Julia Muenchhoff

Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis

ABSTRACT Toxic cyanobacterial blooms cause economic losses and pose significant public health threats on a global scale. Characterization of the gene cluster for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 is described, and the complete biosynthetic pathway is proposed. The cyr gene cluster spans 43 kb and is comprised of 15 open reading frames containing genes required for the biosynthesis, regulation, and export of the toxin. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions and subsequent reductions, and rings are formed via Michael additions in a stepwise manner. The uracil ring is formed by a novel pyrimidine biosynthesis mechanism and tailoring reactions, including sulfation and hydroxylation that complete biosynthesis. These findings enable the design of toxic strain-specific probes and allow the future study of the regulation and biological role of cylindrospermopsin.

Environmental conditions that influence toxin biosynthesis in cyanobacteria

Over the past 15 years, the genetic basis for production of many cyanobacterial bioactive compounds has been described. This knowledge has enabled investigations into the environmental factors that regulate the production of these toxins at the molecular level. Such molecular or systems level studies are also likely to reveal the physiological role of the toxin and contribute to effective water resource management. This review focuses on the environmental regulation of some of the most relevant cyanotoxins, namely the microcystins, nodularin, cylindrospermopsin, saxitoxins, anatoxins and jamaicamides.

A novel prokaryotic l‐arginine:glycine amidinotransferase is involved in cylindrospermopsin biosynthesis

We report the first characterization of an l‐arginine:glycine amidinotransferase from a prokaryote. The enzyme, CyrA, is involved in the pathway for biosynthesis of the polyketide‐derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically distinct from other amidinotransferases, and structural alignment shows differences between the active site residues of CyrA and the well‐characterized human l‐arginine:glycine amidinotransferase (AGAT). Overexpression of recombinant CyrA in Escherichia coli enabled biochemical characterization of the enzyme, and we confirmed the predicted function of CyrA as an l‐arginine:glycine amidinotransferase by 1H NMR. As compared with AGAT, CyrA showed narrow substrate specificity when presented with substrate analogs, and deviated from regular Michaelis–Menten kinetics in the presence of the non‐natural substrate hydroxylamine. Studies of initial reaction velocities and product inhibition, and identification of intermediate reaction products, were used to probe the kinetic mechanism of CyrA, which is best described as a hybrid of ping‐pong and sequential mechanisms. Differences in the active site residues of CyrA and AGAT are discussed in relation to the different properties of both enzymes. The enzyme had maximum activity and maximum stability at pH 8.5 and 6.5, respectively, and an optimum temperature of 32 °C. Investigations into the stability of the enzyme revealed that an inactivated form of this enzyme retained an appreciable amount of secondary structure elements even on heating to 94 °C, but lost its tertiary structure at low temperature (Tmax of 44.5 °C), resulting in a state reminiscent of a molten globule. CyrA represents a novel group of prokaryotic amidinotransferases that utilize arginine and glycine as substrates with a complex kinetic mechanism and substrate specificity that differs from that of the eukaryotic l‐arginine:glycine amidinotransferases.

Comparative genomics of Cylindrospermopsis raciborskii strains with differential toxicities

BackgroundCylindrospermopsis raciborskii is an invasive filamentous freshwater cyanobacterium, some strains of which produce toxins. Sporadic toxicity may be the result of gene deletion events, the horizontal transfer of toxin biosynthesis gene clusters, or other genomic variables, yet the evolutionary drivers for cyanotoxin production remain a mystery. Through examining the genomes of toxic and non-toxic strains of C. raciborskii, we hoped to gain a better understanding of the degree of similarity between these strains of common geographical origin, and what the primary differences between these strains might be. Additionally, we hoped to ascertain why some cyanobacteria possess the cylindrospermopsin biosynthesis (cyr) gene cluster and produce toxin, while others do not. It has been hypothesised that toxicity or lack thereof might confer a selective advantage to cyanobacteria under certain environmental conditions.ResultsIn order to examine the fundamental differences between toxic and non-toxic C. raciborskii strains, we sequenced the genomes of two closely related isolates, CS-506 (CYN+) and CS-509 (CYN-) sourced from different lakes in tropical Queensland, Australia. These genomes were then compared to a third (reference) genome from C. raciborskii CS-505 (CYN+). Genome sizes were similar across all three strains and their G + C contents were almost identical. At least 2,767 genes were shared among all three strains, including the taxonomically important rpoc1, ssuRNA, lsuRNA, cpcA, cpcB, nifB and nifH, which exhibited 99.8-100% nucleotide identity. Strains CS-506 and CS-509 contained at least 176 and 101 strain-specific (or non-homologous) genes, respectively, most of which were associated with DNA repair and modification, nutrient uptake and transport, or adaptive measures such as osmoregulation. However, the only significant genetic difference observed between the two strains was the presence or absence of the cylindrospermopsin biosynthesis gene cluster. Interestingly, we also identified a cryptic secondary metabolite gene cluster in strain CS-509 (CYN-) and a second cryptic cluster common to CS-509 and the reference strain, CS-505 (CYN+).ConclusionsOur results confirm that the most important factor contributing to toxicity in C. raciborskii is the presence or absence of the cyr gene cluster. We did not identify any other distally encoded genes or gene clusters that correlate with CYN production. The fact that the additional genomic differences between toxic and non-toxic strains were primarily associated with stress and adaptation genes suggests that CYN production may be linked to these physiological processes.

