Hsp90 chaperones PPARγ and regulates differentiation and survival of 3T3-L1 adipocytes.

Adipose tissue dysregulation has a major role in various human diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) is a key regulator of adipocyte differentiation and function, as well as a target of insulin-sensitizing drugs. The Hsp90 chaperone stabilizes a diverse set of signaling 'client' proteins, thereby regulates various biological processes. Here we report a novel role for Hsp90 in controlling PPARγ stability and cellular differentiation. Specifically, we show that the Hsp90 inhibitors geldanamycin and novobiocin efficiently impede the differentiation of murine 3T3-L1 preadipocytes. Geldanamycin at higher concentrations also inhibits the survival of both developing and mature adipocytes, respectively. Further, Hsp90 inhibition disrupts an Hsp90-PPARγ complex, leads to the destabilization and proteasomal degradation of PPARγ, and inhibits the expression of PPARγ target genes, identifying PPARγ as an Hsp90 client. A similar destabilization of PPARγ and a halt of adipogenesis also occur in response to protein denaturing stresses caused by a single transient heat-shock or proteasome inhibition. Recovery from stress restores PPARγ stability and adipocyte differentiation. Thus, our findings reveal Hsp90 as a critical stress-responsive regulator of adipocyte biology and offer a potential therapeutic target in obesity and the metabolic syndrome.

Spondyloarthropathies and bone resorption: a possible role of heat shock protein (Hsp70).

Spondyloarthropathies consist of chronic inflammatory disorders genetically linked with each other through HLA-B27 molecules, and are connected with the destruction of periarticular bone and also with systemic bone loss in many cases. Expected molecular mechanisms behind these conditions overlap the functions of Hsp70s, a group of major molecular chaperones and cytokines. Hsp70s may control disease progression via inhibition of unfolded HLA-B27 protein accumulation and alteration of ER stress signaling. Further, Hsp70s may improve disease related malfunction of antigen presentation, and may induce nitric oxide (NO) release from macrophages which probably protective against spondyloarthropathies as well. Considering premised possible influence of Hsp70s on core mechanisms of spondyloarthropathies it may be expected that, increased expression of Hsp70s advantageously retards disease progress, or may lead to remission. On the other hand Hsp70s as danger signals induce the secretion of proinflammatory cytokines playing major role in the progression of spondyloarthropathy induced bone loss. Consequently, the effect of Hsp70s on the progression of spondyloarthropathic bone loss is "Janus-faced" in some respect: increase of Hsp70s' level is likely advantageous regarding to the core of disorder; but it may facilitate existing bone resorption processes.

Salivary Genomics, Transcriptomics and Proteomics: The Emerging Concept of the Oral Ecosystem and their Use in the Early Diagnosis of Cancer and other Diseases.

There is an increasingly growing interest world-wide for the genomics, transcriptomics and proteomics of saliva and the oral cavity, since they provide a non-invasive source of unprecedently rich genetic information. The complexity of oral systems biology goes much beyond the human genome, transcriptome and proteome revealed by oral mucosal cells, gingival crevicular fluid, and saliva, and includes the complexity of the oral microbiota, the symbiotic assembly of bacterial, fungal and other microbial flora in the oral cavity. In our review we summarize the recent information on oral genomics, transcriptomics and proteomics, of both human and microbial origin. We also give an introduction and practical advice on sample collection, handling and storage for analysis. Finally, we show the usefulness of salivary and oral genomics in early diagnosis of cancer, as well as in uncovering other systemic diseases, infections and oral disorders. We close the review by highlighting a number of possible exploratory pathways in this emerging, hot research field.

Aging cellular networks: chaperones as major participants.

We increasingly rely on the network approach to understand the complexity of cellular functions. Chaperones (heat shock proteins) are key "networkers", which sequester and repair damaged proteins. In order to link the network approach and chaperones with the aging process, we first summarize the properties of aging networks suggesting a "weak link theory of aging". This theory suggests that age-related random damage primarily affects the overwhelming majority of the low affinity, transient interactions (weak links) in cellular networks leading to increased noise, destabilization and diversity. These processes may be further amplified by age-specific network remodelling and by the sequestration of weakly linked cellular proteins to protein aggregates of aging cells. Chaperones are weakly linked hubs (i.e., network elements with a large number of connections) and inter-modular bridge elements of protein-protein interaction, signalling and mitochondrial networks. As aging proceeds, the increased overload of damaged proteins is an especially important element contributing to cellular disintegration and destabilization. Additionally, chaperone overload may contribute to the increase of "noise" in aging cells, which leads to an increased stochastic resonance resulting in a deficient discrimination between signals and noise. Chaperone- and other multi-target therapies, which restore the missing weak links in aging cellular networks, may emerge as important anti-aging interventions.

