Protein aggregation and aggregate toxicity: new insights into protein folding,misfolding diseases and biological evolution

The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of more than 20 degenerative conditions affecting either the central nervous system or a variety of peripheral tissues. As these conditions include Alzheimer's, Parkinson's and the prion diseases, several forms of fatal systemic amyloidosis, and at least one condition associated with medical intervention (haemodialysis), they are of enormous importance in the context of present-day human health and welfare. Much remains to be learned about the mechanism by which the proteins associated with these diseases aggregate and form amyloid structures, and how the latter affect the functions of the organs with which they are associated. A great deal of information concerning these diseases has emerged, however, during the past 5 years, much of it causing a number of fundamental assumptions about the amyloid diseases to be re-examined. For example, it is now apparent that the ability to form amyloid structures is not an unusual feature of the small number of proteins associated with these diseases but is instead a general property of polypeptide chains. It has also been found recently that aggregates of proteins not associated with amyloid diseases can impair the ability of cells to function to a similar extent as aggregates of proteins linked with specific neurodegenerative conditions. Moreover, the mature amyloid fibrils or plaques appear to be substantially less toxic than the pre-fibrillar aggregates that are their precursors. The toxicity of these early aggregates appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increases in free Ca²⁺ that eventually lead to apoptotic or necrotic cell death. The 'new view' of these diseases also suggests that other degenerative conditions could have similar underlying origins to those of the amyloidoses. In addition, cellular protection mechanisms, such as molecular chaperones and the protein degradation machinery, appear to be crucial in the prevention of disease in normally functioning living organisms. It also suggests some intriguing new factors that could be of great significance in the evolution of biological molecules and the mechanisms that regulate their behaviour.Abbreviations ER Endoplasmic reticulum - Hsp Heat-shock protein - HypF-N N-terminal domain of hydrogenase maturation factor HypF - ROS Reactive oxygen species - SH3 Src-homology 3     

Involvement of bivalent cations and arachidonic acid in neutrophil aggregation

Chemotactic factors and arachidonic acid aggregate neutrophils; indomethacin and 5,8,11,14-eicosatetraynoic acid, two blockers of cellular arachidonate metabolism, inhibit these responses. Additionally, A23187, an ionophore which specifically transports bivalent cations into cells, also aggregates the neutrophils, and this response, as well as the response to chemotactic factors, requires the presence of extracellular calcium and magnesium. In this report these relationships were further studied. It was found that human neutrophil aggregation stimulated by arachidonic acid also required calcium and magnesium. Furthermore, cells preincubated with arachidonate for 4 min before exposure to the bivalent cations did not aggregate before or after this exposure and, following this exposure, were refractory to subsequent stimulation by more arachidonate, a chemotactic tripeptide (formylmethionylleucylphenylalanine), or A23187, i.e., these cells had become nonselectively desensitized to the aggregating agents. Finally, indomethacin and 5,8,11,14-eicoasatetraynoic acid inhibited the aggregation response stimulated by A23187 and their potency in doing so paralleled their potency in inhibiting the responses induced by arachidonate or the chemotactic tripeptide. Thus, the neutrophil aggregation responses induced by arachidonate, chemotactic factor, and A23187 were similarly influenced by preincubating the cells with arachidonate, had similar requirements for calcium and magnesium, and were similarly inhibited by blockers of arachidonate metabolism. It appears that bivalent cations and arachidonic acid play essential and, perhaps, interacting roles in the aggregation response to diverse stimuli.Publication 1877 of the Research Institute of Scripps Clinic, La Jolla, California 92037.Supported by research grants: A1 07007 (NIH); Office of Naval Research; Council for Tobacco Research.     

Involvement of protein phosphatase 2A in the interleukin-3-stimulated Jak2-Stat5 signaling pathway.

In this study, we report that the tyrosine kinase, Janus kinase 2 (Jak2), associates with the serine/threonine protein phosphatase 2A (PP2A) in 32Dcl3 myeloid progenitor cells. The association between Jak2 and PP2A transiently increases following interleukin-3 (IL-3) stimulation and activation of Jak2. The catalytic subunit of PP2A is tyrosine phosphorylated by Jak2 in vitro and in vivo, resulting in inhibition of phosphatase activity. PP2A also associates with Stat5 in 32Dcl3 cells in an IL-3-dependent manner. Pretreatment of 32Dcl3 cells with okadaic acid (OA), an inhibitor of PP2A, resulted in increased tyrosine phosphorylation and nuclear translocation of Stat5. Our results suggest that PP2A plays a negative regulatory role in regulating the IL-3 signaling pathway via formation of complexes with Jak2 and Stat5.     