Plasma Protein Profiling of Mild Cognitive Impairment and Alzheimer's Disease Across Two Independent Cohorts

To unlock the full potential of disease modifying treatments, it is essential to develop early biomarkers for Alzheimers disease (AD). For practical reasons, blood-based markers that could provide a signal at the stage of mild cognitive impairment (MCI) or even earlier would be ideal. Using the proteomic approach of isobaric tagging for relative and absolute quantitation (iTRAQ), we compared the plasma protein profiles of MCI, AD, and cognitively normal control subjects from two independent cohorts: the Sydney Memory and Ageing Study (261 MCI subjects, 24 AD subjects, 411 controls) and the Hunter Community Study (180 MCI subjects, 153 controls). The objective was to identify any proteins that are differentially abundant in MCI and AD plasma in both cohorts, since they might be of interest as potential biomarkers, or could help direct future mechanistic studies. Proteins representative of biological processes relevant to AD pathology, such as the complement system, the coagulation cascade, lipid metabolism, and metal and vitamin D and E transport, were found to differ in abundance in MCI. In particular, levels of complement regulators C1 inhibitor and factor H, fibronectin, ceruloplasmin, and vitamin D-binding protein were significantly decreased in MCI participants from both cohorts. Several apolipoproteins, including apolipoprotein AIV, B-100, and H were also significantly decreased in MCI. Most of these proteins have previously been reported as potential biomarkers for AD; however, we show for the first time that a significant decrease in plasma levels of two potential biomarkers (fibronectin and C1 inhibitor) is evident at the MCI stage.

Identification of two residues essential for the stringent substrate specificity and active site stability of the prokaryotic l‐arginine:glycine amidinotransferase CyrA

A novel prokaryotic l‐arginine:glycine amidinotransferase (CyrA; EC2.1.4.1) is involved in the biosynthesis of the polyketide‐derived cytotoxin cylindrospermopsin in the cyanobacterium Cylindrospermopsis raciborskii AWT250, and was previously characterized with regard to kinetic mechanism and substrate specificity [Muenchhoff J et al. (2010) FEBS J277, 3844–3860]. In order to elucidate the structure–function–stability relationship of this enzyme, two residues in its active site were replaced with the residues that occur in the human l‐arginine:glycine amidinotransferase (h‐AGAT) at the corresponding positions (F245N and S247M), and a double variant carrying both substitutions was also created. In h‐AGAT, both of these residues are critical for the function of this enzyme with regard to substrate binding, ligand‐induced structural changes, and stability of the active site. In this study, we demonstrated that both single residue replacements resulted in a dramatic broadening of substrate specificity, but did not affect the kinetic mechanism. Experiments with substrate analogues indicate that donor substrates require a carboxylate group for binding. Evidence from initial velocity studies suggests that CyrA undergoes ligand‐induced structural changes that involve Phe245. Stability parameters (Topt and Tmax) of the CyrA variants differed from those of wild‐type CyrA. Structural flexibilities of the wild type and all three variants were comparable on the basis of dynamic fluorescence quenching, indicating that changes in Topt are most likely attributable to localized effects within the active site. Overall, the results indicated that these two residues are essential for both stringent substrate specificity and the active site stability and flexibility of this unique cyanobacterial enzyme.

