A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Megalania) priscus.

The predatory ecology of Varanus komodoensis (Komodo Dragon) has been a subject of long-standing interest and considerable conjecture. Here, we investigate the roles and potential interplay between cranial mechanics, toxic bacteria, and venom. Our analyses point to the presence of a sophisticated combined-arsenal killing apparatus. We find that the lightweight skull is relatively poorly adapted to generate high bite forces but better adapted to resist high pulling loads. We reject the popular notion regarding toxic bacteria utilization. Instead, we demonstrate that the effects of deep wounds inflicted are potentiated through venom with toxic activities including anticoagulation and shock induction. Anatomical comparisons of V. komodoensis with V. (Megalania) priscus fossils suggest that the closely related extinct giant was the largest venomous animal to have ever lived.

Amyloid-beta peptide (Abeta) neurotoxicity is modulated by the rate of peptide aggregation: Abeta dimers and trimers correlate with neurotoxicity.

Alzheimer's disease is an age-related neurodegenerative disorder with its toxicity linked to the generation of amyloid-beta peptide (Abeta). Within the Abeta sequence, there is a systemic repeat of a GxxxG motif, which theoretical studies have suggested may be involved in both peptide aggregation and membrane perturbation, processes that have been implicated in Abeta toxicity. We synthesized modified Abeta peptides, substituting glycine for leucine residues within the GxxxG repeat motif (GSL peptides). These GSL peptides undergo beta-sheet and fibril formation at an increased rate compared with wild-type Abeta. The accelerated rate of amyloid fibril formation resulted in a decrease in the presence of small soluble oligomers such as dimeric and trimeric forms of Abeta in solution, as detected by mass spectrometry. This reduction in the presence of small soluble oligomers resulted in reduced binding to lipid membranes and attenuated toxicity for the GSL peptides. The potential role that dimer and trimer species binding to lipid plays in Abeta toxicity was further highlighted when it was observed that annexin V, a protein that inhibits Abeta toxicity, specifically inhibited Abeta dimers from binding to lipid membranes.

Dimeric structures of alpha-synuclein bind preferentially to lipid membranes.

There is substantial evidence which implicates alpha-synuclein and its ability to aggregate and bind vesicle membranes as critical factors in the development of Parkinson's disease. In order to investigate the interaction between alpha-synuclein wild type (Wt) and its familial mutants, A53T and A30P with lipid membranes, we developed a novel lipid binding assay using surface enhanced laser desorption/ionisation-time of flight-mass spectrometry (SELDI-TOF MS). Wt and A53T exhibited similar lipid binding profiles; monomeric species and dimers bound with high relative affinity to the lipid surface, the latter of which exhibited preferential binding. Wt and A53T trimers and tetramers were also detected on the lipid surface. A30P exhibited a unique lipid binding profile; monomeric A30P bound with a low relative affinity, however, the dimeric species of A30P exhibited a higher binding ability. Larger order A30P oligomers were not detected on the lipid surface. Tapping mode atomic force microscopy (AFM) imaging was conducted to further examine the alpha-synuclein-lipid interaction. AFM analysis revealed Wt and its familial mutants can penetrate lipid membranes or disrupt the lipid and bind the hydrophobic alkyl self-assembled monolayer (SAM) used to form the lipid layer. The profile of these studied proteins revealed the presence of 'small features' consistent with the presence of monomeric and dimeric forms of the protein. These data collectively indicate that the dimeric species of Wt and its mutants can bind and cause membrane perturbations.

SELDI-TOF mass spectrometry-based protein profiling of kidney tissue.

Protein profiling has numerous applications in renal research including the detection of protein biomarkers with aberrant expression levels during disease development. Such information is essential for early diagnosis and will aid the improvement of patient management and minimise the progression of disease. Further to this, data generated from these studies will assist the elucidation of the precise mechanisms of disease development and can lead to the discovery of potential drug targets. Surface enhanced laser desorption/ionisation time of flight mass spectrometry (SELDI-TOF MS), is emerging as a popular profiling tool for such studies. It incorporates the methods of solid-phase chromatography and TOF-MS in a single platform. This chapter provides a guide for establishing kidney profiling experiments using SELDI-TOF MS and will cover the following topics: 1) preparation of tissue extracts; 2) array processing, including optimisation of conditions for biomarker discovery; and 3) data acquisition/analysis.

Serum amyloid a is a biomarker of acute exacerbations of chronic obstructive pulmonary disease.

