Challenging protein purification from anammox bacteria

The anaerobic ammonium oxidation (anammox) is a fascinating microbial pathway contributing to the global biogeochemical nitrogen cycle. The anammox pathway of nitrogen conversion can only be elucidated after the responsible proteins have been purified and characterised. The anammox bacteria have a complex cell envelope consisting of protein and lipopolysaccharide and they grow in dense cell aggregates. Preparing cell extract and purifying proteins from the cell aggregates is hampered by the extracellular polymeric material and by gel formation. It was demonstrated that protein-protein (i.e. disulfide formation) as well as protein-polysaccharide interaction caused this gel formation in extracts. Cell extract gelled upon freezing/thawing and boiling. Additionally, proteins aggregated on various chromatography media upon concentration and during desalting. The polysaccharides clogged the matrix of chromatographic materials and the pores of ultrafiltration membranes. The precipitation of proteins and polysaccharides caused very low resolution and streaking on SDS- and two-dimensional polyacrylamide gels. The present work describes the potential causes for gel formation in anammox cell extracts. Optimized protocols for sample preparation for polyacrylamide gel electrophoresis and ion exchange chromatography are presented. High-resolution gel electrophoresis of the cell extract was achieved after clarification from polymeric substances with denaturating phenol extraction and the purification of a 10 kDa cytochrome c is presented as an example.     

The expanded octarepeat domain selectively binds prions and disrupts homomeric prion protein interactions

Insertion of additional octarepeats into the prion protein gene has been genetically linked to familial Creutzfeldt Jakob disease and hence to de novo generation of infectious prions. The pivotal event during prion formation is the conversion of the normal prion protein (PrPC) into the pathogenic conformer PrPSc, which subsequently induces further conversion in an autocatalytic manner. Apparently, an expanded octarepeat domain directs folding of PrP toward the PrPSc conformation and initiates a self-replicating conversion process. Here, based on three main observations, we have provided a model on how altered molecular interactions between wild-type and mutant PrP set the stage for familial Creutzfeldt Jakob disease with octarepeat insertions. First, we showed that wild-type octarepeat domains interact in a copper-dependent and reversible manner, a "copper switch." This interaction becomes irreversible upon domain expansion, possibly reflecting a loss of function. Second, expanded octarepeat domains of increasing length gradually form homogenous globular multimers of 11-21 nm in the absence of copper ions when expressed as soluble glutathione S-transferase fusion proteins. Third, octarepeat domain expansion causes a gain of function with at least 10 repeats selectively binding PrPSc in a denaturant-resistant complex in the absence of copper ions. Thus, the combination of both a loss and gain of function profoundly influences homomeric interaction behavior of PrP with an expanded octarepeat domain. A multimeric cluster of prion proteins carrying expanded octarepeat domains may therefore capture and incorporate spontaneously arising short-lived PrPSc-like conformers, thereby providing a matrix for their conversion.     

Effect of vertical sleeve gastrectomy in melanocortin receptor 4-deficient rats

Bariatric surgery is currently the most effective treatment for obesity. Vertical sleeve gastrectomy (VSG), a commonly applied bariatric procedure, involves surgically incising most of the volume of the stomach. In humans, partial loss of melanocortin receptor-4 (MC4R) activity is the most common monogenic correlate of obesity regardless of lifestyle. At present it is unclear whether genetic alteration of MC4R signaling modulates the beneficial effects of VSG. Following VSG, we analyzed body weight, food intake, glucose sensitivity, and macronutrient preference of wild-type and MC4R-deficient (Mc4r(+/-) and Mc4r(-/-)) rats compared with sham-operated controls. VSG reduced body weight and fat mass and improved glucose metabolism and also shifted preference toward carbohydrates and away from fat. All of this occurred independently of MC4R activity. In addition, MC4R was resequenced in 46 human subjects who underwent VSG. We observed common genetic variations in the coding sequence of MC4R in five subjects. However, none of those variations appeared to affect the outcome of VSG. Taken together, these data suggest that the beneficial effect of VSG on body weight and glucose metabolism is not mediated by alterations in MC4R activity.     

