Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.     

Expression of (pro)renin receptor in human erythroid cell lines and its increased protein accumulation by interferon-γ

The renin-angiotensin system is known to enhance erythropoiesis. (Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, has recently been identified. However, expression of (P)RR in erythroid cells has not been studied. The aim of the present study is to clarify expression of (P)RR in erythroid cells, and the effects of erythropoietin, angiotensin II, transforming growth factor-β1 (TGF-β1), interferon-γ (IFN-γ) and interleukin-1β (IL-1β) on its expression. Western blot analysis showed that (P)RR protein was expressed in human cultured erythroid cell lines, YN-1 and YN-1-0-A (a clonal variant cell line of YN-1). Erythropoietin (1IU/ml) increased (P)RR mRNA expression levels in YN-1-0-A cells (1.7-fold increase compared with control), but angiotensin II did not. Treatment of YN-1-0-A cells with IFN-γ (10ng/ml) for 48h increased the expression levels of (P)RR protein significantly (1.4-fold increase compared with control), whereas it had no significant effects on expression levels of (P)RR mRNA. Treatment of YN-1-0-A cells with TGF-β1 or IL-1β for 24 or 48h had no significant effects on expression levels of (P)RR. The present study has shown for the first time expression of (P)RR in erythroid cells, raising the possibility that (P)RR may have a role in erythropoiesis and the pathophysiology of certain types of anemia.     

Expression of a neuromodulin-β-galactosidase fusion protein in primary cultured neurons and its accumulation in growth cones

Cultured embryonic neurons share a number of characteristic morphological and physiological properties with their counterparts in vivo. For example, differentiating hippocampal neurons in culture develop two distinct classes of processes that serve as dendrites and axons. It has also been shown that the microtubule organization and composition in axons differs from those in dendrites, which may contribute to differential transport of macromolecules into axons or dendrites. We have expressed a neuromodulin--galactosidase fusion gene in cultured mesencephalic neurons in order to study the transport of the neurospecific protein neuromodulin into neurite growth cones. When -galactosidase alone was expressed in neurons, it was found in the cell bodies with diffuse neurite staining. In marked contrast, the neuromodulin--galactosidase fusion protein was rapidly transported into neurites and was concentrated in the growth cones. This system may provide a useful model for studying the structural domain(s) of neuromodulin that are required for transport and accumulation of neuromodulin in the growth cones of neurons.     

Assessment of α-synuclein secretion in mouse and human brain parenchyma

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease.     

Biochemical characterization of tau protein and its associated syndapin 1 and protein kinase Cɛ for their functional regulation in rat brain

Background

We recently reported that both sulfatide and cholesterol-3-sulfate (SCS) function as potent stimulators for the GSK-3β-mediated phosphorylation of tau protein (TP) in vitro [J. Biochem. 143 (2008) 359–367].

Methods

By means of successive gel filtration on a Superdex 200 pg column and three distinct ion-exchange column chromatographies, TP and its associated proteins were highly purified from the extract of rat brain.

Results

We found that (i) syndapin 1 and novel protein kinase C? (nPKC?) were identified as the TP-associated proteins; (ii) SCS highly stimulated the phosphorylation of TP and syndapin 1 by nPKC? as well as CK1; (iii) the full phosphorylation of TP and syndapin 1 by nPKC? in the presence of sulfatide resulted in their dissociation; (iv) TP primed by CK1 functioned as an effective phosphate acceptor for GSK-3β; (v) syndapin 1 highly stimulated the GSK-3β-mediated phosphorylation of TP; and (vi) TP isoforms were highly expressed in aged brain, whereas syndapin 1 was consistently detected in adult brain, but not in newborn brain.

General significance

These results provided here suggest that (i) TP-associated nPKC? suppresses the GSK-3β-mediated phosphorylation of TP through the phosphorylation of GSK-3β by the kinase in vitro; and (ii) SCS act as effective sole mediators to induce the GSK-3β-mediated high phosphorylation of both TP and its associated syndapin 1 involved in the biochemical processes of neuronal diseases, including Alzheimer's disease.     

Lysosomal cholesterol accumulation is commonly found in most peroxisomal disorders and reversed by 2-hydroxypropyl-β-cyclodextrin

Peroxisomal disorders(PDs) are a heterogenous group of diseases caused by defects in peroxisome biogenesis or functions. Xlinked adrenoleukodystrophy is the most prevalent form of PDs and results from mutations in the ABCD1 gene, which encodes a transporter mediating the uptake of very long-chain fatty acids(VLCFAs). The curative approaches for PDs are very limited.Here, we investigated whether cholesterol accumulation in the lysosomes is a biochemical feature shared by a broad spectrum of PDs. ...     