RATIONALE: Much of the total disease burden and cost of chronic obstructive pulmonary disease (COPD) is associated with acute exacerbations of COPD (AECOPD). Serum amyloid A (SAA) is a novel candidate exacerbation biomarker identified by proteomic screening. OBJECTIVES: To assess SAA as a biomarker of AECOPD. METHODS: Biomarkers were assessed (1) cross-sectionally (stable vs. AECOPD; 62 individuals) and (2) longitudinally with repeated measures (baseline vs. AECOPD vs. convalescence; 78 episodes in 37 individuals). Event severity was graded (I, ambulatory; II, hospitalized; III, respiratory failure) based on consensus guidelines. MEASUREMENTS AND MAIN RESULTS: Presumptively newly acquired pathogens were associated with onset of symptomatic AECOPD. In the cross-sectional study, both SAA and C-reactive protein (CRP) were elevated at AECOPD onset compared with stable disease (SAA median, 7.7 vs. 57.6 mg/L; P < 0.01; CRP median, 4.6 vs. 12.5 mg/L; P < 0.01). Receiver operator characteristics analysis was used to generate area-under-curve values for event severity. SAA discriminated level II/III events (SAA, 0.88; 95% confidence interval, 0.80-0.94 vs. CRP, 0.80; 95% confidence interval, 0.70-0.87; P = 0.05). Combining SAA or CRP with major symptoms (Anthonisen criteria, dyspnea) did not further improve the prediction model for severe episodes. IL-6 and procalcitonin were not informative. CONCLUSIONS: SAA is a novel blood biomarker of AECOPD that is more sensitive than CRP alone or in combination with dyspnea. SAA may offer new insights into the pathogenesis of AECOPD.

Analysis of Abeta interactions using ProteinChip technology.

Abeta peptides are now acknowledged to play a central role in the pathogenesis of Alzheimer's disease. Their generation results from the sequential cleavage of amyloid precursor protein by beta and gamma secretases. The resulting peptide fragments impart toxicity via their ability to form soluble oligomers and bind to cell membranes. In this chapter we describe the use of ProteinChip technology to study the physicochemical behaviour of Abeta and its mechanisms of toxicity. These include analyzing (1) Abeta processing and quantitation of peptide fragments, (2) Abeta aggregation and the quantitation of oligomers, and (3) Abeta-lipid interactions.

Localization of the third heparin-binding site in the human complement regulator factor H1.

Complement factor H (fH) plays a pivotal role in regulating the alternative pathway, allowing complement activation to proceed on foreign surfaces, whilst protecting surrounding host cell surfaces from complement-mediated damage. Host cell recognition is mediated by polyanions such as sialic acid and glycosaminoglycans (GAGs), which promote a high affinity interaction between fH and C3b deposited on host cell surfaces. Factor H is composed of 20 short consensus repeats (SCRs); two heparin-binding sites have been identified within SCR 7 and SCR 20 and a third site is thought to exist within or near SCR 13. Using an extensive series of recombinant fH fragments and heparin affinity chromatography, we have localized the third heparin-binding domain to SCR 9. A recombinant fH fragment containing both SCR 7 and SCR 9 exhibited higher affinity for heparin than SCR 7 alone, suggesting that the individual heparin-binding sites interact simultaneously with heparin to create a higher avidity interaction. Recombinant fragments containing SCR 9 bound to endothelial cells, indicating that this domain is capable of interacting with polyanions within a physiologically relevant environment. In addition, the three heparin-binding sites exhibited differences in their specificity for certain GAGs, suggesting that the individual binding domains may possess separate GAG recognition functions.

Studies on soluble ectodomain proteins of relaxin (LGR7) and insulin 3 (LGR8) receptors.

The ectodomains of both the relaxin (LGR7) and the INSL3 (LGR8) receptors can be expressed on the cell surface using only a single transmembrane domain. These membrane-anchored proteins retain the ability to bind relaxin and can be cleaved from the cell surface. The subsequent LGR7 protein, 7BP, binds relaxin and can act as a functional relaxin antagonist. By contrast, the equivalent LGR8 protein 8BP does not bind relaxin or antagonize LGR8 activity. The 7BP protein has been successfully immobilized onto chemically derivatized surfaces for the capture of relaxin peptides and subsequent identification via SELDI-MS analysis.

Evaluation of relaxin's antifibrotic action by SELDI-TOF mass spectrometry-based profiling of relaxin knockout mice, a model of progressive fibrosis.

