Bone morphogenetic proteins and their antagonists: current and emerging clinical uses

Bone morphogenetic proteins (BMPs) are members of the TGFβ superfamily of secreted cysteine knot proteins that includes TGFβ₁, nodal, activins and inhibins. BMPs were first discovered by Urist in the 1960s when he showed that implantation of demineralized bone into intramuscular tissue of rabbits induced bone and cartilage formation. Since this seminal discovery, BMPs have also been shown to play key roles in several other biological processes, including limb, kidney, skin, hair and neuronal development, as well as maintaining vascular homeostasis. The multifunctional effects of BMPs make them attractive targets for the treatment of several pathologies, including bone disorders, kidney and lung fibrosis, and cancer. This review will summarize current knowledge on the BMP signalling pathway and critically evaluate the potential of recombinant BMPs as pharmacological agents for the treatment of bone repair and tissue fibrosis in patients.

Enrichment and analysis of nonenzymatically glycated peptides: Boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance posttranslational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation ( CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.

Mammalian triokinase and dihydroxyacetone kinase are the same enzyme

It is widely accepted that the ATP-dependent phosphorylation of D-glyceraldehyde in the fructokinase pathway of fructose metabolism requires the enzyme “triokinase”. However, experimental data on this enzyme are remarkably scarce. The enzyme has been purified from a variety of sources and peptides derived from the pig kidney enzyme show high similarity to human dihydroxyacetone kinase – an enzyme which also has FMN cyclase activity in high manganese ion concentrations. The properties of the two enzymes are also highly similar. Therefore it is proposed that mammalian triokinase and dihydroxyacetone kinase are, in fact, the same enzyme. This has consequences for investigations of normal and aberrant fructose metabolism and for the teaching of biochemistry in medical and science courses.

Cyclic AMP production and insulin releasing activity of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide

Synthetic fragment peptides of glucose-dependent insulinotropic polypeptide (GIP) were evaluated for their ability to elevate cellular cAMP production and stimulate insulin secretion. In GIP receptor transfected CHL cells, GIP(4-42) and GIP(17-30) dose-dependently inhibited GIP-stimulated cAMP production (40 +/- 8%; p <0.01 and 15 +/- 6%; p <0.05, respectively), while GIP(1-16) exerted very weak agonist effects on cAMP production. In the clonal pancreatic beta-cell line, BRIN-BD11, GIP(1-16) demonstrated weak insulin releasing activity compared with native GIP. In contrast, GIP(4-42) and GIP (17-30) weakly antagonized the insulin releasing activity of the native peptide (23 +/- 6%; p <0.05 and 11 +/- 3%, respectively). These data demonstrate the critical role of the N-terminus and the involvement of regions of the C-terminal domain in generating full biological potency of GIP.

The repertoire of G protein-coupled receptors in the human parasite Schistosoma mansoni and the model organism Schmidtea mediterranea.

Background

G protein-coupled receptors (GPCRs) constitute one of the largest groupings of eukaryotic proteins, and represent a particularly lucrative set of pharmaceutical targets. They play an important role in eukaryotic signal transduction and physiology, mediating cellular responses to a diverse range of extracellular stimuli. The phylum Platyhelminthes is of considerable medical and biological importance, housing major pathogens as well as established model organisms. The recent availability of genomic data for the human blood fluke Schistosoma mansoni and the model planarian Schmidtea mediterranea paves the way for the first comprehensive effort to identify and analyze GPCRs in this important phylum.

Results

Application of a novel transmembrane-oriented approach to receptor mining led to the discovery of 117 S. mansoni GPCRs, representing all of the major families; 105 Rhodopsin, 2 Glutamate, 3 Adhesion, 2 Secretin and 5 Frizzled. Similarly, 418 Rhodopsin, 9 Glutamate, 21 Adhesion, 1 Secretin and 11 Frizzled S. mediterranea receptors were identified. Among these, we report the identification of novel receptor groupings, including a large and highly-diverged Platyhelminth-specific Rhodopsin subfamily, a planarian-specific Adhesion-like family, and atypical Glutamate-like receptors. Phylogenetic analysis was carried out following extensive gene curation. Support vector machines (SVMs) were trained and used for ligand-based classification of full-length Rhodopsin GPCRs, complementing phylogenetic and homology-based classification.

