Phosphorylation of Drosophila heat shock transcription factor.

The role that phosphorylation plays in regulating heat shock factor (HSF) function and activity has been the subject of several studies. Here, we demonstrate that Drosophila melanogaster HSF (DmHSF) is a phosphoprotein that is multiply phosphorylated at some sites and is dephosphorylated at others upon heat shock. However, the steady-state level of phosphorylation of Drosophila HSF remains unchanged after heat shock. Phosphoamino-acid analysis reveals that predominantly serine residues are phosphorylated for both the non-shocked and heat shocked molecules. Gel mobility shift assays using extracts from SL2 cells treated with a variety of phosphatase and kinase inhibitors show little or no effect on the heat shock induced DNA binding activity of HSF or on its recovery. We conclude that phosphorylation plays no significant role in regulating the heat induced DNA binding activity of Drosophila HSF.     

Hsp85 conformational change within the heat shock temperature range.

One of the major mammalian heat shock proteins, hsp85, aggregates extensively when heated in the presence of non-ionic detergents (J Cell. Physiol. 140: 601-607, 1989). The present study used intrinsic fluorescence and susceptibility to tryptic proteolysis to probe hsp85 conformation within the physiological and heat shock temperature ranges. Fluorescence intensity decreased and the emission spectrum was red-shifted (2.5 nm) as hsp85 was heated from 15 degrees to 50 degrees C. Upon heating in the absence of detergent, the red shift, monitored by the ratio of fluorescence emission at 330 nm to that at 350 nm, began at 38 degrees-45 degrees C with a transition midpoint at 45 degrees-50 degrees C, depending on the rate of temperature increase. This transition was masked by 1% n-octyl-O-glucoside - a detergent previously shown to promote aggregation. The spectral changes were not reversible upon cooling to 15 degrees C. Susceptibility to proteolysis in the absence of detergent, measured by the degradation of characteristic large fragments, increased sharply between 40 degrees C and 45 degrees C. These findings suggest that hsp85 undergoes a major conformational change within the range of temperatures known to induce hsp synthesis. This change is consistent with partial unfolding which exposes additional sites to the aqueous environment and influences detergent binding.     

Gamma-secretase activity is associated with a conformational change of nicastrin

Gamma-secretase is a high molecular weight multicomponent protein complex with an unusual intramembrane-cleaving aspartyl protease activity. Gamma-secretase is intimately associated with Alzheimer disease because it catalyzes the proteolytic cleavage, which leads to the liberation of amyloid beta-peptide. At least presenilin (PS), Nicastrin (Nct), APH-1, and PEN-2 are constituents of the gamma-secretase complex, with PS apparently providing the active site of gamma-secretase. Expression of gamma-secretase complex components is tightly regulated, however little is known about the assembly of the complex. Here we demonstrate that Nct undergoes a major conformational change during the assembly of the gamma-secretase complex. The conformational change is directly associated with gamma-secretase function and involves the entire Nct ectodomain. Loss of function mutations generated by deletions failed to undergo the conformational change. Furthermore, the conformational alteration did not occur in the absence of PS. Our data thus suggest that gamma-secretase function critically depends on the structural "activation" of Nct.     

Cooperative binding of Drosophila heat shock factor to arrays of a conserved 5 bp unit

Drosophila heat shock factor (HSF) exists as a multimer in solution and when bound to its regulatory element (HSE). We have previously reported evidence that subunits of HSF associate to form homotrimers and that each subunit contacts a conserved 5 bp DNA sequence repeated within an HSE. Here we show that HSF binding is highly cooperative at two distinct levels: between subunits of the HSF multimer, and between multimers. The binding of HSF to one of a pair of adjacent trimeric binding sites facilitates HSF binding to the second by over 2000-fold. This cooperativity is particularly important in binding HSF at 37 degrees C, and could account for the requirement for multiple binding sites in vivo and, in part, for the differential expression of heat shock genes.     

An alteration in molecular form associated with activation of human heat shock factor

In higher eucaryotes, heat shock factor (HSF) exists in a cryptic form in unstressed cells. We investigated molecular forms of human HSF before and after activation by sucrose density gradient centrifugation and by gel mobility shift assay using a 32P-labeled heat shock element (HSE). We found that the in vivo or in vitro activated HSF, which is capable of binding to HSE, and its inactive form present in unstressed cells have different sedimentation coefficient; the former is 8 S whereas the latter is 4-5 S. Both the 8 S and 4-5 S forms contain the HSF polypeptide which has the ability to bind to HSE upon activation. The inactive 4-5 S form acquires HSE-binding ability when activated by heat shock or other stimuli. This HSF activity was greatly reduced, however, during recentrifugation in sucrose density gradient and, in addition, the residual activity was not recovered in 8 S fractions. Transformation of the inactive 4-5 S form of HSF to the stable, active 8 S form was achieved when the inactive form was activated and mixed with cytosols of unstressed cells.