A statistical mechanical model for β-hairpin kinetics

Understanding the mechanism of protein secondary structure formation is an essential part of the protein-folding puzzle. Here, we describe a simple statistical mechanical model for the formation of a β-hairpin, the minimal structural element of the antiparallel β-pleated sheet. The model accurately describes the thermodynamic and kinetic behavior of a 16-residue, β-hairpin-forming peptide, successfully explaining its two-state behavior and apparent negative activation energy for folding. The model classifies structures according to their backbone conformation, defined by 15 pairs of dihedral angles, and is further simplified by considering only the 120 structures with contiguous stretches of native pairs of backbone dihedral angles. This single sequence approximation is tested by comparison with a more complete model that includes the 215 possible conformations and 15 × 215 possible kinetic transitions. Finally, we use the model to predict the equilibrium unfolding curves and kinetics for several variants of the β-hairpin peptide.

A mouse model for adenovirus gene delivery

The cellular attachment receptor for adenovirus (Ad), Coxsackie adenovirus receptor (CAR), required for delivery of Ad into primary cells, is not present on all cell types, thus restricting Ad-gene delivery systems. To circumvent this constrain, a transgenic mouse has been generated that expresses a truncated human CAR in all tissues analyzed. These mice allowed efficient in vitro infections at low multiplicities into lymphoid, myeloid, and endothelial cells. Furthermore, in vivo administration of Ad-vectors results in infection of macrophages, lymphocytes, and endothelial cells. In addition, tail vein injection resulted in targeting of virus into previously inaccessible areas, such as the lung and the capillaries of the brain. The CAR transgenic mice will be useful for rapid functional genomic analysis in vivo, for testing the efficacy of gene therapy procedures or as a source of easily transducible cells.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus.

RNA synthesis by the paramyxovirus respiratory syncytial virus, a ubiquitous human pathogen, was found to be more complex than previously appreciated for the nonsegmented negative-strand RNA viruses. Intracellular RNA replication of a plasmid-encoded "minigenome" analog of viral genomic RNA was directed by coexpression of the N, P, and L proteins. But, under these conditions, the greater part of mRNA synthesis terminated prematurely. This difference in processivity between the replicase and the transcriptase was unanticipated because the two enzymes ostensively shared the same protein subunits and template. Coexpression of the M2 gene at a low level of input plasmid resulted in the efficient production of full-length mRNA and, in the case of a dicistronic minigenome, sequential transcription. At a higher level, coexpression of the M2 gene inhibited transcription and RNA replication. The M2 mRNA contains two overlapping translational open reading frames (ORFs), which were segregated for further analysis. Expression of the upstream ORF1, which encoded the previously described 22-kDa M2 protein, was associated with transcription elongation. A model involving this protein in the balance between transcription and replication is proposed. ORF2, which lacks an assigned protein, was associated with inhibition of RNA synthesis. We propose that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions.

Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers.

A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.

Truncated WT1 mutants alter the subnuclear localization of the wild-type protein.

WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors. Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins. The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus. In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1 (+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing. Localization to subnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments. Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures.

min K channels form by assembly of at least 14 subunits.

Injection of min K mRNA into Xenopus oocytes results in expression of slowly activating voltage-dependent potassium channels, distinct from those induced by expression of other cloned potassium channels. The min K protein also differs in structure, containing only a single predicted transmembrane domain. While it has been demonstrated that all other cloned potassium channels form by association of four independent subunits, the number of min K monomers which constitute a functional channel is unknown. In rat min K, replacement of Ser-69 by Ala (S69A) causes a shift in the current-voltage (I-V) relationship to more depolarized potentials; currents are not observed at potentials negative to 0 mV. To determine the subunit stoichiometry of min K channels, wild-type and S69A subunits were coexpressed. Injections of a constant amount of wild-type mRNA with increasing amounts of S69A mRNA led to potassium currents of decreasing amplitude upon voltage commands to -20 mV. Applying a binomial distribution to the reduction of current amplitudes as a function of the different coinjection mixtures yielded a subunit stoichiometry of at least 14 monomers for each functional min K channel. A model is presented for how min K subunits may form a channel.

A model for the transcriptional regulation of the CYP2B1/B2 gene in rat liver.

