Geldanamycin induces Hsp70 and prevents alpha-synuclein aggregation and toxicity in vitro

Geldanamycin (GA) is a naturally occurring benzoquinone ansamycin that induces heat shock protein 70 (Hsp70). GA has been shown to reduce alpha-synuclein induced neurotoxicity in a fly model of Parkinson's disease. We have previously shown that heat shock proteins can prevent alpha-synuclein aggregation and protect against alpha-synuclein induced toxicity in human H4 neuroglioma cells. Here, we hypothesize that GA treatment will reduce alpha-synuclein aggregation and prevent alpha-synuclein induced toxicity and we show that GA can induce Hsp70 in a time- and concentration-dependent manner in H4 cells. Pretreatment with 200nM GA 24h prior to transfection prevented alpha-synuclein aggregation and protected against toxicity. Treatment of cells with pre-existing inclusions with GA did not result in a reduction in the number of cells containing inclusions, suggesting that upregulation of Hsp70 is not sufficient to remove established inclusions. Similarly, Western blot analysis demonstrated that GA treatment could dramatically reduce both total alpha-synuclein and high molecular weight alpha-synuclein aggregates. Taken together, these data suggest that GA is effective in preventing alpha-synuclein aggregation and may represent a pharmacological intervention to therapeutically increase expression of molecular chaperone proteins to treat neurodegenerative diseases where aggregation is central to the pathogenesis.     

TorsinA and heat shock proteins act as molecular chaperones: suppression of alpha-synuclein aggregation

TorsinA, a protein with homology to yeast heat shock protein104, has previously been demonstrated to colocalize with alpha-synuclein in Lewy bodies, the pathological hallmark of Parkinson's disease. Heat shock proteins are a family of chaperones that are both constitutively expressed and induced by stressors, and that serve essential functions for protein refolding and/or degradation. Here, we demonstrate that, like torsinA, specific molecular chaperone heat shock proteins colocalize with alpha-synuclein in Lewy bodies. In addition, using a cellular model of alpha-synuclein aggregation, we demonstrate that torsinA and specific heat shock protein molecular chaperones colocalize with alpha-synuclein immunopositive inclusions. Further, overexpression of torsinA and specific heat shock proteins suppress alpha-synuclein aggregation in this cellular model, whereas mutant torsinA has no effect. These data suggest that torsinA has chaperone-like activity and that the disease-associated GAG deletion mutant has a loss-of-function phenotype. Moreover, these data support a role for chaperone proteins, including torsinA and heat shock proteins, in cellular responses to neurodegenerative inclusions.     

Alpha-synuclein has structural and functional similarities to small heat shock proteins

The aggregation and fibrillization of α-synuclein, a major component of Lewy bodies, is a key event in Parkinson’s disease. Although the mechanisms of fibrils formation are largely investigated, physiological function of α-synuclein is not yet clearly elucidated. Here, we showed that C-terminal region of α-synuclein is similar to α-crystalline domain of small heat shock proteins. In our experiments, α-synuclein, like small heat shock proteins, protected cellular proteins from denaturation, and confer Escherichia coli cellular tolerances against thermal- and oxidative-stresses.     

Small heat shock proteins differentially affect Abeta aggregation and toxicity

beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and has been implicated in neurotoxicity associated with the disease. Abeta aggregates readily in vitro and in vivo, and its toxicity has been linked to its aggregation state. Prevention of Abeta aggregation has been investigated as a means to prevent Abeta toxicity associated with AD. Recently we found that Hsp20 from Babesia bovis prevented both Abeta aggregation and toxicity [S. Lee, K. Carson, A. Rice-Ficht, T. Good, Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity, Protein Sci. 14 (2005) 593-601.]. In this work, we examined the mechanism of Hsp20 interaction with Abeta1-40 and compared its activity to that of other small heat shock proteins, carrot Hsp17.7 and human Hsp27. While all three small heat shock proteins were able to prevent Abeta aggregation, only Hsp20 was able to attenuate Abeta toxicity in cultured SH-SY5Y cells. Understanding the mechanism of the Hsp20-Abeta interaction may provide insights into the design of the next generation of Abeta aggregation and toxicity inhibitors.     

Challenges and complexities of alpha-synuclein toxicity: new postulates in unfolding the mystery associated with Parkinson's disease

The discovery of two missense mutations in alpha-synuclein gene and the identification of the alpha-synuclein as the major component of Lewy bodies and Lewy neurites have imparted a new direction in understanding Parkinson's disease. Now that alpha-synuclein has been implicated in several neurodegenerative disorders makes it increasingly clear that aggregation of alpha-synuclein is a hallmark feature in neurodegeneration. Although little has been learned about its normal function, alpha-synuclein appears to be associated with membrane phospholipids and may therefore participate in a number of cell signaling pathways. Here, we review the localization, structure, and function of alpha-synuclein and provide a new hypothesis on, (a) the disruption in the membrane binding ability of synuclein which may be the major culprit leading to the alpha-synuclein aggregation and (b) the complexity associated with nuclear localization of alpha-synuclein and its possible binding property to DNA. Further, we postulated the three possible mechanisms of synuclein induced neuronal degeneration in Parkinson's disease.     

