Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system.

Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^