The phenobarbitone-responsive minimal promoter has been shown to lie between nt -179 and nt + 1 in the 5' (upstream) region of the CYP2B1/B2 gene in rat liver, on the basis of the drug responsiveness of the sequence linked to human growth hormone gene as reporter and targeted to liver as an asialoglycoprotein-DNA complex in vivo. Competition analyses of the nuclear protein-DNA complexes formed in gel shift assays with the positive (nt -69 to -98) and negative (nt -126 to -160) cis elements (PE and NE, respectively) identified within this region earlier indicate that the same protein may be binding to both the elements. The protein species purified on PE and NE affinity columns appear to be identical based on SDS/PAGE analysis, where it migrates as a protein of 26-28 kDa. Traces of a high molecular weight protein (94-100 kDa) are also seen in the preparation obtained after one round of affinity chromatography. The purified protein stimulates transcription of a minigene construct containing the 179 nt on the 5' side of the CYP2B1/B2 gene linked to the I exon in a cell-free system from liver nuclei. The purified protein can give rise to all the three complexes (I, II, and III) with the PE, just as the crude nuclear extract, under appropriate conditions. Manipulations in vitro indicate that the NE has a significantly higher affinity for the dephosphorylated form than for the phosphorylated form of the protein. The PE binds both forms. Phenobarbitone treatment of the animal leads to a significant increase in the phosphorylation of the 26- to 28-kDa and 94-kDa proteins in nuclear labeling experiments followed by isolation on a PE affinity column. We propose that the protein binding predominantly to the NE in the dephosphorylated state characterizes the basal level of transcription of the CYP2B1/B2 gene. Phenobarbitone treatment leads to phosphorylation of the protein, shifting the equilibrium toward binding to the PE. This can promote interaction with an upstream enhancer through other proteins such as the 94-kDa protein and leads to a significant activation of transcription.

Negative activation enthalpies in the kinetics of protein folding.

Although the rates of chemical reactions become faster with increasing temperature, the converse may be observed with protein-folding reactions. The rate constant for folding initially increases with temperature, goes through a maximum, and then decreases. The activation enthalpy is thus highly temperature dependent because of a large change in specific heat (delta Cp). Such a delta Cp term is usually presumed to be a consequence of a large decrease in exposure of hydrophobic surfaces to water as the reaction proceeds from the denatured state to the transition state for folding: the hydrophobic side chains are surrounded by "icebergs" of water that melt with increasing temperature, thus making a large contribution to the Cp of the denatured state and a smaller one to the more compact transition state. The rate could also be affected by temperature-induced changes in the conformational population of the ground state: the heat required for the progressive melting of residual structure in the denatured state will contribute to delta Cp. By examining two proteins with different refolding mechanisms, we are able to find both of these two processes; barley chymotrypsin inhibitor 2, which refolds from a highly unfolded state, fits well to a hydrophobic interaction model with a constant delta Cp of activation, whereas barnase, which refolds from a more structured denatured state, deviates from this ideal behavior.

Modulation of c-Myb-induced transcription activation by a phosphorylation site near the negative regulatory domain.

The c-myb protooncogene encodes a highly conserved transcription factor that functions as both an activator and a repressor of transcription. The v-myb oncogenes of E26 leukemia virus and avian myeloblastosis virus encode proteins that are truncated at both the amino and the carboxyl terminus, deleting portions of the c-Myb DNA-binding and negative regulatory domains. This has led to speculation that the deleted regions contain important regulatory sequences. We previously reported that the 42-kDa mitogen-activated protein kinase (p42mapk) phosphorylates chicken and murine c-Myb at multiple sites in the negative regulatory domain in vitro, suggesting that phosphorylation might provide a mechanism to regulate c-Myb function. We now report that three tryptic phosphopeptides derived from in vitro phosphorylated c-Myb comigrate with three tryptic phosphopeptides derived from metabolically labeled c-Myb immunoprecipitated from murine erythroleukemia cells. At least two of these peptides are phosphorylated on serine-528. Replacement of serine-528 with alanine results in a 2- to 7-fold increase in the ability of c-Myb to transactivate a Myb-responsive promoter/reporter gene construct. These findings suggest that phosphorylation serves to regulate c-Myb activity and that loss of this phosphorylation site from the v-Myb proteins may contribute to their transforming potential.

Dominant-negative action of the jimpy mutation in mice complemented with an autosomal transgene for myelin proteolipid protein.

Mutations in genes encoding membrane proteins have been associated with cell death of unknown cause from invertebrate development to human degenerative diseases. A point mutation in the gene for myelin proteolipid protein (PLP) underlies oligodendrocyte death and dysmyelination in jimpy mice, an accurate model for Pelizaeus-Merzbacher disease. To distinguish the loss of PLP function from other effects of the misfolded protein, we took advantage of the X chromosomal linkage of the gene and have complemented jimpy with a wild-type PLP transgene. In this artificial heterozygous situation, the jimpy mutation emerged as genetically dominant. At the cellular level oligodendrocytes showed little increase in survival although endogenous PLP gene and autosomal transgene were truly coexpressed. In surviving oligodendrocytes, wild-type PLP was functional and immunodetectable in myelin. Moreover, compacted myelin sheaths regained their normal periodicity. This strongly suggests that, despite the presence of functional wild-type PLP, misfolded jimpy PLP is by itself the primary cause of abnormal oligodendrocyte death.

Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene.

