DnaJA1 antagonizes constitutive Hsp70-mediated stabilization of tau

Tau aggregation and amyloidogenesis are common hallmarks for neurodegenerative disorders called tauopathies. The molecular chaperone network constitutes the cellular defense against insults such as tau aggregation. However, chaperone effects on tau are dichotomous. Loss of tau's microtubule-binding activity facilitates an inappropriate chaperone interaction that promotes an amyloidogenic tau conformation. Conversely, other chaperones are capable of promoting tau clearance. Here, we demonstrate that a critical contributor to tau triage is the DnaJ-binding domain of Hsp70 proteins. In particular, over-expression of the constitutive DnaJ, DnaJA1, mediated tau clearance, while knockdown facilitated tau accumulation. This clearance was not specific to distinct pathogenic tau species. The activity of DnaJA1 was attenuated by concomitant increases in Hsp70. Tau reductions facilitated by DnaJA1 were dependent on the integrity of lysines known to be poly-ubiquitinated in human Alzheimer's brain. In vivo, DnaJA1 and tau levels were inversely correlated. The effects of DnaJA1 were partially specific: DnaJA1 reduced the levels of a polyQ protein but had no significant effect on α-synuclein levels. These data suggest that DnaJA1 triages all tau species for ubiquitin-dependent clearance mechanisms. Moreover, the levels of DnaJA1 and Hsp70 seem to play against each other with regard to tau: as DnaJA1 levels increase, tau levels are reduced, but this can be prevented if Hsp70 levels are simultaneously induced. Thus, the DnaJ repertoire possibly represents a powerful set of genetic modifiers for tau pathogenesis. Further investigations could provide new insights about triage decisions that facilitate or prevent amyloidogenesis of tau and other proteins associated with neurodegenerative disease.     

Mitochondrial chaperone DnaJA3 induces Drp1-dependent mitochondrial fragmentation

Mitochondrial morphology is dynamic and controlled by coordinated fusion and fission pathways. The role of mitochondrial chaperones in mitochondrial morphological changes and pathology is currently unclear. Here we report that altered levels of DnaJA3 (Tid1/mtHsp40) a mitochondrial member of the DnaJ protein family, and heat shock protein (Hsp) co-chaperone of matrix 70 kDa Hsp70 (mtHsp70/mortalin/HSPA9), induces mitochondrial fragmentation. Suppression of DnaJA3 induced mitochondrial fragmentation in HeLa cells. Elevated levels of DnaJA3 in normal Hs68 fibroblast cells and HeLa, SKN-SH, U87 and U251 cancer cell lines induces mitochondrial fragmentation. Mitochondrial fragmentation induction was not observed in HeLa cells when other DnaJA family members, or mitochondrial DnaJ protein HSC20, were ectopically expressed, indicating that the effects on mitochondrial morphology were specific to DnaJA3. We show that the DnaJ domain (amino acids 88-168) of DnaJA3 is sufficient for the induction of mitochondrial fragmentation. Furthermore, an H121Q point mutation of the DnaJ domain, which abrogates interaction and activation of mtHsp70 ATPase, eliminates fragmentation induced by DnaJA3. This suggests that DnaJA3 interaction with mtHsp70 may be critical in mitochondrial morphological changes. DnaJA3-induced mitochondrial fragmentation was dependent on fission factor dynamin-related protein 1 (Drp1). Ectopic expression of the mitofusins (Mfn1 and Mfn2), however, does not rescue DnaJA3-induced mitochondrial fragmentation. Lastly, elevated levels of DnaJA3 inducing mitochondrial fragmentation were associated with reduction in cell viability. Taken together, elevated DnaJA3 induces Drp1-depedendent mitochondrial fragmentation and decreased cell viability.     

Activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase during high fat diet feeding

The liver plays a central role in regulating cholesterol homeostasis. High fat diets have been shown to induce obesity and hyperlipidemia. Despite considerable advances in our understanding of cholesterol metabolism, the regulation of liver cholesterol biosynthesis in response to high fat diet feeding has not been fully addressed. The aim of the present study was to investigate mechanisms by which a high fat diet caused activation of liver 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) leading to increased cholesterol biosynthesis. Mice were fed a high fat diet (60% kcal fat) for 5 weeks. High fat diet feeding induced weight gain and elevated lipid levels (total cholesterol and triglyceride) in both the liver and serum. Despite cholesterol accumulation in the liver, there was a significant increase in hepatic HMG-CoA reductase mRNA and protein expression as well as enzyme activity. The DNA binding activity of sterol regulatory element binding protein (SREBP)-2 and specific protein 1 (Sp1) were also increased in the liver of mice fed a high fat diet. To validate the in vivo findings, HepG2 cells were treated with palmitic acid. Such a treatment activated SREBP-2 as well as increased the mRNA and enzyme activity of HMG-CoA reductase leading to intracellular cholesterol accumulation. Inhibition of Sp1 by siRNA transfection abolished palmitic acid-induced SREBP-2 and HMG-CoA reductase mRNA expression. These results suggest that Sp1-mediated SREBP-2 activation contributes to high fat diet induced HMG-CoA reductase activation and increased cholesterol biosynthesis. This may play a role in liver cholesterol accumulation and hypercholesterolemia.     

Diurnal and dietary-induced changes in cholesterol synthesis correlate with levels of mRNA for HMG-CoA reductase

We determined the extent to which diurnal variation in cholesterol synthesis in liver is controlled by steady-state mRNA levels for the rate-limiting enzyme in the pathway, hydroxymethylglutaryl (HMG)-CoA reductase. Rats 30 days of age and maintained on a low-cholesterol diet since weaning were injected intraperitoneally with (3)H(2)O. The specific radioactivity of the whole-body water pool soon became constant, allowing for expression of values for incorporation of label into cholesterol as absolute rates of cholesterol synthesis. In liver, there was a peak of cholesterol synthesis from 8 pm to midnight, a 4-fold increase over synthesis rates from 8 am to noon. Increases in synthesis were quantitatively in lock step with increases in mRNA levels for HMG-CoA reductase occurring 4 h earlier. In a parallel experiment, rats received 1% cholesterol in the diet from weaning to 30 days of age. Basal levels of hepatic cholesterol synthesis were greatly diminished and there was little diurnal variation of cholesterol synthesis or of levels of mRNA for HMG-CoA reductase. Levels of mRNA for the low density lipoprotein receptor and scavenger receptor-B1 (putative high density lipoprotein receptor) showed little diurnal variation, regardless of diet. This suggests that diurnal variation of hepatic cholesterol synthesis is driven primarily by varying the steady-state mRNA levels for HMG-CoA reductase. Other tissues were also examined. Adrenal gland also showed a 4-fold diurnal increase in accumulation of recently synthesized cholesterol. In contrast to liver, however, there was little corresponding change in mRNA expression for HMG-CoA reductase. Much of this newly synthesized cholesterol may be of hepatic origin, imported into adrenal by SR-B1, whose mRNA was up-regulated 2-fold. In brain, there was no diurnal variation in either cholesterol synthesis or mRNA expression, and no influence of high- or low-cholesterol diets on synthesis rates or HMG-CoA reductase mRNA levels.     

A Trypanosoma cruzi heat shock protein 40 is able to stimulate the adenosine triphosphate hydrolysis activity of heat shock protein 70 and can substitute for a yeast heat shock protein 40

The process of assisted protein folding, characteristic of members of the heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) molecular chaperone families, is important for maintaining the structural integrity of cellular protein machinery under normal and stressful conditions. Hsp70 and Hsp40 cooperate to bind non-native protein conformations in a process of adenosine triphosphate (ATP)-regulated assisted protein folding. We have analysed the molecular chaperone activity of the cytoplasmic inducible Hsp70 from Trypanosoma cruzi (TcHsp70) and its interactions with its potential partner Hsp40s (T. cruzi DnaJ protein 1 [Tcj1] and T. cruzi DnaJ protein 2 [Tcj2]). Histidine-tagged TcHsp70 (His-TcHsp70), Tcj1 (Tcj1-His) and Tcj2 (His-Tcj2) were over-produced in Escherichia coli and purified by nickel affinity chromatography. The in vitro basal specific ATP hydrolysis activity (ATPase activity) of His-TcHsp70 was determined as 40 nmol phosphate/min/mg protein, significantly higher than that reported for other Hsp70s. The basal specific ATPase activity was stimulated to a maximal level of 60 nmol phosphate/min/mg protein in the presence of His-Tcj2 and a model substrate, reduced carboxymethylated alpha-lactalbumin. In vivo complementation assays showed that Tcj2 was able to overcome the temperature sensitivity of the ydj1 mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 homologue (yeast DnaJ protein 1, Ydj1). These data suggest that Tcj2 is involved in cytoprotection in a similar fashion to Ydj1, and that TcHsp70 and Tcj2 may interact in a nucleotide-regulated process of chaperone-assisted protein folding.     

Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes

Recently, a novel oxysterol, 5-cholesten-3beta, 25-diol 3-sulfate (25HC3S) was identified in primary rat hepatocytes following overexpression of the cholesterol transport protein, StarD1. This oxysterol was also detected in human liver nuclei. In the present study, 25HC3S was chemically synthesized. Addition of 25HC3S (6 microM) to human hepatocytes markedly inhibited cholesterol biosynthesis. Quantitative RT-PCR and Western blot analysis showed that 25HC3S markedly decreased HMG-CoA reductase mRNA and protein levels. Coincidently, 25HC3S inhibited the activation of sterol regulatory element binding proteins (SREBPs), suggesting that inhibition of cholesterol biosynthesis occurred via blocking SREBP-1 activation, and subsequently by inhibiting the expression of HMG CoA reductase. 25HC3S also decreased SREBP-1 mRNA levels and inhibited the expression of target genes encoding acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS). In contrast, 25-hydroxycholesterol increased SREBP1 and FAS mRNA levels in primary human hepatocytes. The results imply that 25HC3S is a potent regulator of SREBP mediated lipid metabolism.     

Regulation of hepatic lanosterol 14 alpha-demethylase gene expression by dietary cholesterol and cholesterol-lowering agents.

Binding of sterol response element binding protein 1a to sterol response element-1 (SRE-1) in the promoter region of lanosterol 14 alpha-demethylase (14DM) has been demonstrated previously. Decreased 14DM activity has been shown to result in accumulation of the intermediate, 3 beta-hydroxy-lanost-8-en-32-al, a known translational downregulator of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Since it has also been demonstrated that feedback regulation of hepatic HMG-CoA reductase occurs primarily at the level of translation, the effects of dietary cholesterol and cholesterol lowering agents on levels of hepatic 14DM mRNA and immunoreactive protein were investigated. Addition of 1% cholesterol to a chow diet markedly decreased hepatic 14DM mRNA and protein levels in Sprague-Dawley rats. The extent and time course of this decrease in 14DM immunoreactive protein closely paralleled that of HMG-CoA reductase. Supplementation of the diet with the HMG-CoA reductase inhibitor, Lovastatin, to a level of 0.02%, raised 14DM mRNA and protein levels 2- to 3-fold. Addition of 2% Colestipol, a bile acid binding resin, to the chow diet caused smaller increases. The highest level of 14DM protein expression was observed in liver, the major site of feedback regulation of HMG-CoA reductase by cholesterol. Taken together, these observations suggest a critical role for 14DM in the feedback regulation of hepatic HMG-CoA reductase.     

8-bromoadenosine cyclic 3',5'-phosphate rapidly increases 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in human granulosa cells: role of cellular sterol balance in controlling the response to tropic stimulation

Exposure of cultured human granulosa cells to 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP) resulted in a rapid increase in the content of the mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the de novo synthesis of cholesterol. HMG-CoA reductase mRNA levels increased within 2 h of stimulation and remained elevated for at least 6 h. Treatment of granulosa cells with 25-hydroxycholesterol, a soluble cholesterol analogue, in combination with aminoglutethimide to block conversion of cellular sterols to pregnenolone, resulted in suppression of HMG-CoA reductase mRNA. When cells were stimulated with 8-bromo-cAMP in the presence of 25-hydroxycholesterol and aminoglutethimide, the increase in HMG-CoA reductase mRNA provoked by the tropic agent was markedly attenuated. This indicates that 8-bromo-cAMP raises HMG-CoA reductase mRNA levels indirectly by accelerating steroidogenesis and depleting cellular sterol pools, thus relieving sterol-mediated negative feedback of HMG-CoA reductase gene expression. 25-Hydroxycholesterol in the presence of aminoglutethimide suppressed low-density lipoprotein (LDL) receptor mRNA, but 8-bromo-cAMP effected a significant stimulation of LDL receptor mRNA levels when added with hydroxysterol and aminoglutethimide. These findings reveal differential regulation of HMG-CoA reductase and LDL receptor mRNAs in the presence of sterol negative feedback.     

