Micheal E. Barnett

Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB

ClpB is a member of the bacterial protein‐disaggregating chaperone machinery and belongs to the AAA+ superfamily of ATPases associated with various cellular activities. The mechanism of ClpB‐assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of nucleotides, ClpB from Escherichia coli undergoes reversible self‐association that involves protein concentration‐dependent populations of monomers, heptamers, and intermediate‐size oligomers. Under low ionic strength conditions, a heptamer becomes the predominant form of ClpB. In contrast, ATPγS, a nonhydrolyzable ATP analog, as well as ADP stabilize hexameric ClpB. Consistently, electron microscopy reveals that ring‐type oligomers of ClpB in the absence of nucleotides are larger than those in the presence of ATPγS. Thus, the binding of nucleotides without hydrolysis of ATP produces a significant change in the self‐association equilibria of ClpB: from reactions supporting formation of a heptamer to those supporting a hexamer. Our results show how ClpB and possibly other related AAA+ proteins can translate nucleotide binding into a major structural transformation and help explain why previously published electron micrographs of some AAA+ ATPases detected both six‐ and sevenfold particle symmetry.

The N-terminal domain of Escherichia coli ClpB enhances chaperone function

ClpB/Hsp104 collaborates with the Hsp70 system to promote the solubilization and reactivation of proteins that misfold and aggregate following heat shock. In Escherichia coli and other eubacteria, two ClpB isoforms (ClpB95 and ClpB80) that differ by the presence or absence of a highly mobile 149‐residues long N‐terminus domain are synthesized from the same transcript. Whether and how the N‐domain contributes to ClpB chaperone activity remains controversial. Here, we show that, whereas fusion of a 20‐residues long hexahistidine extension to the N‐terminus of ClpB95 interferes with its in vivo and in vitro activity, the same tag has no detectable effect on ClpB80 function. In addition, ClpB95 is more effective than ClpB80 at restoring the folding of the model protein preS2‐β‐galactosidase as stress severity increases, and is superior to ClpB80 in improving the high temperature growth and low temperature recovery of dnaK756 ΔclpB cells. Our results are consistent with a model in which the N‐domain of ClpB95 maximizes substrate processing under conditions where the cellular supply of free DnaK–DnaJ is limiting.

Synergistic Cooperation between Two ClpB Isoforms in Aggregate Reactivation

Bacterial AAA+ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in Escherichia coli after heat shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation, and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers, which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their heteroassociation. Our results suggest that the functionality of other AAA+ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.

PKCγ knockout mouse lenses are more susceptible to oxidative stress damage

SUMMARY Cataracts, or lens opacities, are the leading cause of blindness worldwide. Cataracts increase with age and environmental insults, e.g. oxidative stress. Lens homeostasis depends on functional gap junctions. Knockout or missense mutations of lens gap junction proteins, Cx46 or Cx50, result in cataractogenesis in mice. We have previously demonstrated that protein kinase Cγ (PKCγ) regulates gap junctions in the lens epithelium and cortex. In the current study, we further determined whether PKCγ control of gap junctions protects the lens from cataractogenesis induced by oxidative stress in vitro, using PKCγ knockout and control mice as our models. The results demonstrate that PKCγ knockout lenses are normal at 2 days post-natal when compared to control. However, cell damage, but not obvious cataract, was observed in the lenses of 6-week-old PKCγ knockout mice, suggesting that the deletion of PKCγ causes lenses to be more susceptible to damage. Furthermore, in vitro incubation or lens oxidative stress treatment by H2O2 significantly induced lens opacification (cataract) in the PKCγ knockout mice when compared to controls. Biochemical and structural results also demonstrated that H2O2 activation of endogenous PKCγ resulted in phosphorylation of Cx50 and subsequent inhibition of gap junctions in the lenses of control mice, but not in the knockout. Deletion of PKCγ altered the arrangement of gap junctions on the cortical fiber cell surface, and completely abolished the inhibitory effect of H2O2 on lens gap junctions. Data suggest that activation of PKCγ is an important mechanism regulating the closure of the communicating pathway mediated by gap junction channels in lens fiber cells. The absence of this regulatory mechanism in the PKCγ knockout mice may cause those lenses to have increased susceptibility to oxidative damage.