Chemical chaperones: mechanisms of action and potential use.

An increasing number of studies indicate that low-molecular-weight compounds can help correct conformational diseases by inhibiting the aggregation or enable the mutant proteins to escape the quality control systems, and thus their function can be rescued. The small molecules were named chemical chaperones and it is thought that they nonselectively stabilize the mutant proteins and facilitate their folding. Chemical chaperones are usually osmotically active, such as DMSO, glycerol, or deuterated water, but other compounds, such as 4-phenylbutiric acid, are also members of the chemical chaperone group. More recently, compounds such as receptor ligands or enzyme inhibitors, which selectively recognize the mutant proteins, were also found to rescue conformational mutants and were termed pharmacological chaperones. An increasing amount of evidence suggests that the action of pharmacological chaperones could be generalized to a large number of misfolded proteins, representing new therapeutic possibilities for the treatment of conformational diseases. A new and exciting strategy has recently been developed, leading to the new chemical group called folding agonist. These small molecules are designed to bind proteins and thus restore their native conformation.

Pharmacological modulation of the heat shock response.

Life presents a continuous series of stresses. Increasing the adaptation capacity of the organism is a long-term survival factor of various organisms and has become an attractive field of intensive therapeutic research. Induction of the heat shock response promotes survival after a wide variety of environmental stresses. Preclinical studies have proven that physiological and pharmacological chaperone inducers and co-inducers are an efficient therapeutic approach in different acute and chronic diseases. In this chapter, we summarize current knowledge of the current state of chaperone modulation and give a comprehensive list of the main drug candidates.

Pharmacological attenuation of apoptosis in reoxygenated endothelial cells.

BRX-235 (Iroxanadine), a novel drug developed by Biorex (Hungary), was previously characterized as a vasculoprotector against atherosclerosis, an activator of p38 kinase, and an enhancer of stress-responsive heat shock protein (Hsp) expression. The present data demonstrate that BRX-235 may improve survival of vascular endothelial cells (ECs) following ischemia/reperfusion stress. ECs cultured from human umbilical veins were exposed to hypoxia/reoxygenation to mimic ischemia/reperfusion. Caspase activation and apoptosis were monitored in the reoxygenated cells. Addition of BRX-235 (0.1-1 microM) to culture medium prior to hypoxia or at start of reoxygenation significantly reduced the caspase-dependent apoptosis. The cytoprotection conferred by the pre-hypoxic drug administration was sensitive to quercetin and seems to be based on enhanced Hsp accumulation in stressed ECs. In the case of post-hypoxic drug administration, the cytoprotection was strongly inhibited by SB202190 and SB203580 and appears to be associated with enhanced p38 kinase activation in reoxygenated ECs.

The effect of treatment with BRX-220, a co-inducer of heat shock proteins, on sensory fibers of the rat following peripheral nerve injury.

In this study, we examined the effect BRX-220, a co-inducer of heat shock proteins, in injury-induced peripheral neuropathy. Following sciatic nerve injury in adult rats and treatment with BRX-220, the following features of the sensory system were studied: (a) expression of calcitonin gene-related peptide (CGRP); (b) binding of isolectin B4 (IB4) in dorsal root ganglia (DRG) and spinal cord; (c) stimulation-evoked release of substance P (SP) in an in vitro spinal cord preparation and (d) nociceptive responses of partially denervated rats. BRX-220 partially reverses axotomy-induced changes in the sensory system. In vehicle-treated rats there is a decrease in IB4 binding and CGRP expression in injured neurones, while in BRX-220-treated rats these markers were better preserved. Thus, 7.0 +/- 0.6% of injured DRG neurones bound IB4 in vehicle-treated rats compared to 14.4 +/- 0.9% in BRX-220-treated animals. Similarly, 4.5 +/- 0.5% of DRG neurones expressed CGRP in the vehicle-treated group, whereas 9.0 +/- 0.3% were positive in the BRX-220-treated group. BRX-220 also partially restored SP release from spinal cord sections to electrical stimulation of primary sensory neurones. Behavioural tests carried out on partially denervated animals showed that BRX-220 treatment did not prevent the emergence of mechanical or thermal hyperalgesia. However, oral treatment for 4 weeks lead to reduced pain-related behaviour suggesting either slowly developing analgesic actions or enhancement of recovery processes. Thus, the morphological improvement seen in sensory neurone markers was accompanied by restored functional activity. Therefore, treatment with BRX-220 promotes restoration of morphological and functional properties in the sensory system following peripheral nerve injury.