Conclusions

Genome-wide investigation of GPCRs in two platyhelminth genomes reveals an extensive and complex receptor signaling repertoire with many unique features. This work provides important sequence and functional leads for understanding basic flatworm receptor biology, and sheds light on a lucrative set of anthelmintic drug targets.

The Plasmodium falciparum Malaria M1 Alanyl Aminopeptidase (PfA-M1): Insights of Catalytic Mechanism and Function from MD Simulations

Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1-3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 angstrom-long channel and at the opening of the 30 angstrom-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed.

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of ‘Pathogenic’, ‘Commensal’ and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two ‘putative virulence’ genes (eMLST) that provides improved high resolution
typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA₁ clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA₂ strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA₁ and IA₂ strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.

The role of salt and shear on the storage and assembly of spider silk proteins

Major ampullate silk fibers of orb web-weaving spiders have impressive mechanical properties due to the fact that the underlying proteins partially fold into helical/amorphous structures, yielding relatively elastic matrices that are toughened by anisotropic nanoparticulate inclusions (formed from stacks of beta-sheets of the same proteins). In vivo the transition from soluble protein to solid fibers involves a combination of chemical and mechanical stimuli (such as ion exchange, extraction of water and shear forces). Here we elucidate the effects of such stimuli on the in vitro aggregation of engineered and recombinantly produced major ampullate silk-like proteins (focusing on structure-function relationships with respect to their primary structures), and discuss their relevance to the storage and assembly of spider silk proteins in vivo. (C) 2009 Elsevier Inc. All rights reserved.

Production and Processing of Spider Silk Proteins

Natural spider silk fibers have impressive mechanical properties (outperforming many man-made fibers) and are, moreover, biocompatible, biodegradable, and produced under benign conditions (using water as a solvent at ambient temperature). The problems associated with harvesting natural spider silks inspired us to devise a method to produce spider silk-like proteins biotechnologically (the first subject tackled in this highlight); we subsequently discuss their processing into various materials morphologies, and some potential technical and biomedical applications.

Structure and bioactivity of neuropeptide F from the human parasites Schistosoma mansoni and Schistosoma japonicum

The blood flukes Schistosoma mansoni and Schistosoma japonicum inflict immense suffering as agents of human schistosomiasis. Previous investigations have found the nervous systems of these worms contain abundant immunoreactivity to antisera targeting invertebrate neuropeptide Fs (NPFs) as well as structurally similar neuropeptides of the mammalian neuropeptide Y (NPY) family. Here, cDNAs encoding NPF in these worms were identified, and the mature neuropeptides from the two species differed by only a single amino acid. Both neuropeptides feature the characteristics common among NPFs; they are 36 amino acids long with a carboxyl-terminal Gly-Arg-X-Arg-Phe-amide and Tyr residues at positions 10 and 17 from the carboxyl terminus. Synthetic S. mansoni NPF potently inhibits the forskolin-stimulated accumulation of cAMP in worm homogenates, with significant effects at 10(-11) M. This is the first demonstration of an endogenous inhibition of cAMP by an NPF, and because this is the predominant pathway associated with vertebrate NPY family peptides, it demonstrates a conservation of downstream signaling pathways used by NPFs and NPY peptides.

A strategy for designing inhibitors of alpha-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders.

Convergent biochemical and genetic evidence suggests that the formation of alpha-synuclein (alpha-syn) protein deposits is an important and, probably, seminal step in the development of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been reported that transgenic animals overexpressing human alpha-syn develop lesions similar to those found in the brain in PD, together with a progressive loss of dopaminergic cells and associated abnormalities of motor function. Inhibiting and/or reversing alpha-syn self-aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. We synthesized a library of overlapping 7-mer peptides spanning the entire alpha-syn sequence, and identified amino acid residues 64-100 of alpha-syn as the binding region responsible for its self-association. Modified short peptides containing alpha-syn amino acid sequences from part of this binding region (residues 69-72), named alpha-syn inhibitors (ASI), were found to interact with full-length alpha-syn and block its assembly into both early oligomers and mature amyloid-like fibrils. We also developed a cell-permeable inhibitor of alpha-syn aggregation (ASID), using the polyarginine peptide delivery system. This ASID peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-syn(A53T), a familial PD-associated mutation. ASI peptides without this delivery system did not reverse levels of Fe(II)-induced DNA damage. Furthermore, the ASID peptide increased (P