type and mutant. The results are interpreted using the Monod-Wyman-Changeux (MWC) model for co-operativity.

The N terminus of the molecular chaperonin GroEL is a crucial structural element for its assembly.

The Escherichia coli heat-shock protein GroEL is a member of the highly conserved family of tetradecameric chaperonins 60, which assist in the folding and assembly of other proteins. Using site-directed mutagenesis, it is shown that replacement of the absolutely conserved amino acid residue Lys-3 by arginine or isoleucine destabilizes the GroEL particle and that the replacement Lys-3-->Glu completely blocks its formation. The rank order of effects of these mutations on the stability of the GroEL particle correlates with the associated changes in net charge at that position. Our results show that the N terminus of GroEL is a crucial structural element for its assembly.

The strongly conserved carboxyl-terminus glycine-methionine motif of the Escherichia coli GroEL chaperonin is dispensable.

The universally distributed heat-shock proteins (HSPs) are divided into classes based on molecular weight and sequence conservation. The members of at least two of these classes, the HSP60s and the HSP70s, have chaperone activity. Most HSP60s and many HSP70s feature a striking motif at or near the carboxyl terminus which consists of a string of repeated glycine and methionine residues. We have altered the groEL gene (encoding the essential Escherichia coli HSP60 chaperonin) so that the protein produced lacks its 16 final (including nine gly, and five met) residues. This truncated product behaves like the intact protein in several in vitro tests, the only discernible difference between the two proteins being in the rate at which ATP is hydrolysed. GroELtr can substitute for GroEL in vivo although cells dependent for survival on the truncated protein survive slightly less well during the stationary phase of growth. Elevated levels of the wild-type protein can suppress a number of temperature-sensitive mutations; the truncated protein lacks this ability.

A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL.

To facilitate folding and assembly of different proteins, chaperonin GroEL requires the presence of its helper protein GroES. Using a photochemical cross-linking approach, we show that GroES and newly synthesized pre-beta-lactamase (pre-beta lac) contact with each other only within the ternary complex with GroEL. Possibly owing to this contact GroES is able to directly influence the pre-beta lac/GroEL interaction. Furthermore, the cross-linking of pre-beta lac to GroES suggests that the binding of the protein ligands to GroEL occurs near the GroES binding site, known to be in the central hole space of GroEL.

Positive cooperativity in the functioning of molecular chaperone GroEL.

In the presence of its partner, GroES, the tetradecameric molecular chaperone GroEL binds 14 ATP molecules, half of which are hydrolyzed in a cooperative manner. Moreover GroEL can bind, with a positive cooperativity, more than two molecules of nonfolded protein rhodanese. The role of the cooperative mechanism in the functioning of GroEL is discussed.

(Mg-ATP)-dependent self-assembly of molecular chaperone GroEL.

The important Escherichia coli heat-shock protein GroEL of relative molecular mass 57,259 is a typical molecular chaperone. It possesses ATPase activity and interacts in ATP-driven reactions with non-folded proteins to stimulate their correct folding and/or assembly by preventing the formation of improper protein structures or aggregates. As GroEL is isolated and functions as a 20-25S tetradecameric particle (GroELp), the question arises--what is the mechanism of its own assembly? Here we show the (Mg-ATP)-dependent self-stimulation ('self-chaperoning') in vitro of GroELp reassembly from its monomeric state.

Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein.

It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion. Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli. Dissociation of these complexes is ATP-dependent. The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.

The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle.

Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides.

Localization of elongation factor Tu on the ribosome.

The EF-Tu-binding center of the E. coli ribosome has been localized by immunoelectron microscopy after cross-linking of the specific EF-Tu X 70 S ribosomal complex with dimethylsuberimidate. EF-Tu has been found to be in contact with the 50 S subunit in the region of the L7/L12 stalk and with the 30 S subunit in the upper part of its body on the side opposite the top of the ledge (the platform). The EF-Tu position on a model of the 70 S ribosome is presented.