Surface-enhanced laser desorption ionization-time-of-flight mass spectrometry was employed to identify potential biomarkers for early-onset fibrosis. These biomarkers were then used to evaluate the efficacy of relaxin to reverse or ameliorate the development of the condition.

Aberrant protein expression in plasma and kidney tissue during experimental obstructive nephropathy.

Kidney failure is a major health problem worldwide. Patients with end-stage renal disease require intensive medical support by dialysis or kidney transplantation. Current methods for diagnosis of kidney disease are either invasive or insensitive, and renal function may decline by as much as 50% before it can be detected using current techniques. The goal of this study was, therefore, to identify biomarkers of kidney disease (associated with renal fibrosis) that can be used for the development of a non-invasive clinical test for early disease detection. We utilized two protein-profiling technologies (SELDI-TOF MS and 2-D) to screen the plasma and kidney proteome for aberrantly expressed proteins in an experimental mouse model of unilateral uretric obstruction, which mimics the pathology of human renal disease. Several differentially regulated proteins were detected at the plasma level of day-3-obstructed animals, which included serum amyloid A1, fibrinogen α, haptoglobin precursor protein, haptoglobin and major urinary proteins 11 and 8. Differentially expressed proteins detected at the tissue level included ras-like activator protein 2, haptoglobin precursor protein, malate dehydrogenase, α enolase and murine urinary protein (all p<0.05 versus controls). Immunohistochemistry was used to confirm the up-regulation of fibrinogen. Interestingly, these proteins are largely separated into four major classes: (i) acute-phase reactants (ii) cell-signaling molecules (iii) molecules involved in cell growth and metabolism and (iv) urinary proteins. These results provide new insights into the pathology of obstructive nephropathy and may facilitate the development of specific assay(s) to detect and monitor renal fibrosis.

Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain.

Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.

A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein.

The complement inhibitor factor H (fH) interacts via its seventh short consensus repeat (SCR) domain with multiple ligands including heparin, streptococcal M protein and C-reactive protein (CRP). The aim of this study was to localize the residues in SCR 7 required for these interactions. We initially built a homology model of fH SCR 6-7 using the averaged NMR structures of fH SCR 15-16 and vaccinia control protein SCR 3-4 as templates. Electrostatic potentials of the model's surface demonstrated a co-localization of three clusters of positively charged residues on SCR 7, labeled site A (R369 and K370), site B (R386 and K387) and site C (K392). These residues, localized to the linker region preceding SCR 7 and to the end of a "hypervariable loop" in SCR 7, were systematically replaced with uncharged alanine residues in an fH construct containing SCR 1-7. The resulting proteins were expressed in the methylotrophic yeast, Pichia pastoris. By ELISA analysis we demonstrated: first, that substituting site A inhibited heparin and CRP binding; secondly, that substituting site B inhibited binding to heparin, CRP and M protein; and thirdly, that substituting site C clearly inhibited only heparin binding.

The human complement regulator factor H binds pneumococcal surface protein PspC via short consensus repeats 13 to 15.

The innate ability of Streptococcus pneumoniae to resist complement activation and complement-mediated phagocytosis may be a direct consequence of the ability of the bacteria to bind components of the complement regulatory system. One such component, factor H (fH), is a crucial fluid-phase negative regulator of the alternative pathway of complement and is utilized by a number of pathogenic organisms to resist complement attack. The pneumococcal surface protein C (PspC [also known as CbpA] and SpsA) has been shown to bind fH, although the exact binding site within one or more of the 20 short consensus repeats (SCRs) of the molecule is not known. The purpose of the current study was to map specific SCRs on fH responsible for this binding. Initial experiments utilizing type 2 pneumococcal strain D39 and its isogenic PspC-negative derivative (D39/pspC mutant) showed that fH binding was PspC dependent. A purified recombinant protein derivative of PspC that lacked the proline-rich region (PspCDeltaPro) had a reduced binding efficiency for fH, thereby directly showing the importance of this region for the fH interaction. We have specifically shown by inhibition experiments that SCRs responsible for heparin and C3b binding of fH are not involved in binding PspC and the interaction between fH and PspC is largely hydrophobic, since no inhibition was observed in the presence of high concentrations of NaCl. Construction of SCR proteins encompassing the whole fH molecule showed that SCRs 8 to 15 (SCR 8-15) mediated binding to PspC. Further localization experiments revealed that SCR 13 and SCR 15 were required for full binding, although partial binding was retained when either SCR was removed.

Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46).

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.