Stereospecific interactions of proline residues in protein structures and complexes

The constrained backbone torsion angle of a proline (Pro) residue has usually been invoked to explain its three-dimensional context in proteins. Here we show that specific interactions involving the pyrrolidine ring atoms also contribute to its location in a given secondary structure and its binding to another molecule. It is adept at participating in two rather non-conventional interactions, C-H...pi and C-H...O. The geometry of interaction between the pyrrolidine and aromatic rings, vis-à-vis the occurrence of the C-H...pi interactions has been elucidated. Some of the secondary structural elements stabilized by Pro-aromatic interactions are beta-turns, where a Pro can interact with an adjacent aromatic residue, and in antiparallel beta-sheet, where a Pro in an edge strand can interact with an aromatic residue in the adjacent strand at a non-hydrogen-bonded site. The C-H groups at the Calpha and Cdelta positions can form strong C-H...O interactions (as seen from the clustering of points) and such interactions involving a Pro residue at C' position relative to an alpha-helix can cap the hydrogen bond forming potentials of the free carbonyl groups at the helix C terminus. Functionally important Pro residues occurring at the binding site of a protein almost invariably engage aromatic residues (with one of them being held by C-H...pi interaction) from the partner molecule in the complex, and such aromatic residues are highly conserved during evolution.     

NF-kappa B hyperactivation has differential effects on the APC function of nonobese diabetic mouse macrophages

Type 1 diabetes is characterized by a chronic inflammatory response resulting in the selective destruction of the insulin-producing beta cells. We have previously demonstrated that dendritic cells (DCs) prepared from nonobese diabetic (NOD) mice, a model for spontaneous type 1 diabetes, exhibit hyperactivation of NF-kappaB resulting in an increased capacity to secrete proinflammatory cytokines and stimulate T cells compared with DCs of nondiabetic strains of mice. In the current study, the activational status of NF-kappaB and its role in regulating the APC function of macrophages (Mphi) prepared from NOD, nonobese resistant (NOR), and BALB/c mice was investigated. Independent of the stimulus, splenic and bone marrow-derived Mphi prepared from NOD mice exhibited increased NF-kappaB activation relative to NOR and BALB/c Mphi. This hyperactivation was detected for different NF-kappaB complexes and correlated with increased IkappaBalpha degradation. Furthermore, increased NF-kappaB activation resulted in an enhanced capacity of NOD vs NOR or BALB/c Mphi to secrete IL-12(p70), TNF-alpha, and IL-1alpha, which was inhibited upon infection with an adenoviral recombinant encoding a modified form of IkappaBalpha. In contrast, elevated NF-kappaB activation had no significant effect on the capacity of NOD Mphi to stimulate CD4(+) or CD8(+) T cells in an Ag-specific manner. These results demonstrate that in addition to NOD DCs, NOD Mphi exhibit hyperactivation of NF-kappaB, which correlates with an increased ability to mediate a proinflammatory response. Furthermore, NF-kappaB influences Mphi APC function by regulating cytokine secretion but not T cell stimulation.     

Glutamate counteracts the denaturing effect of urea through its effect on the denatured state

The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 m potassium l-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of l-glutamate, suggesting that l-glutamate has the ability to counteract the denaturing effect of urea. d-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of l-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 m potassium l-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N lambda-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.     

Mutation of an N-terminal acidic-rich region of p115-RhoGEF dissociates alpha13 binding and alpha13-promoted plasma membrane recruitment

The Ras homology (Rho) guanine nucleotide exchange factor p115-RhoGEF couples the alpha(13) heterotrimeric guanine nucleotide binding protein (G protein) subunit to Rho GTPase. Alpha(13) binds to a regulator of G protein signaling (RGS) domain in p115-RhoGEF, but the mechanism of alpha(13) activation of p115-RhoGEF is poorly understood. In this report, we demonstrate in cell-based assays that the acidic-rich N-terminus, adjacent to the RGS domain, is required for binding to activated alpha(13), and refine the importance of this region by showing that mutation of glutamic acids 27 and 29 in full-length p115-RhoGEF is sufficient to prevent interaction with activated alpha(13). However, alpha(13)-interacting deficient N-terminal mutants of p115-RhoGEF retain alpha(13)-dependent plasma membrane recruitment. Overall, these findings demonstrate a critical role for the N-terminal extension of p115-RhoGEF in mediating binding to alpha(13) and dissociate two activities of p115-RhoGEF: binding to activated alpha(13) and translocation to the PM in response to activated alpha(13).     

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.     