Upregulation of heat shock proteins rescues motoneurones from axotomy-induced cell death in neonatal rats.

Heat shock proteins (hsps) are induced in a variety of cells following periods of stress, where they promote cell survival. In this study, we examined the effect of upregulating hsp expression by treatment with BRX-220, a co-inducer of hsps, on the survival of injured motoneurones. Following sciatic nerve crush at birth, rat pups were treated daily with BRX-220. The expression of hsp70 and hsp90, motoneurone survival, and muscle function was examined at various intervals later and the number of functional motor units was assessed by in vivo isometric tension recordings. Fourteen days after injury, significantly more motoneurones survived in the BRX-220-treated group (39 +/- 2.8%) compared to the saline-treated group (21 +/- 1.7%). Moreover, in the BRX-220-treated group no further loss of motoneurones occurred, so that at 10 weeks 42 +/- 2.1% of motoneurones survived compared to 15 +/- 0.6% in the untreated group. There were also more functional motor units in the hindlimb muscles of BRX-220-treated animals. In addition, treatment with BRX-220 resulted in a significant increase in the expression of hsp70 and hsp90 in glia and neurones. Thus, treatment with BRX-220, a co-inducer of hsps, protects motoneurones from axotomy-induced cell death.

Water and cellular folding processes.

Proteins require a unique, native structure to perform their functions. Water molecules play an important role to develop and maintain this three-dimensional structure. Water is also necessary for several forms of enzyme catalysis, and is a constituent of many protein-protein, protein-DNA, or protein-RNA interfaces. Larger proteins acquire their native structure in a complicated folding pathway having several folding traps. Recent data indicated a key role of water molecules in this process. Protein flexibility, structural rearrangements, conformational transitions all require the fluctuating changes in hydrogen bond structure provided by interacting water molecules. Besides proteins, RNA and DNA structure is also heavily influenced by the presence of water. This review summarizes the important aspects of these fields, and draws attention to several open questions and hypotheses.

Chaperone overload is a possible contributor to 'civilization diseases'.

Molecular chaperones dampen the effect of damaging mutations that would otherwise be removed from the population by natural selection. Here, I propose that the development of modern medical practice depressed this process, leading to a rise of phenotypically silent mutations in the genome. The background of misfolded proteins increases during ageing and, by competition, prevents the chaperone-mediated buffering of silent mutations. Phenotypically exposed mutations contribute to a more-abundant manifestation of multigene-diseases. This 'chaperone overload' hypothesis emphasizes the need for efficient ways to enhance chaperone capacity in ageing subjects, and will hopefully lead to the identification and 'repair' of silent mutations.

The somatostatin analogue TT-232 induces apoptosis in A431 cells: sustained activation of stress-activated kinases and inhibition of signalling to extracellular signal-regulated kinases.

TT-232 is a somatostatin analogue containing a five-residue ring structure. The present report describes TT-232-induced signalling events in A431 cells, where a 4-h preincubation with the peptide irreversibly induced a cell death program, which involves DNA-laddering and the appearance of shrunken nuclei, but is unrelated to somatostatin signalling. Early intracellular signals of TT-232 include a transient two-fold activation of the extracellular signal-regulated kinase (ERK2) and a strong and sustained activation of the stress-activated protein kinases c-Jun NH(2)-terminal kinase (JNK)/SAPK and p38MAPK. Blocking the signalling to ERK or p38MAPK activation had no effect on the TT-232-induced cell killing. At the commitment time for inducing cell death, TT-232 decreased EGFR-tyrosine phosphorylation and prevented epidermial growth factor (EGF)-induced events like cRaf-1 and ERK2 activation. Signalling to ERK activation by FCS, phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF) was similarly blocked. Our data suggest that TT-232 triggers an apoptotic type of cell death, concomitant with a strong activation of JNK and a blockade of cellular ERK2 activation pathways.