Changes in the plasma proteome at asymptomatic and symptomatic stages of autosomal dominant Alzheimer's disease

The autosomal dominant form of Alzheimer’s disease (ADAD) is far less prevalent than late onset Alzheimer’s disease (LOAD), but enables well-informed prospective studies, since symptom onset is near certain and age of onset is predictable. Our aim was to discover plasma proteins associated with early AD pathology by investigating plasma protein changes at the asymptomatic and symptomatic stages of ADAD. Eighty-one proteins were compared across asymptomatic mutation carriers (aMC, n = 15), symptomatic mutation carriers (sMC, n = 8) and related noncarriers (NC, n = 12). Proteins were also tested for associations with cognitive measures, brain amyloid deposition and glucose metabolism. Fewer changes were observed at the asymptomatic than symptomatic stage with seven and 16 proteins altered significantly in aMC and sMC, respectively. This included complement components C3, C5, C6, apolipoproteins A-I, A-IV, C-I and M, histidine-rich glycoprotein, heparin cofactor II and attractin, which are involved in inflammation, lipid metabolism and vascular health. Proteins involved in lipid metabolism differed only at the symptomatic stage, whereas changes in inflammation and vascular health were evident at asymptomatic and symptomatic stages. Due to increasing evidence supporting the usefulness of ADAD as a model for LOAD, these proteins warrant further investigation into their potential association with early stages of LOAD.

Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System

Sirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.

Plasma apolipoproteins and physical and cognitive health in very old individuals

Apolipoproteins play a crucial role in lipid metabolism with implications in cardiovascular disease, obesity, diabetes, Alzheimers disease, and longevity. We quantified 7 apolipoproteins in plasma in 1067 individuals aged 56-105 using immunoassays and explored relationships with APOE polymorphism ε2/3/4, vascular health, frailty, and cognition. ApoA1, ApoA2, ApoB, ApoC3, ApoE, ApoH, and ApoJ decreased from mid-life, although ApoE and ApoJ had U-shaped trends. Centenarians had the highest ApoE levels and the lowest frequency of APOE ε4 allele relative to younger groups. Apolipoprotein levels trended lower in APOE ε4 homozygotes and heterozygotes compared with noncarriers, with ApoE and ApoJ being significantly lower. Levels of all apolipoproteins except ApoH were higher in females. Sex- and age-related differences were apparent in the association of apolipoproteins with cognitive performance, as only women had significant negative associations of ApoB, ApoE, ApoH, and ApoJ in mid-life, whereas associations at older age were nonsignificant or positive. Our findings suggest levels of some apolipoproteins, especially ApoE, are associated with lifespan and cognitive function in exceptionally long-lived individuals.

PLASMA APOLIPOPROTEINS AND PHYSICAL AND COGNITIVE HEALTH IN VERY OLD INDIVIDUALS

jection induces CRMP2 phosphorylation in mouse model, suggesting a possible relationship between Abeta and CRMP2 phosphorylation. However, CRMP2 phosphorylation status in LBD has not previously been investigated. Given that there is a significantly higher burden of Abeta plaque in DLB compared to PDD,we hypothesize that CRMP2 phosphorylation may be selectively altered in DLB. Methods: Postmortem neocortical tissues were from the Brains for Dementia Research network, well characterized patients with PDD (N1⁄420) and DLB (N1⁄420), together with age-matched controls (N1⁄420). Samples from inferior parietal lobe neocortex (BA40) were processed for western blot and ELISA. CRMP2 phosphorylation as well as beta-IIItubulin of axonal marker were assessed by western blot. Soluble Abeta concentrations were assessed by ELISA. Results:We found that (1) CRMP2 phosphorylation at Thr514 is significantly elevated only in DLB but not PDD in BA40 (p<0.01 for all comparisons). (2) Within the dementia groups, increased CRMP2 phosphorylation at Thr514 significantly correlated with increased Abeta42/Abeta40 ratio (rho1⁄40.38, p<0.05 in LBD and rho1⁄40.63, p<0.01 in DLB) as well as decreased beta-III-tubulin levels (rho1⁄40.38, p<0.05 in LBD). Conclusions: Our results showed that increased CRMP2 phosphorylation at Thr514 is only present in DLB but not in PDD, indicating different underlying neuropathological mechanisms in the two diseases. CRMP2 phosphorylation at Thr514 is found to correlate with Abeta42/ Abeta40ratio, suggesting future investigation of Thr514 as a putative underlying mechanisms of axonal impairment. References: 1. Ip, J.P. et al.2014. CRMP2: Functional Roles in Neural Development and Therapeutic Potential in Neurological Diseases. The Neuroscientist 2. Isono, T. et al. 2013. Abeta25-35 induces impairment of cognitive function and long-term potentiation through phosphorylation of CRMP2. Neuroscience research 3. Howlett, D.R. et al. 2014. Regional Multiple Pathology Scores Are Associated with Cognitive Decline in Lewy Body Dementias. Brain Pathology.