Molecular genetics of preeclampsia and HELLP syndrome — A review

Preeclampsia is characterised by new onset hypertension and proteinuria and is a major obstetrical problem for both mother and foetus. Haemolysis elevated liver enzymes and low platelets (HELLP) syndrome is an obstetrical emergency and most cases occur in the presence of preeclampsia. Preeclampsia and HELLP are complicated syndromes with a wide variety in severity of clinical symptoms and gestational age at onset. The pathophysiology depends not only on periconceptional conditions and the foetal and placental genotype, but also on the capability of the maternal system to deal with pregnancy. Genetically, preeclampsia is a complex disorder and despite numerous efforts no clear mode of inheritance has been established. A minor fraction of HELLP cases is caused by foetal homozygous LCHAD deficiency, but for most cases the genetic background has not been elucidated yet. At least 178 genes have been described in relation to preeclampsia or HELLP syndrome. Confined placental mosaicism (CPM) is documented to cause early onset preeclampsia in some cases; the overall contribution of CPM to the occurrence of preeclampsia has not been adequately investigated yet. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

Europrotomus (Mollusca: Caenogastropoda: Strombidae): a new Middle Miocene European strombid genus (Revision of Euprotomus Gill, 1870. Part 4)

The medium-sized, strongly ornamented nominal species Strombus schroeckingeri H?rnes in Hoernes & Auinger, 1884 is well known from Langhian deposits of the Paratethys Sea. Its traditional generic affiliation in the literature with Euprotomus Gill, 1870, implicated a biogeographic relation of the Paratethys and the proto-Mediterranean with the Neogene Indo-West-Pacific region. This relation, however, is problematic because the Tethys Seaway was already closed or a very shallow, strongly evaporitic passage at that time. Despite its superficial similarity with extant genera of the IWP region, the genus is unrelated to all known strombid genera and represents a new genus, which is introduced herein as Europrotomus nov. gen. The genus comprises only one or maybe two species, which occur in the European Middle Miocene geological record. No ancestors are known so far. This sudden occurrence is discussed as immigration from an adjacent bioprovince rather than as a result of autochthonous evolution. Hypothetically, such a biogeographic source area might have been established along the coasts of tropical West Africa??a scenario that is already documented for Pleistocene times.     

Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process

An anaerobic microbial community was enriched in a chemostat that was operated for more than 8 years with benzene and nitrate as electron acceptor. The coexistence of multiple species in the chemostat and the presence of a biofilm, led to the hypothesis that benzene-degrading species coexist in a syntrophic interaction, and that benzene can be degraded in syntrophy by consortia with various electron acceptors in the same culture. The benzene-degrading microorganisms were identified by DNA-stable isotope probing with [U-(13) C]-labelled benzene, and the effect of different electron donors and acceptors on benzene degradation was investigated. The degradation rate constant of benzene with nitrate (0.7 day(-1) ) was higher than reported previously. In the absence of nitrate, the microbial community was able to use sulfate, chlorate or ferric iron as electron acceptor. Bacteria belonging to the Peptococcaceae were identified as dominant benzene consumers, but also those related to Rhodocyclaceae and Burkholderiaceae were found to be associated with the anaerobic benzene degradation process. The benzene degradation activity in the chemostat was associated with microbial growth in biofilms. This, together with the inhibiting effect of hydrogen and the ability to degrade benzene with different electron acceptors, suggests that benzene was degraded via a syntrophic process.     

Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.     

SWI/SNF complex in disorder: SWItching from malignancies to intellectual disability

Heterozygous germline mutations in components of switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes were recently identified in patients with non-syndromic intellectual disability, Coffin-Siris syndrome and Nicolaides-Baraitser syndrome. The common denominator of the phenotype of these patients is severe intellectual disability and speech delay. Somatic and germline mutations in SWI/SNF components were previously implicated in tumor development. This raises the question whether patients with intellectual disability caused by SWI/SNF mutations in the germline are exposed to an increased risk of developing cancer. Here we compare the mutational spectrum of SWI/SNF components in intellectual disability syndromes and cancer, and discuss the implications of the results of this comparison for the patients.     

Structure and functional analysis of the IGF-II/IGF2R interaction

Embryonic development and normal growth require exquisite control of insulin-like growth factors (IGFs). In mammals the extracellular region of the cation-independent mannose-6-phosphate receptor has gained an IGF-II-binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF-II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11-12, 11-12-13-14 and domains 11-12-13/IGF-II complex. A distinctive juxtaposition of these domains provides the IGF-II-binding unit, with domain 11 directly interacting with IGF-II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF-II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF-II 'binding-hotspot', revealing that IGF-binding proteins and IGF2R have converged on the same high-affinity site.     

Protease-activated receptor-2 exerts contrasting model-specific effects on acute experimental pancreatitis

Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.