Regulation of pyridoxal-5′-phosphate level by biogenic amines in mouse brain

We investigated the relationship between the concentration of pyridoxal-5′-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.     

NirF is a periplasmic protein that binds d₁ heme as part of its essential role in d₁ heme biogenesis

The cytochrome cd? nitrite reductase from Paracoccus pantotrophus catalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors. The site of nitrite reduction is the d? heme, which is synthesized under anaerobic conditions by using nirECFD-LGHJN gene products. In vivo studies with an unmarked deletion strain, ΔnirF, showed that this gene is essential for cd? assembly and consequently for denitrification, which was restored when the ΔnirF strain was complemented with wild-type, plasmid-borne, nirF. Removal of a signal sequence and deletion of a conserved N-terminal Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo NirF activity. We demonstrate here that the product of the nirF gene is a periplasmic protein and, hence, must be involved in a late stage of the cofactor biosynthesis. In vitro studies with purified NirF established that it could bind d? heme. It is concluded that His41 of NirF, which aligns with His200 of the d? heme domain of cd?, is essential both for this binding and for the production of d? heme; replacement of His41 by Ala, Cys, Lys and Met all gave nonfunctional proteins. Potential functions of NirF are discussed.     

Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit

Cellular senescence is a process wherein proliferating cells undergo permanent cell cycle arrest while remaining viable. Senescence results in enhanced secretion of proteins that promote cancer and inflammation. We report here that the structure of the Golgi complex which regulates secretion is altered in senescent cells. In cells where senescence is achieved by replicative exhaustion or in cells wherein senescence has been induced with BrdU treatment dependent stress, the Golgi complex is dispersed. The expression of a G protein γ subunit, γ11, capable of translocation from the plasma membrane to the Golgi complex on receptor activation increases with senescence. Knockdown of γ11 or overexpression of a dominant negative γ3 subunit inhibits Golgi dispersal induced by senescence. Overall these results suggest that in cellular senescence an upregulated G protein gamma subunit mediates alterations in the structure of the Golgi.     

Promotion of β-amyloid production by C-reactive protein and its implications in the early pathogenesis of Alzheimer's disease

C-reactive protein (CRP) and β-amyloid protein (Aβ) are involved in the development of Alzheimer's disease (AD). However, the relationship between CRP and Aβ production is unclear. In vitro and in vivo experiments were performed to investigate the association of CRP with Aβ production. Using the rat adrenal pheochromocytoma cell line (PC12 cells) to mimic neurons, cytotoxicity was evaluated by cell viability and supernatant lactate dehydrogenase (LDH) activity. The levels of amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE-1), and presenilins (PS-1 and PS-2) were investigated using real-time polymerase chain reaction and Western blotting analysis. Aβ1-42 was measured by enzyme-linked immunosorbent assay. The relevance of CRP and Aβ as well as potential mechanisms were studied using APP/PS1 transgenic (Tg) mice. Treatment with 0.5-4.0 μM CRP for 48 h decreased cell viability and increased LDH leakage in PC12 cells. Incubation with CRP at a sub-toxic concentration of 0.2 μM increased the mRNA levels of APP, BACE-1, PS-1, and PS-2, as well as Aβ1-42 production. CRP inhibitor reversed the CRP-induced upregulations of the mRNA levels of APP, BACE-1, PS-1, and PS-2, and the protein levels of APP, BACE-1, PS-1, and Aβ1-42, but did not reversed Aβ1-42 cytotoxicity. The cerebral levels of CRP and Aβ1-42 in APP/PS1 Tg mice were positively correlated, accompanied with the elevated mRNA expressions of serum amyloid P component (SAP), complement component 1q (C1q), and tumor necrosis factor-α (TNF-α). These results suggest that CRP cytotoxicity is associated with Aβ formation and Aβ-related markers expressions; CRP and Aβ were relevant in early-stage AD; CRP may be an important trigger in AD pathogenesis.     