Regulation of protein turnover by heat shock proteins

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin–proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.     

Plasminogen and angiostatin interact with heat shock proteins

Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases, binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15–20%. Collectively, these data indicate that (i) while plasminogen interacts specifically with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen only partially displaces angiostatin’s binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells like ECs.     

人口腔螺旋体热休克蛋白的初步研究

为分析牙周病的发病与人口爱螺旋体的热休克蛋白的关系。通过ＳＤＳ－ＰＡＧＥ电泳将菌体蛋白分离,转移电泳（Ｗｅｓｔｅｒｎ ｂｌｏｔ）检查螺旋体的热休克蛋白抗原,牙周病患者血清与螺旋体的热休克蛋白进行免疫印迹试验检查灯克蛋白抗体。结果为实验所用的螺旋体有４种可诱导产生质变休克蛋白,患者血清中有多种对于口腔螺旋体蛋白能起作用的本,其中有两名患者的血清对Ｔ．ｓｏｃｒａｎｓｋｉｉ３５５３５菌株的６０ｋＤ或６５     

The heat shock proteins: Their roles as multi-component machines for protein folding

The roles played by heat shock proteins in fungi have been subjected to intense scrutiny. Presuming they only played roles in tolerance to stress gave way to the realization that many of them were essential for maintenance of cell physiology under all conditions. Recent progress has revealed their action as multi-component machines, playing roles in signalling and expansion of phenotypic plasticity, as well as their well-established function as molecular chaperones.     

Acceleration of alpha-synuclein aggregation by homologous peptides

alpha-Synuclein (alpha-Syn), amyloid beta-protein and prion protein are among the amyloidogenic proteins that are associated with the neurodegenerative diseases. These three proteins share a homologous region with a consensus sequence mainly consisting of glycine, alanine and valine residues (accordingly named as the GAV motif), which was proposed to be the critical core for the fibrillization and cytotoxicity. To understand the role of the GAV motif in protein amyloidogenesis, we studied the effects of the homologous peptides corresponding to the sequence of GAV motif region (residues 66-74) on alpha-Syn aggregation. The result shows that these peptides can promote fibrillization of wild-type alpha-Syn and induce that of the charge-incorporated mutants but not the GAV-deficient alpha-Syn mutant. The acceleration of alpha-Syn aggregation by the homologous peptides is under a sequence-specific manner. The interplay between the GAV peptide and the core regions in alpha-Syn may accelerate the aggregation process and stabilize the fibrils. This finding provides clues for developing peptide mimics that could promote transforming the toxic oligomers or protofibrils into the inert mature fibrils.     

Genetic analysis of heat shock proteins in maize

A genetic analysis of heat shock protein (HSP) synthesis was performed in seedling leaf tissue of two maize inbred lines, their F₁ hybrid and F₂ progeny. Protein synthesis following a high temperature treatment was visualized by [³⁵S]-methionine in vivo labelling and two-dimensional gel electrophoresis. The parental lines' HSP synthesis patterns revealed both qualitative and quantitative polymorphisms implicative of differences in HSP structural genes and regulatory factors. The F₁ hybrid HSP profile indicated that synthesis of all parental HSPs conformed to dominant inheritance patterns, including complete dominance, over-dominance and co-dominance. Alleles for six low-molecularweight HSPs in F₂ progeny assorted according to typical 31 Mendelian ratios for dominant gene expression. There is evidence for unlinked gene loci of four different HSP gene pairs, but data for three other HSP gene pairs were inconclusive, perhaps reflecting linkage for one pair and complex regulatory factor interactions for the other two pairs of genes. These results clearly indicate the existence of genetic variability in HSP synthesis and emphasize the potential of partitioning their roles in thermal tolerance using genetic and molecular analyses.     

Secreted heat shock proteins in sunflower suspension cell cultures

Sunflower suspension cell cultures were subjected to different heat treatments and the electrophoretic patterns of heat-induced endocellular and secreted proteins were analyzed. In response to heat shock (3 h at 40°C), sunflower cells synthesized new polypeptides and secreted them into the medium, while the synthesis of other polypeptides was suppressed. Two major polypeptides of about 50 and 32 kDa were strongly induced. The two-dimensional electrophoretic analysis showed that the 32-kDa band is composed of at least four different polypeptides. Western blotting hybridizations of secreted proteins with various lectins were performed. The 32-kDa band gave a positive signal with concanavalin A. Received: 8 March 1996 / Revision received: 30 September 1996 / Accepted: 15 October 1996     

Small heat shock proteins and adaptation of various Drosophila species to hyperthermia

The dynamics and the level of accumulation of small heat shock proteins (sHSP group 21–27) after a heat exposure were studied in three Drosophila species differing in thermotolerance. The southern species Drosophila virilis, having the highest thermotolerance, surpassed thermosensitive D. lummei and D. melanogaster in the level of sHSPs throughout the temperature range tested. The results suggest an important role of sHSPs in the molecular mechanisms of adaptation to adverse environmental conditions, particularly to hyperthermia.     

