Understanding protein folding from globular to amyloid state: Aggregation: Darker side of protein

Folding and unfolding are crucial ways of modulating biological activity and targeting proteins to different cellular locations. In the living system, protein folding occurs in a very crowded environment, often assisted with helper proteins. In some cases this pathway can go off beam and the protein can either misfold or aggregate or form structures of elongated-unbranched morphology known as amyloid fibrils. Protein folding is not just an academic matter. Recombinant biotechnology and pharmaceutical industries are some of the fields where both theoretical and practical knowledge of protein folding is required. Misfolded protein and amyloid fibrils that escape the cellular quality control check are the basic reason of a number of increasingly widespread neurodegenerative diseases such as Alzheimer's and variant Creutzfeldt-Jakob etc. Thus, protein folding study also emerges as an interesting area in the field of biomedical research. This review deals with basic concepts related to protein folding and misfolding forming toxic aggregates and amyloid fibrils as well as disease associated with them. A more practical approach will be revealed to the early diagnosis of aggregation-prone diseases and amyloid states and their balanced therapeutics.     

FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability

Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.     

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked xenopus laevis embryos

In the present study we have characterized the synthesis of members of the HSP30 family during Xenopus laevis development using a polyclonal antipeptide antibody derived from the carboxyl end of HSP30C. Two-dimensional PAGE/immunoblot analysis was unable to detect any heat-inducible small HSPs in cleavage, blastula, gastrula, or neurula stage embryos. However, heat-inducible accumulation of a single protein was first detectable in early tailbud embryos with an additional 5 HSPs at the late tailbud stage and a total of 13 small HSPs at the early tadpole stage. In the Xenopus A6 kidney epithelial cell line, a total of eight heat-inducible small HSPs were detected by this antibody. Comparison of the pattern of protein synthesis in embryos and somatic cells revealed a number of common and unique heat inducible proteins in Xenopus embryos and cultured kidney epithelial cells. To specifically identify the protein product of the HSP30C gene, we made a chimeric gene construct with the Xenopus HSP30C coding sequence under the control of a constitutive promoter. This construct was microinjected into fertilized eggs and resulted in the premature and constitutive synthesis of the HSP30C protein in gastrula stage embryos. Through a series of mixing experiments, we were able to specifically identify the protein encoded by the HSP30C gene in embryos and somatic cells and to conclude that HSP30C synthesis was first heat-inducible at the early tailbud stage of development. The differential pattern of heat-inducible accumulation of members of the HSP30 family during Xenopus development suggests that these proteins may have distinct functions at specific embryonic stages during a stress response.     

Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules

Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane.     

Temporal patterns of protein synthesis inManduca epidermis during the change to pupal commitment in vitro: their modulation by 20-hydroxyecdysone and juvenile hormone

Summary The epidermis of final instar tobacco hornworm larvae,Manduca sexta, becomes committed to pupal differentiation in response to ecdysteroid in the absence of juvenile hormone (JH). Many changes in protein synthetic patterns have been noted during this time (Kiely and Riddiford 1985). To determine which of these changes are caused by ecdysteroid and which are important for the change of commitment, we have incubated larvally-committed epidermis for 24 h with 1 g/ml 20-hydroxyecdysone (20HE) and 3 g/ml epoxygeranylsesamole (EGS) (a JH mimic), with 3 g/ml EGS alone, or in hormone-free medium. Synthesis of larval-specific proteins such as insecticyanin and larval cuticular proteins was reduced to trace amounts or was undetectable after culture with 20HE for 24 h. The larval cuticular proteins that are greatly increasedin vivo on day 3 were not synthesized after exposure to 20HEin vitro. Ecdysteroid increased the synthesis of many of the proteins first seenin vivo on day 3 or during the wandering stage. The synthesis of about half of these latter proteins was inhibited by JH, indicating that they were likely part of the change of commitment. Other proteins that appear at this stagein vivo showed increased synthesis also in hormone-free medium and therefore were independent of the change of commitment.     

The role of aryl hydrocarbon receptor and the reactive oxygen species in the modulation of glutathione transferase by heavy metals in murine hepatoma cell lines

Glutathione transferase (GST) is a phase II detoxifying enzyme that plays a protective mechanism against oxidizing substances and toxic contaminants. Among these contaminants, heavy metals and polycyclic and halogenated aromatic hydrocarbons (PHAHs) have been shown to exert their toxic effects through the modulation of detoxifying enzymes, including the GSTs. Recently, we showed that heavy metals particularly Hg2+, Pb2+, and Cu2+ modulate the expression of phase II detoxifying enzymes such as NAD(P)H:quinone oxidoreductase 1 and Gsta1 in a concentration- and time-dependent manner. However, the effect of heavy metals and their potential interactions with aryl hydrocarbon receptor (AhR) ligands, PHAHs, on total Gst activity is still unknown. In the current study, we have investigated the effects of Hg2+, Pb2+, and Cu2+ in the absence and presence of four AhR ligands on the total Gst activity and reactive oxygen species (ROS) production in wild-type and AhR-deficient Hepa 1c1c7 cells. Our results showed that Hg2+ and Cu2+, but not Pb2+, significantly induced Gst activity in wild-type cells, whereas all metals induced the Gst activity in AhR-deficient cells. The induction of Gst activity by heavy metals was strongly correlated with an increase in the ROS production in wild-type, but not in AhR-deficient cells. Co-administration of heavy metals with AhR ligands differentially modulated Gst activity, in that co-exposure to Hg2+ plus AhR ligands could be beneficial in protecting against cytotoxicity as demonstrated by the increase in Gst activity with a proportional decrease in ROS production. Whereas co-exposure to Cu2+ plus AhR ligands was more toxic in that a decrease in Gst activity and an increase in oxidative stress of the cell were observed. We concluded that heavy metals differentially modulate the Gst activity through oxidative stress- and AhR-mediated mechanisms.