Physical and functional interactions between protein tyrosine phosphatase alpha, PI 3-kinase, and PKCdelta.

The somatostatin analogue, TT-232 inhibits cell proliferation and induces apoptosis in a variety of tumor cells both in vivo and in vitro. While the early transient activation of Erk/MAPK was found to be important for the induction of cell cycle arrest, the signaling pathway leading to the activation of Erk/MAPK had not been fully established. Here we present evidence that activation of the Erk/MAPK pathway by TT-232 involves PI 3-kinase, PKCdelta and the protein tyrosine phosphatase alpha (PTPalpha). We show a physical interaction of PI 3-kinase and PKCdelta with PTPalpha and show that the tyrosine phosphatase plays a role in the activation of MAPK. In this process, PTPalpha Ser-180 and Ser-204 phosphorylation is critical for the induction of phosphatase activity, which is required for dephosphorylation of pp60(c-src). Taken together, we demonstrate the physical and functional association between PI 3-kinase, PKCdelta and PTPalpha in a signaling complex that mediates the antitumor activity of the somatostatin analogue TT-232.

[Diabetes mellitus as a general membrane disease and its consequences]. / Diabetes mellitus mint általános membránbetegség és ennek következményei.

The metabolic disturbances and their consequences in diabetes mellitus are well known more or less in details too. However, our knowledge on the diabetic disorders in membrane functions are limited. These damages are connected mostly with the disregulation of the membrane protein syntheses due to deficiency of insulin. In this review the impairments of the Na(+)-pump and the Ca(2+)-transport mechanisms as well as the insulin-dependent glucose transporter GLUT4 will be discussed in diabetes. The capacity of these transporters could be decreased even more than 50 percent in diabetes. This is the reason why using the same dose of cardioactive steroids as if in not diabetic subjects--can cause toxic alterations on the heart in diabetic patients. Insulin regulates not only the expression of some membrane proteins but it can initiate the translocation of the Na(+)-pump and GLUT4 from the intracellular membrane compartments to the plasma membrane in muscle, heart and adipose tissue. Therefore the uptake of K+ and glucose into these tissues will increase significantly under the acute influence of insulin. Untreated diabetic patients generally show hyperkalemia. Forceful treatment with insulin of these subjects often causes severe hypokalemia as a consequence of sudden translocation of the Na(+)-pump. Different Ca(2+)-transport systems are also impaired in diabetes. These changes may result significantly higher free Ca2+ concentration in the cytoplasma of cardiomyocytes. This is one of the most important reason for the Ca2+ overloading and ultimately for heart death. According to authors opinion, beside the dangerous metabolic disorders, general membrane damage and extended disturbances in membrane functions are also very characteristic for diabetes. The acknowledgement of these alterations are very important for the exact planning of the up to date treatment of diabetes.

[Thiazolidinediones--a new class of oral antidiabetic drugs]. / Tiazolidindionok--az oralis antidiabeticumok új hatástani csoportja.

The discovery of a new class of oral antidiabetic drugs was stimulated by difficulties with the treatment currently available for patients with type 2 diabetes mellitus. Thiazolidinediones can lower blood glucose values due to their special insulin-sensitiser effect. In this way, these drugs seem to be very effective in the treatment of type 2 diabetic patients with characteristics of metabolic syndrome. The intracellular action caused by thiazolidinediones differs markedly from that of other oral antidiabetic drugs available. Apart from antihyperglycaemic effect, thiazolidinediones have further beneficial effects in experimental diabetes which require corroboration by clinical studies. Troglitazone was the first drug which reached the market. Unfortunately, this drug was withdrawn soon due to its hepatotoxicity. Rosiglitazone proved to be much safer in clinical studies. Pioglitazone is being tested nowadays in clinical studies. Thiazolidinediones have been already listed among oral antidiabetic drugs in international therapeutical guidelines. Nevertheless, further clinical studies and experiences are needed to determine the final exact indication of thiazolidinediones for the treatment of type 2 diabetic patients.

A nonconventional role of molecular chaperones: involvement in the cytoarchitecture.