Involvement of the ubiquitin-proteasome pathway and molecular chaperones in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant muscular dystrophy that results from small expansions of a polyalanine tract in the PABPN1 gene. Intranuclear inclusions are the pathological hallmark of OPMD. The mechanism by which protein aggregation in OPMD might relate to a toxic gain-of-function has so far remained elusive. Whether protein aggregates themselves are pathogenic or are the consequence of an unidentified underlying molecular mechanism is still unclear. Here, we report that protein aggregation in a cell model of OPMD directly impaires the function of the ubiquitin-proteasome pathway (UPP) as well as molecular chaperone functions. The proteasome inhibitor lactacystin causes significant increase of protein aggregation and toxicity. Moreover, overexpression of molecular chaperones (HSP40 and HSP70) suppressed protein aggregation and toxicity. We also provide evidence that mPABPN1-ala17 protein aggregation proportionally correlates with toxicity. Furthermore, we show that co-expression of chaperones in our OPMD cell model increases the solubility of mPABPN1-ala17 and transfected cell survival rate. Our studies suggest that molecular regulators of polyalanine protein solubility and degradation may provide insights into new mechanisms in OPMD pathogenesis. Further analysis of the cellular and molecular mechanisms by which UPP and molecular chaperones influence the degradation of misfolded proteins could provide novel concepts and targets for the treatment and understanding of the pathogenesis of OPMD and neurodegenerative diseases.     

Mispaired P3 region in the hierarchical folding pathway of the Tetrahymena ribozyme

BACKGROUND: The Tetrahymena group I ribozyme folds into a complex three-dimensional structure for performing catalytic reactions. The catalysis depends on its catalytic core consisting of two helical domains, P4-P6 and P3-P7, connected by single stranded regions. In the folding process, most of this ribozyme folds in a hierarchical manner in which a kinetically stable intermediate determines the overall folding rate. RESULTS: Although the nature of this intermediate has not yet been elucidated, a mispaired P3 stem (alt-P3) appears a likely candidate. To examine the effects of the alt-P3 structure on the kinetic and thermodynamic properties of the active structure of the ribozyme or its P3-P7 domain formation, we prepared and analysed variant ribozymes in which relative stabilities of the original P3 and alt-P3 structure were altered systematically. CONCLUSION: The results indicate that the alt-P3 structure is not the major rate-limiting factor in the folding process.     

Involvement of the mannose receptor and p38 mitogen-activated protein kinase signaling pathway of the microdomain of the integral membrane protein after enteropathogenic Escherichia coli infection

The microdomain of the integral membrane protein (MIMP) has been shown to adhere to mucin and to antagonize the adhesion of enteropathogenic Escherichia coli (EPEC) to epithelial cells; however, the mechanism has not been fully elucidated. In this study, we further identified the receptor of MIMP on NCM460 cells and investigated the mechanism (the p38 mitogen-activated protein kinase [MAPK] pathway) following the interaction of MIMP and its corresponding receptor, mannose receptor. We first identified the target receptor of MIMP on the surfaces of NCM460 cells using immunoprecipitation-mass spectrometry technology. We also verified the mannose receptor and examined the degradation and activation of the p38 MAPK signaling pathway. The results indicated that MIMP adhered to NCM460 cells by binding to the mannose receptor and inhibited the phosphorylation of p38 MAPK stimulated after EPEC infection via inhibition of the Toll-like receptor 5 pathway. These findings indicated that MIMPs relieve the injury of NCM460 cells after enteropathogenic E. coli infection through the mannose receptor and inhibition of the p38 MAPK signaling pathway, both of which may therefore be potential therapeutic targets for intestinal diseases, such as inflammatory bowel disease.     

Involvement of brain ketone bodies and the noradrenergic pathway in diabetic hyperphagia in rats

Uncontrolled type 1 diabetes leads to hyperphagia and severe ketosis. This study was conducted to test the hypothesis that ketone bodies act on the hindbrain as a starvation signal to induce diabetic hyperphagia. Injection of an inhibitor of monocarboxylate transporter 1, a ketone body transporter, into the fourth ventricle normalized the increase in food intake in streptozotocin (STZ)-induced diabetic rats. Blockade of catecholamine synthesis in the hypothalamic paraventricular nucleus (PVN) also restored food intake to normal levels in diabetic animals. On the other hand, hindbrain injection of the ketone body induced feeding, hyperglycemia, and fatty acid mobilization via increased sympathetic activity and also norepinephrine release in the PVN. This result provides evidence that hyperphagia in STZ-induced type 1 diabetes is signaled by a ketone body sensed in the hindbrain, and mediated by noradrenergic inputs to the PVN.     