Control of ER stress by a chemical chaperone counteracts apoptotic signals in IFN-γ-treated murine hepatocytes

Apoptosis of hepatocytes plays a key role in the pathogenesis of immune-mediated hepatitis. However, the detailed mechanisms of apoptotic signaling remain unclear. In this study, we investigated the involvement of ER stress in a model of IFN-γ-induced apoptosis of hepatocytes in vitro, using a chemical chaperone reagent, glycerol. IFN-γ-induced apoptotic events (mitochondrial release of cytochrome c, enzymatic activation of caspase-3 and -9) were markedly inhibited by glycerol. Glycerol induced partial inhibition of cytotoxicity indicated by lactate dehydrogenase release from the cytosol but had no inhibitory effect on the induction of IRF-1 gene expression and reactive oxygen species, required for hepatocyte apoptosis by IFN-γ. Induction of caspase-4 and -12 gene expression, positively correlated with ER stress, was attenuated by glycerol. Gene analysis revealed that induction of ER stress-related genes, C/EBP homologue protein (CHOP/GADD153) and TRB3, was suppressed completely by glycerol treatment. These results suggest that ER stress plays a crucial role in mediating apoptosis of hepatocytes induced by IFN-γ, and a chemical chaperone is an effective inhibitor of the ER stress.     

Modulation of the conformation,fibrillation, and fibril morphologies of human brain α-, β-, and γ-synuclein proteins by the disaccharide chemical chaperone trehalose

Human α-, β-, and γ-synuclein (syn) are natively unfolded proteins present in the brain. Deposition of aggregated α-syn in Lewy bodies is associated with Parkinson's disease (PD) and γ-syn is known to be involved in both neurodegeneration and breast cancer. At physiological pH, while α-syn has the highest propensity for fibrillation followed by γ-syn, β-syn does not form any fibrils. Fibril formation in these proteins could be modulated by protein structure stabilizing osmolytes such as trehalose which has an exceptional stabilizing effect for globular proteins. We present a comprehensive study of the effect of trehalose on the conformation, aggregation, and fibril morphology of α-, β-, and γ-syn proteins. Rather than stabilizing the intrinsically disordered state of the synucleins, trehalose accelerates the rate of fibril formation by forming aggregation-competent partially folded intermediate structures. Fibril morphologies are also strongly dependent on the concentration of trehalose with ≤ 0.4M favoring the formation of mature fibrils in α-, and γ-syn with no effect on the fibrillation of β-syn. At ≥ 0.8M, trehalose promotes the formation of smaller aggregates that are more cytotoxic. Live cell imaging of preformed aggregates of a labeled A90C α-syn shows their rapid internalization into neural cells which could be useful in reducing the load of aggregated species of α-syn. The findings throw light on the differential effect of trehalose on the conformation and aggregation of disordered synuclein proteins with respect to globular proteins and could help in understanding the effect of osmolytes on intrinsically disordered proteins under cellular stress conditions.     

Expression and function of heregulin-α and its receptors in the mouse mammary gland

Heregulin-α (HRGα) is a cytokine secreted by the mammary mesenchyme, adjacent to lobuloalveolar structures. To understand the role of HRGα and its receptors in mammary glands, and the underlying mechanisms, we performed this study to determine the expression and localization of HRGα and its receptors ErbB2 and ErbB3. We also determined the role of HRGα in the development of mammary glands, β-casein expression and secretion, Rab3A protein expression and the phosphorylation of HRGα signaling molecules using confocal laser scanning microscopy, tissue culture, capillary electrophoresis, Western blotting and enzyme-linked immunosorbent assays. We found that a peak was on pregnancy day 15. Changes of ErbB2 and ErbB3 expression were positively and linearly correlated with HRGα, indicating that HRGα positively regulates ErbB2 and ErbB3 expression. During pregnancy, HRGα enhanced the phosphorylation of STAT5, p42/p44, p38, PKC and Rab3A protein expression, stimulated the proliferation and differentiation of the ductal epithelial cells of mammary glands, and increased and maintained the expression and secretion of β-casein. During lactation, HRGα enhanced the phosphorylation of STAT5 and p38, inhibited the phosphorylation of PKC and Rab3A protein expression, maintained the morphology of the mammary glands and increased the secretion of lactoprotein to reduce the expression of β-casein in mammary epithelial cells. During involution, HRGα induced the phosphorylation of STAT3 and Rab3A protein expression, and inhibited the phosphorylation of PKC to stimulate the degeneration of mammary epithelial cells. It also inhibited the secretion of β-casein, resulting in increased levels of β-casein in mammary epithelial cells.     

