A null mutation in the inflammation-associated S100 protein S100A8 causes early resorption of the mouse embryo

S100A8 (also known as CP10 or MRP8) was the first member of the S100 family of calcium-binding proteins shown to be chemotactic for myeloid cells. The gene is expressed together with its dimerization partner S100A9 during myelopoiesis in the fetal liver and in adult bone marrow as well as in mature granulocytes. In this paper we show that S100A8 mRNA is expressed without S100A9 mRNA between 6.5 and 8.5 days postcoitum within fetal cells infiltrating the deciduum in the vicinity of the ectoplacental cone. Targeted disruption of the S100A8 gene caused rapid and synchronous embryo resorption by day 9.5 of development in 100% of homozygous null embryos. Until this point there was no evidence of developmental delay in S100A8(-/-) embryos and decidualization was normal. The results of PCR genotyping around 7.5-8.5 days postcoitum suggest that the null embryos are infiltrated with maternal cells before overt signs of resorption. This work is the first evidence for nonredundant function of a member of the S100 gene family and implies a role in prevention of maternal rejection of the implanting embryo. The S100A8 null provides a new model for studying fetal-maternal interactions during implantation.

Protein fold recognition score functions: Unusual construction strategies

We describe two ways of optimizing score functions for protein sequence to structure threading. The first method adjusts parameters to improve sequence to structure alignment. The second adjusts parameters so as to improve a score function's ability to rank alignments calculated in the first score function. Unlike those functions known as knowledge-based force fields, the resulting parameter sets do not rely on Boltzmann statistics, have no claim to representing free energies and are purely constructions for recognizing protein folds. The methods give a small improvement, but suggest that functions can be profitably optimized for very specific aspects of protein fold recognition, Proteins 1999;36:454-461. (C) 1999 Wiley-Liss, Inc.

A rapid selection/amplification procedure for high-level expression of recombinant protein in a metal-amplifiable mammalian expression system

We describe a strategy for the selection and amplification of foreign gene expression in Chinese hamster ovary (CHO) cells employing a metallothionein gene-containing expression vector. This report describes an amplification procedure that results in an enrichment of clones exhibiting high levels of recombinant protein production and reduces the labour required for screening recombinant cell lines.

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

Protein sequence threading, the alignment problem, and a two-step strategy

Conventionally, protein structure prediction via threading relies on some nonoptimal method to align a protein sequence to each member of a library of known structures. We show how a score function (force field) can be modified so as to allow the direct application of a dynamic programming algorithm to the problem. This involves an approximation whose damage can be minimized by an optimization process during score function parameter determination. The method is compared to sequence to structure alignments using a more conventional pair-wise score function and the frozen approximation. The new method produces results comparable to the frozen approximation, but is faster and has fewer adjustable parameters. It is also free of memory of the template's original amino acid sequence, and does not suffer from a problem of nonconvergence, which can be shown to occur with the frozen approximation. Alignments generated by the simplified score function can then be ranked using a second score function with the approximations removed. (C) 1999 John Wiley & Sons, Inc.

MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins

MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of:Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear (N-15) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action. (C) 1999 Academic Press.

Active site-directed protein regulation

Regulation of protein function is vital for the control of cellular processes. Proteins are often regulated by allosteric mechanisms, in which effecters bind to regulatory sites distinct from the active sites and alter protein function. Intrasteric regulation, directed at the active site and thus the counterpart of allosteric control, is now emerging as an important regulatory mechanism.

What the evolution of the amyloid protein precursor supergene family tells us about its function

The Alzheimer's disease amyloid protein precursor (APP) gene is part of a multi-gene super-family from which sixteen homologous amyloid precursor-like proteins (APLP) and APP species homologues have been isolated and characterised. Comparison of exon structure (including the uncharacterised APL-1 gene), construction of phylogenetic trees, and analysis of the protein sequence alignment of known homologues of the APP super-family were performed to reconstruct the evolution of the family and to assess the functional significance of conserved protein sequences between homologues. This analysis supports an adhesion function for all members of the APP super family, with specificity determined by those sequences which are not conserved between APLP lineages, and provides evidence for an increasingly complex APP superfamily during evolution. The analysis also suggests that Drosophila APPL and Caenorhabdotids elegans APL-1 may be a fourth APLP lineage indicating that these proteins, while not functional homologues of human APP, are similarly likely to regulate cell adhesion. Furthermore, the beta A4 sequence is highly conserved only in APP orthologues, strongly suggesting this sequence is of significant functional importance in this lineage. (C) 2000 Elsevier Science Ltd. All rights reserved.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.

Sausage: protein threading with flexible force fields

Sausage is a protein sequence threading program, but with remarkable run-time flexibility. Using different scripts, it can calculate protein sequence-structure alignments, search structure libraries, swap force fields, create models form alignments, convert file formats and analyse results. There are several different force fields which might be classed as knowledge-based, although they do not rely on Boltzmann statistics. Different force fields are used for alignment calculations and subsequent ranking of calculated models.

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D H-1 NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the H-1 NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Her chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and IID-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.

Acyclic permutants of naturally occurring cyclic proteins - Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the p-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.