Caspase cleavage product lacking amino-terminus of IkappaBalpha sensitizes resistant cells to TNF-alpha and TRAIL-induced apoptosis

In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity.     

Development of a novel diffusion-based method to estimate the size of the aggregated Abeta species responsible for neurotoxicity

beta-Amyloid peptide (Abeta) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration associated with the disease. Abeta is toxic only when aggregated, however, the size and structure of the aggregated species associated with toxicity is unknown. In the present study, we developed a diffusion-based method to simultaneously separate and detect the biological activity of toxic Abeta oligomers and used the method to examine the relationship between size of aggregated protein and toxicity to SH-SY5Y cells. From these measurements, the effective diffusivity and hydrodynamic radius of the toxic oligomeric species of Abeta could be determined. A sensitivity analysis was performed to examine the effects of model assumptions used in data analysis on the effective diffusivity calculated. The method provides a new estimate of the size of small toxic Abeta species associated with fibril formation. This work contributes to our understanding of the relationship between Abeta structure and toxicity and with further refinements may aid in our ability to design agents which alter the Abeta aggregation/dissociation processes associated with neurotoxicity.     

Tissue distribution of cytosolic beta-elimination reactions of selenocysteine Se-conjugates in rat and human

Selenocysteine Se-conjugates (e.g. methylselenocysteine) have been shown to be potent chemopreventive and chemoprotective agents, and inducers of apoptosis. Although the mechanism of action remains to be elucidated, beta-elimination of these compounds by beta-lyase enzymes into corresponding selenols, pyruvate and ammonia is thought to be critical. This study describes in vitro beta-lyase activity in nine rat organs using three selenocysteine Se-conjugates and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine. For all substrates the highest beta-elimination rates were found in kidney, followed by liver, while brain, spleen, heart, large and small intestine, thyroid and lung were of minor importance. Since liver plays an important role in beta-elimination, hepatic beta-lyase activity was extensively studied using 23 selenocysteine Se-conjugates and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and was compared with previously obtained renal beta-lyase data. The results showed that hepatic beta-lyase activities were 4-25-fold lower than the corresponding renal beta-lyase activities. Hepatic beta-elimination of the substrates appeared to be exclusively catalyzed by the pyridoxal 5'-phosphate-dependent beta-lyase enzyme kynureninase. Studies performed with human hepatic cytosols of three individuals showed that hepatic beta-lyase activity was 2-5-fold higher when compared with the previously obtained human renal activity. Significant correlation was obtained between human hepatic beta-lyase activities of three individuals. The relevance of this data for using SeCys-conjugates as chemopreventive and a chemoprotective agent is discussed. Based on the large differences in organ-selective beta-elimination and specific beta-lyase activity between rat and humans, the rat might not be a good model to investigate nephrotoxicity of cysteine S-conjugates, and chemoprevention and chemoprotection of SeCys-conjugates in man.     

Keratinocyte apoptosis on type I collagen gel caused by lack of laminin 5/10/11 deposition and Akt signaling

Cultured human foreskin keratinocytes (HFKs) adhere to and grow on nonfibrous collagen via integrin alpha2beta1. During incubation, the receptors used for adhesion are changed to integrins alpha3beta1 and alpha6beta4 and those receptors bind to laminin 5 which is deposited by keratinocytes themselves. In this report, we examined the behaviors of HFKs and transformed keratinocytes on collagen fibril gels. These cells adhered to and spread on collagen gels using integrin alpha2beta1. After several hours on collagen gels, however, cells became round and apoptosis occurred. The behavior of keratinocytes contrasted to that of fibroblasts that grew well even on collagen gel. At the point of apoptosis, integrins alpha2beta1 and alpha3beta1 were not found in the contact region of HFKs. Also, deposition of laminin 5 on collagen gel was not found despite the synthesis of mRNA for laminin 5 and laminin 10/11, while soluble laminin 5 protein is readily detectable. Phosporylation of Akt, which is known as a survival signal, was detected in HFKs cultured on coated collagen; however, the protein level and signals of Akt were dramatically decreased on collagen gel after 1 day of culture. These results indicate that collagen gel has different effects than nonfibrous collagen on HFKs and transformed keratinocytes and the interactions of integrin alpha3beta1 and laminin 5/10/11 are indispensable for maintenance of keratinocyte adhesion and survival.     

