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The insulin and insulin-like growth factor-1 (IGF-1) receptors mediate signaling for energy uptake and growth through insulin receptor substrates (IRSs), which interact with these receptors as well as with downstream effectors. Oxygen is essential not only for ATP production through oxidative phosphorylation but also for many cellular processes, particularly those involved in energy homeostasis. The oxygen tension in vivo is significantly lower than that in the air and can vary widely depending on the tissue as well as on perfusion and oxygen consumption. How oxygen tension affects IRSs and their functions is poorly under- stood. Our findings indicate that transient hypoxia (i% oxygen) leads to caspase-mediated cleavage of IRS-1 without inducing cell death. The IRS-1 protein level rebounds rapidly upon return to normoxia. Protein tyrosine phosphatases (PTPs) appear to be important for the IRS-1 cleavage because tyrosine phosphorylation of the insulin receptor was decreased in hypoxia and IRS-1 cleavage could be blocked either with H2O2 or with vanadate, each of which inhibits PTPs. Activity of Akt, a downstream effector of insulin and IGF-1 signaling that is known to suppress caspase activation, was suppressed in hypoxia. Overexpression of dominant-negative Akt led to IRS-1 cleavage even in normoxia, and overexpression of constitutively active Akt partially suppressed IRS-1 cleavage in hypoxia, suggesting that hypoxia- mediated suppression of Akt may induce caspase-mediated IRS-1 cleavage. In conclusion, our study elucidates a mechanism by which insulin and IGF-1 signaling can be matched to the oxygen level that is available to support growth and energy metabolism. [ABSTRACT FROM AUTHOR]

The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-lace forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s20,w 7.0 S; Mapp 169, 200) at concentrations ≤3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a Mapp of 1 ⊗ 106. Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles. [ABSTRACT FROM AUTHOR]

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A1AO-ATP synthases use Na+ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A1AO-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80°C and still retained an activity of 2.5 units/mg of protein at 45°C. The high activity of the enzyme at 45°C opened, for the first time, a way to directly measure ion transport in an A1AO-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45°C and coupled ATP hydrolysis to primary and electrogenic Na+ transport. This is the first proof of Na+ transport by an A1AO-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na+ in the bioenergetics of archaea. [ABSTRACT FROM AUTHOR]