Cellular dopamine is increased following exposure to a factor derived from immortalized striatal neurons [corrected].

A factor obtained from an immortalized hybrid monoclonal cell line (X61) of striatal origin is capable of increasing the dopamine content of hybrid, monoclonal cells expressing a dopaminergic phenotype (MN9D) and of aggregate cultures containing primary dopaminergic neurons. The factor is a protein smaller than 100 kDa and appears to be different than a number of other trophic agents with effects on the dopaminergic neuron. The effect on dopamine content appears to be specific to centrally-derived neuronal elements; the protein having no effect on the dopamine content of PC12 cells. Given that parkinsonian symptoms are only apparent following degeneration of a substantial portion of the mesencephalic dopaminergic cell population, activity capable of increasing dopamine content of the surviving cells may represent an interesting candidate therapeutic agent.

Purification of a 38-kDa protein from rabbit reticulocyte lysate which promotes protein renaturation by heat shock protein 70 and its identification as delta-aminolevulinic acid dehydratase and as a putative DnaJ protein.

We reported recently that a rabbit reticulocyte 66-kDa protein (termed RF-hsp 70 by our laboratory and p60 and hop by others) functions as a hsp 70 recycling protein and markedly enhances the renaturation of luciferase by hsp 70 (Gross, M., and Hessefort, S. (1996) J. Biol. Chem. 271, 16833-16841). In this report, we confirm that the ability of RF-hsp 70 to promote the conversion of hsp 70. ADP to hsp 70.ATP, thus enhancing the protein folding activity of hsp 70, is caused by the purified 66-kDa protein and not by a trace DnaJ/hsp 40 protein contaminant. To determine the relationship between RF-hsp 70 and the DnaJ/hsp 40 heat shock protein family, which also enhances protein renaturation by hsp 70, we purified a 38-kDa protein from rabbit reticulocyte lysate based upon its ability to stimulate renaturation of luciferase by hsp 70. Partial amino acid sequencing of this 38-kDa protein has indicated, unexpectedly, that it is the enzyme delta-aminolevulinic acid dehydratase (ALA-D) and that it does not contain detectable sequences corresponding to the DnaJ/hsp 40 protein family. In addition, immunoblot analysis with a polyclonal antibody made to HeLa cell hsp 40 (from StressGen) confirms that our purified ALA-D contains no hsp 40, although hsp 40 is present in relatively crude rabbit reticulocyte protein fractions. Rabbit reticulocyte ALA-D is about as active in converting delta-aminolevulinic acid to porphobilinogen and as Zn2+-dependent as ALA-D purified from other sources. Rabbit reticulocyte ALA-D stimulates the renaturation of luciferase by hsp 70 up to 10-fold at concentrations that are the same as or less than that of hsp 70, and it has no renaturation activity in the absence of hsp 70. The renaturation effect of ALA-D is additive with that of RF-hsp 70 at limiting or saturating concentrations of each, and, unlike RF-hsp 70, ALA-D does not promote the dissociation of hsp 70.ADP in the presence of ATP. The renaturation-enhancing effect of ALA-D may be caused by a region near its carboxyl terminus which has sequence homology to the highly conserved domain of the DnaJ protein family, which is similar to the sequence homology between this domain and a carboxyl-terminal region in auxilin, a DnaJ-like protein that requires this region for its hsp 70-dependent function (Ungewickell, E., Ungewickell, H., Holstein, S. E. H., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L. E., and Eisenberg, E. (1995) Nature 378, 632-635).

Extensive sequencing of tryptic peptides of a rabbit reticulocyte 66-kDa protein that promotes recycling of Hsp 70. Homology To stress-related proteins.