Isolation and characterization of rhodanese intermediates during thermal inactivation and their implications for the mechanism of protein aggregation

The initial steps of heat-induced inactivation and aggregation of the enzyme rhodanese have been studied and found to involve the early formation of modified but catalytically active conformations. These intermediates readily form active dimers or small oligomers, as evident from there being only a small increase in light scattering and an increase in fluorescence energy homotransfer from rhodanese labeled with fluorescein. These species are probably not the domain-unfolded form, as they show activity and increased protection of hydrophobic surfaces. Cross-linking with glutaraldehyde and fractionation by gel filtration show the predominant formation of dimer during heat incubation. Comparison between the rates of aggregate formation at 50 degrees C after preincubation at 25 or 40 degrees C gives evidence of product-precursor relationships, and it shows that these dimeric or small oligomeric species are the basis of the irreversible aggregation. The thermally induced species is recognized by and binds to the chaperonin GroEL. The unfoldase activity of GroEL subsequently unfolds rhodanese to produce an inactive conformation and forms a stable, reactivable complex. The release of 80% active rhodanese upon addition of GroES and ATP indicates that the thermal incubation induces an alteration in conformation, rather than any covalent modification, which would lead to formation of irreversibly inactive species. Once oligomeric species are formed from the intermediates, GroEL cannot recognize them. Based on these observations, a model is proposed for rhodanese aggregation that can explain the paradoxical effect in which rhodanese aggregation is reduced at higher protein concentration.     

High-resolution imaging demonstrates dynein-based vesicular transport of activated Trk receptors

Target-derived neurotrophins signal from nerve endings to the cell body to influence cellular and nuclear responses. The retrograde signal is conveyed by neurotrophin receptors (Trks) themselves. To accomplish this, activated Trks may physically relocalize from nerve endings to the cell bodies. However, alternative signaling mechanisms may also be used. To identify the vehicle wherein the activated Trks are located and transported, and to identify associated motor proteins that would facilitate transport, we use activation-state specific antibodies in concert with immunoelectron microscopy and deconvolution microscopy. We show that the'activated Trks within rat sciatic nerve axons are preferentially localized to coated and uncoated vesicles. These vesicles are moving in a retrograde direction and so accumulate distal to a ligation site. The P-Trk containing vesicles, in turn, colocalize with dynein components, and not with kinesins. Collectively, these results indicate activated Trk within axons travel in vesicles and dynein is the motor that drives these vesicles towards the cell bodies.     

A potentially important excretory-secretory product of Giardia lamblia

In this study we have reported the detailed characterization of a 58 kDa excretory-secretory product (ESP) of Giardia lamblia. The method of purification has been simplified which has improved the purification fold as well as the yield of the ESP. The binding efficacy of disialoganglioside (GD2) to the purified ESP was found to be maximum among all other gangliosides used. The N-terminal sequence of the immunoreactive 29 kDa peptide obtained from partial tryptic digest of the ESP was found to be AD-FVPQVST. The IgG against the purified ESP (IgGES) showed cross-reactivity with the binding subunit of the commercially available cholera toxin and also with two protein bands of western cottonmouth moccasin snake toxin. The ESP could accumulate fluid in the intestine of sealed adult mice and also induce morphological changes in HEp-2 cells. The crude extract of G. lamblia trophozoites preincubated with Escherichia coli revealed 8-fold augmentation in the cytopathic activity on HEp-2 cells as compared to that of crude preparation from trophozoites only.     

Truncated, inactive N-acetylglucosaminyltransferase III (GlcNAc-TIII) induces neurological and other traits absent in mice that lack GlcNAc-TIII

N-Acetylglucosaminyltransferase III (GlcNAc-TIII), the product of the Mgat3 gene, transfers the bisecting GlcNAc to the core mannose of complex N-glycans. The addition of this residue is regulated during development and has functional consequences for receptor signaling, cell adhesion, and tumor progression. Mice homozygous for a null mutation at the Mgat3 locus (Mgat3(Delta)) or for a targeted mutation in the Mgat3 gene (previously called Mgat3(neo), but herein renamed Mgat3(T37) because the allele generates inactive GlcNAc-TIII of approximately 37 kDa) were found to exhibit retarded progression of liver tumors. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of neutral N-glycans from kidneys revealed no significant differences, and both mutants showed the expected lack of N-glycan species with an additional GlcNAc. However, the two mutants differed in several biological traits. Mgat3(T37/T37) homozygotes in a mixed or 129(SvJ) background were retarded in growth rate and exhibited an altered leg clasp reflex, an altered gait, and defective nursing behavior. Pups abandoned by Mgat3(T37/T37) mothers were rescued by wild-type foster mothers. None of these Mgat3(T37/T37) traits were exhibited by Mgat3(Delta/Delta) mice or by heterozygous mice carrying the Mgat3(T37) mutation. Similarly, no dominant-negative effect was observed in Chinese hamster ovary cells expressing truncated GlcNAc-TIII in the presence of wild-type GlcNAc-TIII. However, compound heterozygotes carrying both the Mgat3(T37) and Mgat3(Delta) mutations exhibited a marked leg clasp reflex, indicating that in the absence of wild-type GlcNAc-TIII, truncated GlcNAc-TIII causes this phenotype. The Mgat3 gene was expressed in brain at embryonic day 10.5 and thereafter and in neurons of adult cerebellum. The mutant Mgat3 gene was also highly expressed in Mgat3(T37/T37) brain. This may be the basis of the unexpected neurological phenotype induced by truncated, inactive GlcNAc-TIII in the mouse.