Mutations in the APC (adenomatous polyposis coli) gene appear to be responsible for not only familial adenomatous polyposis but also many sporadic cases of gastrointestinal cancers. Using homologous recombination in mouse embryonic stem cells, we constructed mice that contained a mutant gene encoding a product truncated at a 716 (Apc delta 716). Mendelian transmission of the gene caused most homozygous mice to die in utero before day 8 of gestation. The heterozygotes developed multiple polyps throughout the intestinal tract, mostly in the small intestine. The earliest polyps arose multifocally during the third week after birth, and new polyps continued to appear thereafter. Surprisingly, every nascent polyp consisted of a microadenoma covered with a layer of the normal villous epithelium. These microadenomas originated from single crypts by forming abnormal outpockets into the inner (lacteal) side of the neighboring villi. We carefully dissected such microadenomas from nascent polyps by peeling off the normal epithelium and determined their genotype by PCR: all microadenomas had already lost the wild-type Apc allele, whereas the mutant allele remained unchanged. These results indicate that loss of heterozygosity followed by formation of intravillous microadenomas is responsible for polyposis in Apc delta 716 intestinal mucosa. It is therefore unlikely that the truncated product interacts directly with the wild-type protein and causes the microadenomas by a dominant negative mechanism.

Parenting influences on depression: A moderated mediated model

Little is known about the developmental processes through which parenting factors may influence clinical depression among youth. This study investigated whether parenting influences the onset of clinical depression through the mediating mechanism of negative attributional style, particularly under conditions of high stress, in a community sample of children and adolescents (N = 289). Results supported a moderated mediation model in which low levels of observed parent positive regard and sensitivity to distress during a youth stressor task were indirectly associated with an increased likelihood of experiencing an episode of depression over an18 month period, through the mediating influence of youth negative attributional style, but only for youth who also experienced a high number of peer stressors. These findings elucidate mechanisms through which parenting may contribute to risk for depression during the transition into and across adolescence.

Mathematical approach for predicting non-negative tristimulus values using the CAT02 chromatic adaptation transform

It has been reported that for certain colour samples, the chromatic adaptation transform CAT02 imbedded in the CIECAM02 colour appearance model predicts corresponding colours with negative tristimulus values (TSVs), which can cause problems in certain applications. To overcome this problem, a mathematical approach is proposed for modifying CAT02. This approach combines a non-negativity constraint for the TSVs of corresponding colours with the minimization of the colour differences between those values for the corresponding colours obtained by visual observations and the TSVs of the corresponding colours predicted by the model, which is a constrained non-linear optimization problem. By solving the non-linear optimization problem, a new matrix is found. The performance of the CAT02 transform with various matrices including the original CAT02 matrix, and the new matrix are tested using visual datasets and the optimum colours. Test results show that the CAT02 with the new matrix predicted corresponding colours without negative TSVs for all optimum colours and the colour matching functions of the two CIE standard observers under the test illuminants considered. However, the accuracy with the new matrix for predicting the visual data is approximately 1 CIELAB colour difference unit worse compared with the original CAT02. This indicates that accuracy has to be sacrificed to achieve the non-negativity constraint for the TSVs of the corresponding colours.

Feedbacks between vegetation pattern and resource loss dramatically decrease ecosystem resilience and restoration potential in a simple dryland model

Conceptual frameworks of dryland degradation commonly include ecohydrological feedbacks between landscape spatial organization and resource loss, so that decreasing cover and size of vegetation patches result in higher water and soil losses, which lead to further vegetation loss. However, the impacts of these feedbacks on dryland dynamics in response to external stress have barely been tested. Using a spatially-explicit model, we represented feedbacks between vegetation pattern and landscape resource loss by establishing a negative dependence of plant establishment on the connectivity of runoff-source areas (e.g., bare soils). We assessed the impact of various feedback strengths on the response of dryland ecosystems to changing external conditions. In general, for a given external pressure, these connectivity-mediated feedbacks decrease vegetation cover at equilibrium, which indicates a decrease in ecosystem resistance. Along a gradient of gradual increase of environmental pressure (e.g., aridity), the connectivity-mediated feedbacks decrease the amount of pressure required to cause a critical shift to a degraded state (ecosystem resilience). If environmental conditions improve, these feedbacks increase the pressure release needed to achieve the ecosystem recovery (restoration potential). The impact of these feedbacks on dryland response to external stress is markedly non-linear, which relies on the non-linear negative relationship between bare-soil connectivity and vegetation cover. Modelling studies on dryland vegetation dynamics not accounting for the connectivity-mediated feedbacks studied here may overestimate the resistance, resilience and restoration potential of drylands in response to environmental and human pressures. Our results also suggest that changes in vegetation pattern and associated hydrological connectivity may be more informative early-warning indicators of dryland degradation than changes in vegetation cover.