Trypsinization and sequence analysis of the 66-kDa rabbit reticulocyte protein (RF-hsp 70), shown in the preceding article to function as a recycling protein for hsp 70, demonstrates striking similarity to the transformation-sensitive human protein IEF SSP 3521 (Honoré, B., Leffers, H., Madsen, P., Rasmussen, H. H., Vandekerckhove, J., and Celis, J. E.(1992) J. Biol. Chem. 267, 8485-8491) and mouse extendin (Blatch, G. L., Lassle, M., Takatori, T., Gandhi, T., Kundra, V., and Zetter, B. R.(1995) Proc. Am. Assoc. Cancer Res. 36, 68). The human and mouse proteins share 97% sequence identity, and sequencing of 20 polypeptides (225 residues) from RF-hsp 70 reveals only 10 differences between the rabbit and human proteins and 13 differences between the rabbit and mouse proteins (96 and 94% identity, respectively). In addition, all three proteins are of similar size, and each contains 11 cysteines. These findings strongly suggest that these three proteins are homologs of the same activity. All differences (but one) between the human and mouse proteins occur within the amino-terminal half of the protein, and there is only one difference among 121 sequenced residues between RF-hsp 70 and the human or mouse protein which occurs within the carboxyl-terminal 70% of the molecule. In addition, where partial sequences of RF-hsp 70 and p60, a chick oviduct protein that shows 70% identity to the human protein (Smith, D. F., Sullivan, W. P., Marion, T. N., Zaitsu, K., Madden, B., McCormick, D. J., and Toft, D. O. (1993) Mol. Cell. Biol. 13, 869-876), overlap (a total of 54 residues), RF-hsp 70 and chick p60 show 78% sequence identity. Studies of the initial digestion of RF-hsp 70 by trypsin indicate that it is first converted to 58- and 54-kDa components, each of which is then converted to a 43-kDa polypeptide. This 43-kDa component is located in the human and mouse proteins at position 124 to about 470. It is converted subsequently to a 31-kDa polypeptide by trypsin hydrolysis at position 207. This 31-kDa component is finally split into 17- and 14-kDa polypeptides that are located at positions 208 to approximately 351 and 352 to approximately 470, respectively. The 14-kDa polypeptide is relatively resistant to further digestion with trypsin, and seven tryptic peptides from other parts of RF-hsp 70 contain internal lysine and/or arginine residues (as do several tryptic peptides produced from IEF SSP 3521 and chick p60). Both features may be due to interference with trypsin action by secondary structure in the protein, since trypsinization of reduced and carboxymethylated RF-hsp 70 results in hydrolysis of the 14-kDa polypeptide and reduces the level of peptides that contain internal lysine and/or arginine, although it does not eliminate them.

Purification and characterization of a 66-kDa protein from rabbit reticulocyte lysate which promotes the recycling of hsp 70.

We have purified to apparent homogeneity a 66-kDa protein from rabbit reticulocyte lysate which is associated with hsp 70. Our characterization of this 66-kDa protein demonstrates that its physiological role is to promote the recycling of hsp 70 by catalyzing the dissociation of hsp 70-bound ADP in exchange for ATP. We have therefore termed the 66-kDa protein RF-hsp 70, a recycling factor for hsp 70. RF-hsp 70 promotes stoichiometric binding of ATP to hsp 70, and it increases about 5-fold the rate of dissociation of hsp 70.ADP in the presence of ATP. This process represents adenine nucleotide exchange, since dissociation of ADP does not occur unless ATP is added; dATP, GTP, and ITP cannot substitute for ATP. The mechanism of action of RF-hsp 70 is to lower the KD of hsp 70 for ATP about 6-7-fold to a value that is close to the KDof hsp 70 for ADP. RF-hsp 70 also stimulates the ATPase activity of hsp 70, including the 42-kDa amino-terminal portion of hsp 70 generated by chymotrypsin, demonstrating that RF-hsp 70 interacts with that part of hsp 70 known to contain the ATP/ADP binding site. Confirming its recycling function, RF-hsp 70 stimulates about 7-10-fold the ability of hsp 70 to reactivate heat-denatured firefly luciferase. In addition, RF-hsp 70 acts catalytically to recycle hsp 70, since, at 0.2 times the molar concentration of hsp 70, RF-hsp 70 increases the rate of renaturation of luciferase by hsp 70 about 3-4-fold. The action of RF-hsp 70 is also partially species-specific since it is most effective with rabbit reticulocyte hsp 70, less effective with bovine brain hsp 70, even less effective with human hsp 70, and ineffective with broad bean hsp 70.