A hallmark of chaperone action is assistance in protein folding. Indeed, folding of nascent prokaryotic proteins proceeds mostly as a chaperone-assisted, posttranslational event. On the contrary, in nonstressed eukaryotic cells folding-related tasks of eukaryotic chaperones are restricted to a subset of proteins, and "jobless" chaperones may form an extension of the cytoarchitecture, facilitating intracellular traffic of proteins and other macromolecules.

The antitumor somatostatin analogue TT-232 induces cell cycle arrest through PKCdelta and c-Src.

The heptapeptide TT-232 is structurally related to the hypothalamic hormone somatostatin and shows promise as an anticancer drug because of its tumor-specific cytotoxic effects. Apart from the ability to induce apoptosis, the synthetic peptide can trigger an alternative pathway that leads to cell cycle arrest in certain tumor cell systems. We found that pulse treatment with TT-232 blocks the cell cycle G(1)/S transition irreversibly in A431 cells. Investigation of the TT-232 signaling pathway yielded results similar to those reported for somatostatin although its affinity to the somatostatin receptor 1 is significantly reduced. We show that functional protein kinase C (PKC) delta as well as c-Src are necessary mediators of the TT-232 cytostatic effect and we propose a signaling pathway that leads to cell cycle arrest.

Streptozotocin-induced diabetes alters the oligomerization pattern of acetylcholinesterase in rat skeletal muscle.

AIMS/HYPOTHESIS: Diabetes mellitus has a serious effect on most of the properties of skeletal muscles. Changes in neuromuscular transmission are also involved in propagating the disease. METHODS: In our experiments, acetylcholinesterase was extracted from the fast extensor digitorum longus and slow soleus muscles of control, non-treated 6-week-diabetic and insulin-treated diabetic rats. The extracts were applied to velocity sedimentation and acetylcholinesterase activity was determined. RESULTS: We observed considerable differences in the distribution of individual acetylcholinesterase molecular forms in diabetic fast muscles. This included a 59% decline in G4 content together with a fivefold increase in A8 and a 53 % increase in A12 activity resulting in a shift of acetylcholinesterase profile characteristically towards slow muscles. These alterations were partly reversed by insulin treatment. CONCLUSION/INTERPRETATION: In slow muscles diabetes caused an increase in G4 activity without affecting the sedimentation profile. Decline in G4 content in fast muscles could contribute to enhanced desensitization of acetylcholine receptors in diabetes.

Protein-disulfide isomerase- and protein thiol-dependent dehydroascorbate reduction and ascorbate accumulation in the lumen of the endoplasmic reticulum.

The transport and intraluminal reduction of dehydroascorbate was investigated in microsomal vesicles from various tissues. The highest rates of transport and intraluminal isotope accumulation (using radiolabeled compound and a rapid filtration technique) were found in hepatic microsomes. These microsomes contain the highest amount of protein-disulfide isomerase, which is known to have a dehydroascorbate reductase activity. The steady-state level of intraluminal isotope accumulation was more than 2-fold higher in hepatic microsomes prepared from spontaneously diabetic BioBreeding/Worcester rats and was very low in fetal hepatic microsomes although the initial rate of transport was not changed. In these microsomes, the amount of protein-disulfide isomerase was similar, but the availability of protein thiols was different and correlated with dehydroascorbate uptake. The increased isotope accumulation was accompanied by a higher rate of dehydroascorbate reduction and increased protein thiol oxidation in microsomes from diabetic animals. The results suggest that both the activity of protein-disulfide isomerase and the availability of protein thiols as reducing equivalents can play a crucial role in the accumulation of ascorbate in the lumen of the endoplasmic reticulum. These findings also support the fact that dehydroascorbate can act as an oxidant in the protein-disulfide isomerase-catalyzed protein disulfide formation.

Interaction of the human DnaJ homologue, HSJ1b with the 90 kDa heat shock protein, Hsp90.

The 90 kDa heat shock protein (Hsp90) is a major cytoplasmic molecular chaperone associating with numerous other proteins. Both genetic and in vitro refolding experiments using reticulocyte lysate have suggested a functional interaction of Hsp90 with yeast human homologues of E. coli DnaJ. Here we present direct evidence using surface plasmon resonance that Hsp90 and the human DnaJ homologue, HSJ1b, bind to each other. We also show that Hsp90 and HSJ1b transfer alpha-lactalbumin to each other in an ATP-dependent manner. The two chaperones have additive effects in preventing rhodanese aggregation.