No abstract

The heat-inducible members of the Hsp100 (or Clp) family of proteins share a common function in helping organisms to survive extreme stress, but the basic mechanism through which these proteins function is not understood. Hsp104 protects cells against a variety of stresses, under many physiological conditions, and its function has been evolutionarily conserved, at least from Saccharomyces cerevisiae to Arabidopsis thaliana. Homology with the Escherichia coli ClpA protein suggests that Hsp104 may provide stress tolerance by helping to rid the cell of heat-denatured proteins through proteolysis. But genetic analysis indicates that Hsp104 may function like Hsp70 as a molecular chaperone. Here we investigate the role of Hsp104 in vivo using a temperature-sensitive Vibrio harveyi luciferase-fusion protein as a test substrate. We find that Hsp104 does not protect luciferase from thermal denaturation, nor does it promote proteolysis of luciferase. Rather, Hsp104 functions in a manner not previously described for other heat-shock proteins: it mediates the resolubilization of heat-inactivated luciferase from insoluble aggregates.

Hsp104 is crucial for stress tolerance in Saccharomyces cerevisiae, and both of its nucleotide-binding domains (NBD1 and NBD2) are required. Here, we characterize the ATPase activity and oligomerization properties of wildtype (WT) Hsp104 and of NBD mutants. In physiological ionic strength buffers (pH 7.5, 37°C) WT Hsp104 exhibits Michaelis-Menten kinetics between 0.5 and 25 mM ATP (K m ϳ5 mM, V max ϳ2 nmol min ؊1 g ؊1). ATPase activity is strongly influenced by factors that vary with cell stress (e.g. temperature, pH, and ADP). Mutations in the P-loop of NBD1 (G217V or K218T) severely reduce ATP hydrolysis but have little effect on oligomerization. Analogous mutations in NBD2 (G619V or K620T) have smaller effects on ATPase activity but impair oligomerization. The opposite relationship was reported for another member of the HSP100 protein family, the Escherichia coli ClpA protein, in studies employing lower ionic strength buffers. In such buffers, the K m of WT Hsp104 for ATP hydrolysis decreased 10-fold and its stability under stress conditions increased, but the effects of the NBD mutations on ATPase activity and oligomerization remained opposite to those of ClpA. Either the functions of the two NBDs in ClpA and Hsp104 have been reversed or both contribute to ATP hydrolysis and oligomerization in a complex manner that can be idiosyncratically affected by such mutations.

No abstract

Using the amino-terminal domain of k repressor as a model system, we show that residues in an unstructured region at the extreme carboxyl terminus of the protein are important for determining its proteolytic susceptibility in Escherichia coil Nonpolar amino acids are destabilizing when placed at the 5 carboxy-terminal residue positions, whereas charged and polar residues are stabilizing. The stabilizing effect of a single charged residue is greatest when it is at the terminal position and diminishes with increasing distance from the carboxyl terminus. The position of destabilizing sequences with respect to the free carboxyl terminus is important for their effect, but their distance from the folded portion of the protein is not important. Specific degradation of proteins with nonpolar carboxyl termini has been reconstituted in vitro using a partially pure, soluble fraction. This degradation is not ATP-dependent. Moreover, amino-terminal domain variants with nonpolar carboxyterminal residues are still rapidly degraded in strains that are deficient in proteolysis of abnormal proteins. These data suggest that the degradation of amino-terminal domain variants with nonpolar carboxy-terminal residues involves proteolytic components distinct from those known to be important for the turnover of unfolded proteins in E. coil

To test the idea that unfolded protein might act as an intracellular signal for induction of the heat shock response in Escbericbia coli, we examined the synthesis of several heat shock proteins after expression of an unfolded variant of the amino-terminal domain of X repressor. These experiments show that expression of a single mutant protein, and not its wild-type counterpart, is sufficient to induce a heat shock-like response. In addition, by measuring the abilities of unfolded variants of differing proteolytic susceptibilities to induce heat shock protein synthesis and by monitoring heat shock protein synthesis as a function of the amount of a single unfolded protein, we show that it is the concentration of unfolded protein in the cell, and not its degradation, that is important for inducing the heat shock-like response.

The phenotypes of single HsplO4 and Hsp7O mutants of the budding yeast Saccharomyces cerevisiae provide no clue that these proteins are functionally related. Mutation of the HSP104 gene severely reduces the ability of cells to survive short exposures to extreme temperatures (thermotolerance) but has no effect on growth rates. On the other hand, mutations in the genes that encode Hsp7O proteins have significant effects on growth rates but do not reduce thermotolerance. The absence of a thermotolerance defect in S. cerevisiae Hsp7O mutants is puzzling, since the protein clearly plays an important role in thermotolerance in a variety of other organisms.

Relaxin is a 6-kDa peptide of the insulin family that is present at increased levels in the circulation during pregnancy. Its functions at that time are thought to include maintenance of myometrial quiescence, regulation of plasma volume, and release of neuropeptides, such as oxytocin and vasopressin. The protein also promotes connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis in various mammalian species. In this report, we provide evidence for a novel target of relaxin, the human monocytic cell line, THP-1. Relaxin bound with high affinity (K d ‫؍‬ 102 pM) to a specific receptor on THP-1 cells. Receptor density was low (ϳ275 receptors/cell), but binding of relaxin triggered intracelluar signaling events. Receptor density was not modulated by pretreatment with estrogen, progesterone, or a number of other agents known to induce differentiation of THP-1 cells. Cross-linking studies showed radiolabeled relaxin bound primarily to cell surface proteins with an apparent molecular mass of >200 kDa. Other members of the insulin-like family of proteins (insulin, insulin-like growth factors I and II, and relaxin-like factor) were unable to displace the binding of relaxin to THP-1 cells, suggesting that a distinct receptor for relaxin exists on this monocyte/macrophage cell line.Relaxin is a 6-kDa polypeptide with basic tertiary structural fold nearly identical to that of insulin (1). However, analysis of chemically synthesized relaxin derivatives has shown that the receptor-binding residues of the two proteins are discrete (2) and, although information is very limited (3, 4), the intracellular signaling events triggered when relaxin binds its receptor appear to be different from those triggered by insulin receptor binding. Consequently, it is not surprising that the physiological functions of relaxin are quite distinct from those of insulin and the other members of the insulin superfamily. Discovered in the 1920s, relaxin has been classically thought of as a "hormone of pregnancy" since it is present in the circulation at increased levels during pregnancy, when its synthesis is primarily directed by the corpus luteum. It serves to promote connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis, and also acts to maintain quiescence of the myometrium (5). In addition, relaxin binding sites identified in specific areas of the brain (6) and heart (7) point to a role for it in cardiovascular function, perhaps in setting the critical thresholds for plasma volume in pregnancy (8). Relaxin is likely to play an important role in male reproductive functions as well. The protein is synthesized in the prostate, and has been shown, in vitro, to increase the motility and egg penetrating ability of sperm (9). Relaxin probably possesses non-reproductive functions as well. The identification of sites of relaxin mRNA synthesis, as well as relaxin binding sites, in the brain and heart of both males and females suggests that in those organs, the pro...