Catalysis of protein folding by protein disulfide isomerase and small-molecule mimics

Protein disulfide isomerase (PDI) catalyzes the formation of native disulfide pairings in secretory proteins. The ability of PDI to act as a disulfide isomerase makes it an essential enzyme in eukaryotes. PDI also fulfills other important roles. Recent studies have emphasized the importance of PDI as an oxidant in the endoplasmic reticulum. Intriguing questions remain regarding how PDI is able to catalyze both isomerization and oxidation in vivo. Studies of PDI and its homologues have led to the development of small-molecule folding catalysts that are able to accelerate disulfide isomerization in vitro and in vivo. PDI will continue to provide both an inspiration for the design of such artificial foldases and a benchmark with which to gauge the success of those designs. Here, we review current understanding of the chemistry and biology of PDI, its homologues, and small molecules that mimic its catalytic activity.     

Neonatal handling and the maternal odor preference in rat pups: Involvement of monoamines and cyclic AMP response element-binding protein pathway in the olfactory bulb

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic–pituitary–adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age.     

Neurodegenerative aspects of protein aggregation

Protein aggregation and amyloid fibril deposits are characteristic features of more than twenty pathologic conditions characterized by plaque deposition in the central nervous system. Recent studies point out relationships between protein misfolding and numerous serious diseases. Despite different origins (sporadic, familial or transmissible), they are sometimes called conformational diseases to emphasize aberrant conformations as the putative cause of deposits that precede or accompany the clinical manifestation of the disease. Neurological disorders such as Alzheimer's disease (AD), Prion disorders (PrD), Parkinson's disease (PD), and Huntington's disease (HD) are the most typical examples of protein-based dementias, characterized by protein conformational transitions (alpha-helix/random coil to beta-sheet) that cause aggregation followed by fibrillization. Although it is very tempting to postulate a common mechanism of toxicity based on conformational and structural analogies, it should be noted that the factors responsible for conformational transition, oligomerization, aggregation, and plaque formation, are still subject of speculation and additional data is required to test the amyloid fibril hypothesis.     

Platelet aggregation in modulation of the pathway L-arginine-NO in rats with cerebral ischemia

Platelet aggregation in the plasma of rats with brain ischemia and modulation of L-arginine-NO pathway were studied in experiments (Born method using analyzer AP 21103AO SOLAR). We used L-arginine and inhibitors of NO-synthase: nonselective inhibitor Nomega-nitro-L-arginine methyl ester, selective inhibitors of neuronal NO-synthase-7-nitro-indazole and selective inhibitor of inducible NO-synthase-S-methylisothiourea. We found that an increase in platelet aggregation in rat plasma observed in both periods is mediated via NO: in an early period of brain ischemia it depends on neuronal NO-synthase and in a late period--on inducible NO-synthase. Moreover, a nonselective inhibitor of all isoforms of NO-synthase (including endothelial NO-synthase) Nomega-nitro-L-arginine methyl ester possesses proaggregant properties. On the other hand, L-arginine in combination with 7-nitro-indazole and S-methylisothiourea induces maximal antiaggregatory effect. These data allows us to suppose that endothelium-derived NO decreases platelet aggregation in brain ischemia-reperfusion in rats.     

The machinery for oxidative protein folding in thermophiles

Disulfide bonds are required for the stability and function of many proteins. A large number of thiol-disulfide oxidoreductases, belonging to the thioredoxin superfamily, catalyze protein disulfide bond formation in all living cells, from bacteria to humans. The protein disulfide isomerase (PDI) is the eukaryotic factor that catalyzes oxidative protein folding in the endoplasmic reticulum; by contrast, in prokaryotes, a family of disulfide bond (Dsb) proteins have an equivalent outcome in the bacterial periplasm. Recently the results from genome analysis suggested an important role for disulfide bonds in the structural stabilization of intracellular proteins from thermophiles. A specific protein disulfide oxidoreductase (PDO) has a key role in intracellular disulfide shuffling in thermophiles. Here we focus on the structural and functional characterization of PDO correlated with the multifunctional eukaryotic PDI. In addition, we highlight the chimeric nature of the machinery for oxidative protein folding in thermophiles in comparison with the mesophilic bacterial and eukaryal counterparts.     

Folding and aggregation of export-defective mutants of the maltose-binding protein

We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31. To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.     

Heat shock proteins in protein folding and membrane translocation.

Constitutively expressed heat-shock proteins of the hsp60 and hsp70 families, classified as 'molecular chaperones', have important functions in the folding and intracellular sorting of newly-synthesized proteins. Recent studies of protein translocation across subcellular membranes have shown: (1) Proteins traverse membranes in extended conformations. (2) Cytosolic hsp70 proteins maintain newly-synthesized precursors destined for translocation in a loosely-folded, translocation-competent state. (3) Hsp 70 proteins on the trans-side of the membrane are required for efficient translocation. (4) In the case of mitochondria, the newly-imported polypeptides are transferred to the 'foldase' hsp60, the homologue of the E. coli groEL, which mediates their folding and oligomeric assembly. Hsp70 and hsp60 fulfill these various functions by their abilities, (1) to recognize an unknown structural element(s) which is transiently exposed in unfolded or incompletely folded polypeptides, and (2) to allow cycles of binding and (step-wise) release of the substrate protein catalyzed by ATP-hydrolysis.     