Amino acid intrinsic alpha-helical propensities III: positional dependence at several positions of C terminus

In this study, we have analyzed experimentally the helical intrinsic propensities of non-charged and non-aromatic residues at different C-terminal positions (C1, C2, C3) of an Ala-based peptide. The effect was found to be complex, resulting in extra stabilization or destabilization, depending on guest amino acid and position under consideration. Polar (Ser, Thr, Cys, Asn, and Gln) amino acids and Gly were found to have significantly larger helical propensities at several C-terminal positions compared with the alpha-helix center (-1.0 kcal/mole in some cases). Some of the nonpolar residues, especially beta-branched ones (Val and Ile) are significantly more favorable at position C3 (-0.3 to -0.4 kcal/mole), although having minor differences at other C-terminal positions compared with the alpha-helix center. Leu has moderate (-0.1 to -0.2 kcal/mole) stabilization effects at position C2 and C3, whereas being relatively neutral at C1. Finally, Met was found to be unfavorable at C1 and C2 ( +0.2 kcal/mole) and favorable at C3 (-0.2 kcal/mole). Thus, significant differences found between the intrinsic helical propensities at the C-terminal positions and those in the alpha-helix center must be accounted for in helix/coil transition theories and in protein design.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

Conserved and nonconserved features of the folding pathway of hisactophilin, a beta-trefoil protein

Based on previous studies of interleukin-1beta (IL-1beta) and both acidic and basic fibroblast growth factors (FGFs), it has been suggested that the folding of beta-trefoil proteins is intrinsically slow and may occur via the formation of essential intermediates. Using optical and NMR-detected quenched-flow hydrogen/deuterium exchange methods, we have measured the folding kinetics of hisactophilin, another beta-trefoil protein that has < 10% sequence identity and unrelated function to IL-1beta and FGFs. We find that hisactophilin can fold rapidly and with apparently two-state kinetics, except under the most stabilizing conditions investigated where there is evidence for formation of a folding intermediate. The hisactophilin intermediate has significant structural similarities to the IL-1beta intermediate that has been observed experimentally and predicted theoretically using a simple, topology-based folding model; however, it appears to be different from the folding intermediate observed experimentally for acidic FGF. For hisactophilin and acidic FGF, intermediates are much less prominent during folding than for IL-1beta. Considering the structures of the different beta-trefoil proteins, it appears that differences in nonconserved loops and hydrophobic interactions may play an important role in differential stabilization of the intermediates for these proteins.     

Charge states rather than propensity for beta-structure determine enhanced fibrillogenesis in wild-type Alzheimer's beta-amyloid peptide compared to E22Q Dutch mutant

The activity of the Alzheimer's amyloid beta-peptide is a sensitive function of the peptide's sequence. Increased fibril elongation rate of the E22Q Dutch mutant of the Alzheimer's amyloid beta-peptide relative to that of the wild-type peptide has been observed. The increased activity has been attributed to a larger propensity for the formation of beta structure in the monomeric E22Q mutant peptide in solution relative to the WT peptide. That hypothesis is tested using four nanosecond timescale simulations of the WT and Dutch mutant forms of the Abeta(10-35)-peptide in aqueous solution. The simulation results indicate that the propensity for formation of beta-structure is no greater in the E22Q mutant peptide than in the WT peptide. A significant measure of "flickering" of helical structure in the central hydrophobic cluster region of both the WT and mutant peptides is observed. The simulation results argue against the hypothesis that the Dutch mutation leads to a higher probability of formation of beta-structure in the monomeric peptide in aqueous solution. We propose that the greater stability of the solvated WT peptide relative to the E22Q mutant peptide leads to decreased fibril elongation rate in the former. Stability difference is due to the differing charge state of the two peptides. The other proposal leads to the prediction that the fibril elongation rates for the WT and the mutant E22Q should be similar under acid conditions.     

Population fragmentation in the southern rock agama, Agama atra: more evidence for vicariance in Southern Africa

Mitochondrial DNA sequence data derived from two genes were used to infer phylogeographical relationships between 13 Agama atra populations. Three distinct geographical assemblages were found among the lizard populations. The first occurs in southern Namibia, the second is restricted to the western dry arid regions of South Africa, whereas the third is distributed throughout the more mesic southern and eastern parts of the subcontinent. Geographically structured differences among populations within Agama clades are probably the result of dispersal and historic isolations among populations. At the broader scale, there were marked congruences between the Agama genetic discontinuities and those described previously in other rock-dwelling vertebrates such as Pronolagus rupestris and Pachydactylus rugosus. This suggests vicariance, probably as a response to natural climatic changes during the past three million years.