Adaptation to heat of cardiomyoblasts in culture protects them against heat shock: role of nitric oxide and heat shock proteins

Dosed adaptation to environmental factors is an efficient non-drug means for increasing the resistance of organs or the body as a whole. We demonstrated earlier that nitric oxide (NO) plays an important role in adaptive defense of the organism, in particular due to activation of heat shock protein (HSP) synthesis. A key question remained open—to what extent the formation of adaptive defense depends on central mechanisms and to what extent on the intracellular mechanisms immediately responding to the adapting factor, and whether the NO-dependent activation of HSP synthesis plays a role in adaptation of isolated cells. In the present study we looked into the possibility of producing a protective effect of adaptation to heat in cell culture. A 6-day adaptation to heat limited to 17% the decrease in metabolic activity induced by heat shock in H9c2 cardiomyoblasts. The development of adaptation was associated with increased NO production. Treatment of cells with the inhibitor of NO synthase L-NNA (100 M) prevented the development of adaptive protection. Adaptation of cell culture enhanced synthesis of HSP70 but not HSP27. Blockade of HSP70 synthesis with quercetin (50 M) left unchanged the protective effect of adaptation. Inhibition of NO synthesis restricted the adaptation-induced HSP70 synthesis. Therefore, the formation of adaptation at the cell level may result from a direct action of an environmental factor without participation of neurohumoral factors. Such adaptation involves NO-dependent mechanisms divorced from the activation of HSP70 synthesis.     

Genetic variability for heat shock proteins in common wheat

Summary The response of the common wheat line Chinese Spring to heat shocks of different time lengths was studied by the two-dimensional (2D) electrophoresis of denatured proteins. After a heat shock of 5 h, 33 heat shock proteins (HSPs) accumulated in an amount sufficient to be revealed by silver stain. Two other wheat lines (Moisson and Selkirk) were then submitted to a heat shock of 5 h, and the responses of the 3 lines were compared: of a total of 35 HSPs, 13 (37.1%) were quantitatively or qualitatively variable. This variability concerns low-molecular-weight and high-molecular-weight HSPs. The three genotypes showed thermal tolerance but Chinese Spring's response to heat treatments was slightly different from those of the other two lines The possibility of a relationship between HSP patterns and thermal sensitivity is discussed.     

Small heat shock proteins in cellular adhesion and migration

Cellular locomotion and adhesion critically depend on regulated turnover of filamentous actin. Biochemical data from diverse model systems support a role for the family of small heat shock proteins (HSPBs) in microfilament regulation. The small chaperones could either act directly, through competition with the motor myosin, or indirectly, through modulation of actin depolymerizing factor/cofilin activity. However, a direct link between HSPBs and actin-based cellular motility remained to be established. In a recent experimental genetics study, we provided evidence for regulation of Plasmodium motility by HSPB6/Hsp20. The infectious forms of malaria parasites, termed sporozoites, display fast and continuous substrate-dependent motility, which is largely driven by turnover of actin microfilaments. Sporozoite gliding locomotion is essential to avoid destruction by host defense mechanisms and to ultimately reach a hepatocyte, the target cell, where to transform and replicate. Genetic ablation of Plasmodium HSP20 dramatically changed sporozoite speed and substrate adhesion, resulting in impaired natural malaria transmission. In this article, we discuss the function of Hsp20 in this fast-moving unicellular protozoan and implications for the roles of HSPBs in adhesion and migration of eukaryotic cells.     

Impairment of redox state and dopamine level induced by alpha-synuclein aggregation and the prevention effect of hsp70

One hypothesis for the etiology of Parkinson's disease (PD) is that the formation of proteinaceous inclusion, which is mainly composed of alpha-synuclein, may contribute to the selective loss of dopaminergic neurons. To further explore the role of alpha-synuclein in neurodegeneration of PD, we examined the possible effects of aggregated alpha-synuclein on the intracellular redox state, dopamine level, and cell death of SK-N-SH cells. Our present studies show that alpha-synuclein aggregation gives rise to both elevated intracellular oxidative state and dopamine level in SK-N-SH cells. Moreover, alpha-synuclein aggregation results in a higher ratio of apoptosis population (55.8%+/-SEM) in cells overexpressing alpha-synuclein aggregation, compared to their normal control groups (8.0%+/-SEM). In contrast, coexpression of hsp70 with alpha-synuclein suppresses the oxidative state shift, restores the normal dopamine levels and blocks neuron cell loss. Therefore, our data provided one possible mechanism by which the alpha-synuclein aggregation may lead to the neurodegeneration in PD via regulating the level of cytoplasmic dopamine and then disturbing the intracellular redox homeostasis. On the other hand, hsp70 can mitigate the degenerative effect conferred by alpha-synuclein, acting as a protective factor in treatment of PD.