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a negative regulator of luteinizing/chorionic gonadotropin hormone-induced steroidogenesis in Leydig cells: central role of steroidogenic acute regulatory protein (StAR)

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells (LCs) and germ cells and is essential for spermatid development and completion of spermatogenesis. Normal basal levels of testosterone in serum and LCs were observed in GRTH null (GRTH(-/-)) mice. However, testosterone production was enhanced in LCs of GRTH(-/-) mice compared with WT mice by both in vivo and in vitro human chorionic gonadotropin stimulation. LCs of GRTH(-/-) mice had swollen mitochondria with a significantly increased cholesterol content in the inner mitochondrial membrane. Basal protein levels of SREBP2, HMG-CoA reductase, and steroidogenic acute regulatory protein (StAR; a protein that transports cholesterol to the inner mitochondrial membrane) were markedly increased in LCs of GRTH(-/-) mice compared with WT mice. Gonadotropin stimulation caused an increase in StAR mRNA levels and protein expression in GRTH(-/-) mice versus WT mice, with no further increase in SREBP2 and down-regulation of HMG-CoA reductase protein. The half-life of StAR mRNA was significantly increased in GRTH(-/-) mice. Moreover, association of StAR mRNA with GRTH protein was observed in WT mice. Human chorionic gonadotropin increased GRTH gene expression and its associated StAR protein at cytoplasmic sites. Taken together, these findings indicate that, through its negative role in StAR message stability, GRTH regulates cholesterol availability at the mitochondrial level. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis has provided insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male.     

Novel inhibitors of heat shock protein Hsp70-mediated luciferase refolding that bind to DnaJ

Inhibitors of both heat shock proteins Hsp90 and Hsp70 have been identified in assays measuring luciferase refolding containing rabbit reticulocyte lysate or purified chaperone components. Here, we report the discovery of a series of phenoxy-N-arylacetamides that disrupt Hsp70-mediated luciferase refolding by binding to DnaJ, the bacterial homolog of human Hsp40. Inhibitor characterization experiments demonstrated negative cooperativity with respect to DnaJ and luciferase concentration, but varying the concentration of ATP had no effect on potency. Thermal shift analysis suggested a direct interaction with DnaJ, but not with Hsp70. These compounds may be useful tools for studying DnaJ/Hsp40 in various cellular processes.     

Inactivation of the PKR protein kinase and stimulation of mRNA translation by the cellular co-chaperone P58(IPK) does not require J domain function

P58(IPK) was discovered as an inhibitor of the interferon-induced, protein kinase, PKR. Upon virus infection, PKR can, as part of the host defense system, inhibit mRNA translation by phosphorylating the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2alpha). We previously found that influenza virus recruits the cellular P58(IPK) co-chaperone to inhibit PKR activity and thus facilitate viral protein synthesis. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain found in all DnaJ chaperone family members. To define the role of molecular chaperones in regulating cell growth in addition to PKR regulation, we performed a detailed analysis of the P58(IPK) J domain. Using growth rescue assays, we found that the P58(IPK) J domain substituted for the J domains of other DnaJ proteins, including DnaJ in Escherichia coli and Ydj1 in Saccharomyces cerevisiae. This is the first time a cellular J domain from a mammalian DnaJ family member was shown to be functional in both prokaryotic DnaJ and eukaryotic Ydj1 constructs. Furthermore, point mutations within the conserved HPD residue cluster of the P58(IPK) J domain disrupted P58(IPK) J function including stimulation of ATPase activity of Hsp70. However, the P58(IPK) HPD mutants still inhibited PKR activity and thus supported cell growth in a yeast rescue assay. Overexpression of the HPD mutants of P58(IPK), similar to their wild-type counterpart, also stimulated mRNA translation in a mammalian cell system. Taken together, our data necessitate a model of P58(IPK) inhibition of PKR kinase activity and stimulation of mRNA translation, which does not require classical J domain function found in the DnaJ molecular chaperone family.