Involvement of Salmonella pathogenicity island 2 in the up-regulation of interleukin-10 expression in macrophages: role of protein kinase A signal pathway

Salmonellae are facultative intracellular bacteria capable of surviving within macrophages. Salmonella pathogenicity island 2 (SPI-2) is required for growth within macrophages and for virulence in mice. In this study, we show the involvement of SPI-2 in a signal transduction pathway that induces cytokine expression in Salmonella-infected macrophages. High levels of interleukin-10 (IL-10) mRNA were induced in macrophages by infection with wild-type salmonellae compared to a strain carrying a mutation in the spiC gene, which is encoded within SPI-2. The two strains had the same effect on the expression of proinflammatory cytokines such as IL-1 alpha, IL-6, and tumor necrosis factor alpha. IL-10 expression was dose dependently blocked by treatment of infected macrophages with the protein kinase A (PKA) inhibitor H-89, while IL-10 expression was increased by the PKA activator dibutyryl cyclic AMP. Cyclic AMP-dependent PKA activity was higher in macrophages infected with wild-type salmonellae compared to the spiC mutant, and Ser(132) phosphorylation of cyclic AMP response element-binding protein (CREB), which is an important mediator of PKA activation, correlated with the levels of PKA activity. Taken together, these results indicate that salmonellae cause an SPI-2-dependent increase in PKA activity that leads to CREB phosphorylation, resulting in up-regulation of IL-10 expression in Salmonella-infected macrophages. Suppression of IL-10 expression by an antisense oligonucleotide did not affect the growth of wild-type salmonellae within macrophages, whereas growth was dose dependently inhibited by H-89, suggesting that the PKA signaling pathway plays a significant role in intramacrophage Salmonella survival.     

Involvement of Fas/FasL pathway in the murine model of atopic dermatitis

Objective and design

The aim of this study was to elucidate the role of apoptosis mediated through Fas/FasL pathway using the mouse model of atopic dermatitis (AD).

Materials and treatment

AD was induced by epicutaneous application of ovalbumin (OVA) in wild-type C57BL/6, B6. MRL-Faslpr/J (Fas?) and B6Smn.C3-Faslgld/J (FasL?) mouse strains.

Methods

Skin samples were subjected to staining for Fas/FasL expression, M30 epitope and assessment of inflammatory response via immunohistochemical staining. Cytokine and chemokine production was assessed by real-time PCR.

Results

In comparison to wild-type mice, OVA sensitization of Fas- and FasL-deficient mice led to increased epidermal and dermal thickness, collagen deposition and local inflammation consisting of macrophages, neutrophils and CD4+?T cells. Fas- and FasL-deficient mice showed increased total counts of regulatory T cells (Tregs) and IgE levels in blood as well as increased expression of IL-1β, IL-4, IL-5, IL-13 and TGF-1β mRNA in comparison to wild-type mice. On the other hand, expression of CXCL9 and CXCL10, IL-17 mRNAs in the skin samples in Fas- and FasL-deficient mice was decreased.

Conclusions

Our results show that lack of the Fas-induced apoptosis leads to exacerbation of AD characteristics such as Th2 inflammation and dermal thickening. Therefore, Fas receptor can play an important role in AD pathogenesis by controlling development of the local inflammation.

     

Involvement of coerulospinal noradrenergic pathway in fentanyl-induced muscular rigidity in rats

Unilateral, site-specific microinjection of fentanyl (2.5 micrograms/50 nl) into the locus coeruleus (LC) in Sprague-Dawley rats anesthetized with ketamine evoked a significant increase in the electromyographic activity recorded from both caudal lateral extensor and gastrocnemius muscles. This correlate of opiate-induced muscular rigidity was appreciably antagonized by a pretreatment with the specific alpha 1-adrenoceptor blocker, prazosin (250 micrograms/kg, i.v.). On the other hand, an equimolar dose (0.65 mumol/kg) of the specific alpha 2-adrenoceptor blocker, yohimbine (0.23 mg/kg, i.v.) failed to prevent the occurrence of fentanyl-induced EMG activation. We suggest that the coerulospinal noradrenergic pathway may be directly involved in the elicitation of muscular rigidity by fentanyl, possibly via alpha 1-adrenoceptors in the spinal cord.