Minor contribution of presenilin 2 for γ-secretase activity in mouse embryonic fibroblasts and adult mouse brain

γ-Secretase plays an important function in the development of Alzheimer disease, since it participates in the production of the toxic amyloid β-peptide (Aβ) from the amyloid precursor protein (APP). Besides APP, γ-secretase cleaves many other substrates resulting in adverse side effects when γ-secretase inhibitors are used in clinical trials. γ-Secretase is a membrane bound protein complex consisting of at least four subunits, presenilin (PS), nicastrin, Aph-1 and Pen-2. PS and Aph-1 exist as different homologs (PS1/PS2 and Aph-1a/Aph-1b, respectively), which generates a variation in complex composition. PS1 and PS2 appears to have distinct roles since PS1 is essential during embryonic development whereas PS2 deficient mice are viable with a mild phenotype. The molecular mechanism behind this diversity is, however, largely unknown. In order to investigate whether PS1 and PS2 show different substrate specificity, we used PS1 or PS2 deficient mouse embryonic fibroblasts to study the processing on the γ-secretase substrates APP, Notch, N-cadherin, and ephrinB. We found that whereas depletion of PS1 severely affected the cleavage of all substrates, the effect of PS2 depletion was minor. In addition, less PS2 was found in active γ-secretase complexes. We also studied the effect of PS2 depletion in adult mouse brain and, in concordance with the results from the mouse embryonic fibroblasts, PS2 deficiency did not alter the cleavage of the two most important substrates, APP and Notch. In summary, this study shows that the contribution of PS2 on γ-secretase activity is of less importance, explaining the mild phenotype of PS2-deficient mice.     

Activation of rat brain adenylate cyclase by proteases: involvement of distinct protein components in the activation by α-chymotrypsin and trypsin

Adenylate cyclase in synaptic plasma membranes from rat brain is activated by α-chymotrypsin or trypsin. These proteases also activate adenylate cyclase reconstituted from the catalytic subunit of adenylate cyclase and the partially purified fraction of the GTP-binding proteins containing both the stimulatory and inhibitory GTP-binding proteins. Properties of the activation of reconstituted adenylate cyclase by the proteases are as follows. (1) The proteases do not directly activate the catalytic subunit. However, the pre-treatment of the partially purified GTP-binding proteins with α-chymotrypsin (100 μg/ml) increases the subsequently reconstituted cyclase activity at least 3-fold. Trypsin (10–30 μg/ml) much more weakly enhances the cyclase activity. (2) α-Chymotrypsin and trypsin synergistically activate the cyclase. (3) Trypsin but not α-chymotrypsin no longer activates the cyclase when the purified stimulatory GTP-binding protein (Gs) replaces the partially purified GTP-binding proteins. (4) The stimulatory effects of α-chymotrypsin and trypsin on the cyclase activity are little or slight unless 5′-guanylylimidodiphosphate (Gpp(NH)p) is present in the reconstitution. (5) The purified βγ-subunits of the GTP-binding proteins markedly inhibit adenylate cyclase. This inhibition is nearly completely attenuated by treating the βα-subunits with α-chymotrypsin (> 10 μg/ml). (6) Trypsin (1–10 μg/ml) inactivates the GTPase of the α-subunit of the inhibitory GTP-binding protein (Gi). This inactivation of the GTPase seems to correlate with the activation of the reconstituted adenylate cyclase by trypsin.We conclude that two distinct protein components are involved in the activation of adenylate cyclase by α-chymotrypsin and trypsin. One component sensitive to α-chymotrypsin is probably the βγ-subunits of the GTP-binding proteins. The other component sensitive to trypsin may be the α-subunit of Gi.     

Activation and competition of lipoylation of H protein and its hydrolysis in a reaction cascade catalyzed by the multifunctional enzyme lipoate–protein ligase A

Protein lipoylation is essential for the function of many key enzymes but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate–protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (a) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate lipoyl-AMP (Lip-AMP); (b) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (c) both the lipoylation of H protein and its hydrolysis is enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles.     

Denitration of peroxynitrite-treated proteins by ‘protein nitratases’ from rat brain and heart

Putative protein nitratases, which catalyze denitration of peroxynitrite (PN)-treated proteins, were detected in the homogenate/crude extract of rat brains and hearts. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained homogenate/crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA or invertase. Enhanced activity of nitratases was noted by preincubating crude extract with Ca²⁺. In addition, at least two types of nitratases may occur: type I, reductant-dependent, and type II, reductant- independent. Furthermore, upon denitration, the activity of PN-treated invertase increased to the same activity level of the untreated invertase. The overall reaction catalyzed by nitratases for denitration of nitrotyrosine residues in protein could be as follows: Protein-Tyr-NO_{_2} + H₂O Protein-Tyr-H + H⁺ + NO₃ ^–. The nitration/denitration of protein-tyrosine may be crucial in regulating signal transduction.     

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.