Control of protein synthesis by hemin. Purification of a rabbit reticulocyte hsp 70 and characterization of its regulation of the activation of the hemin-controlled eIF-2(alpha) kinase.

We have purified a soluble rabbit reticulocyte protein, previously termed the supernatant factor, that reverses the inhibition of protein synthesis in hemin-deficient lysate by promoting the inactivation of the hemin-controlled eIF-2 alpha kinase (HCR) mediating the effect of hemin deficiency. We have identified the supernatant factor as a member of the heat shock protein 70 family, denoted hsp 70(R), based upon its size (72 kDa), specific reaction to a monoclonal antibody against eukaryotic hsp 70, strong binding affinity for ATP, and endogenous ATPase activity. We have investigated the role of hsp 70(R) and hemin in the regulation of the activation of HCR from its latent precursor (ProHCR) and the translational control of protein synthesis in rabbit reticulocyte lysate. We find that autophosphorylation of Pro-HCR is reduced by about 75% by adding saturating hsp 70(R) and almost completely reduced by adding either saturating hemin or limiting hemin plus limiting hsp 70(R). In contrast, autophosphorylation of HCR, which is similar in magnitude to that of ProHCR, is unaffected by adding either saturating hsp 70(R), saturating hemin, or limiting amounts of both. The activation of HCR (measured by inhibition of protein synthesis) from isolated ProHCR is completely prevented by hsp 70(R) in the presence, but not absence, of dithiothreitol. This suppression appears to be due to the association of hsp 70(R) with ProHCR, since hsp 70(R) action is prevented by ATP/Mg2+ and because activation of HCR from less purified ProHCR, that has associated hsp 70(R), is suppressed by dithiothreitol alone. This association is confirmed by sucrose gradient centrifugation, which shows co-sedimentation of some hsp 70(R) with ProHCR following preincubation together that is prevented by ATP/Mg2+ and does not occur after conversion of ProHCR to HCR. Limiting hsp 70(R) reduces the concentration of hemin required to prevent activation of HCR from isolated ProHCR from 0.75 to 0.15 microM and the optimal hemin concentration needed to maintain protein synthesis in reticulocyte lysate from 25 to 10 microM. Limiting hsp 70(R) also allows the delayed addition of hemin to suppress activation of HCR from ProHCR and to reverse inhibition of protein synthesis in hemin deficient lysate. The association of hsp 70(R) with ProHCR also underlies the observation that much more protein is synthesized in reticulocyte lysate in the absence of hemin at 25 degrees C than at temperatures of 30 degrees C or greater. These observed effects may be specific to hsp 70(R), since they are not observed with rabbit reticulocyte eIF-2 or eIF-2B, and since the comparable hsp 70 from bovine brain is incapable of maintaining or restoring protein synthesis in hemin-deficient lysate.

The conversion of eIF-2.GDP to eIF-2.GTP by eIF-2B requires Met-tRNA(fMet).

We have investigated why the recycling of eIF-2.GDP to eIF-2.GTP, mediated by the guanine nucleotide exchange factor eIF-2B, is rapid in rabbit reticulocyte lysate, reconstituted for optimal protein synthesis, but slow in an isolated reaction with purified eIF-2B. We have found that purified eIF-2B dissociates eIF-2.[3H]GDP as efficiently in the presence of GTP as it does in the presence of GDP provided Met-tRNA(fMet) is added. tRNA(fMet) is ineffective, and there is no Met-tRNA(fMet) requirement for exchange with GDP. Exchange of eIF-2 bound GDP for GTP is completely dependent upon Met-tRNA(fMet) in the presence of ATP, suggesting that under physiological conditions efficient recycling of eIF-2.GDP to eIF-2.GTP requires conversion of the latter, a relatively unstable complex, to a more stable Met-tRNA(fMet).eIF-2.GTP complex.