Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis

The anthrax toxin is composed of three independent polypeptide chains. Successful intoxication only occurs when heptamerization of the receptor-binding polypeptide, the protective antigen (PA), allows binding of the two enzymatic subunits before endocytosis. We show that this tailored behavior is caused by two counteracting posttranslational modifications in the cytoplasmic tail of PA receptors. The receptor is palmitoylated, and this unexpectedly prevents its association with lipid rafts and, thus, its premature ubiquitination. This second modification, which is mediated by the E3 ubiquitin ligase Cbl, only occurs in rafts and is required for rapid endocytosis of the receptor. As a consequence, cells expressing palmitoylation-defective mutant receptors are less sensitive to anthrax toxin because of a lower number of surface receptors as well as premature internalization of PA without a requirement for heptamerization

Effects of shear stress on the microalgae Chaetoceros muelleri

The effect of shear stress on the viability of Chaetoceros muelleri was studied using a combination of a rheometer and dedicated shearing devices. Different levels of shear stress were applied by varying the shear rates and the medium viscosities. It was possible to quantify the effect of shear stress over a wide range, whilst preserving laminar flow conditions through the use of a thickening agent. The threshold value at which the viability of algae was negatively influenced was between 1 and 1.3 Pa. Beyond the threshold value the viability decreased suddenly to values between 52 and 66%. The effect of shear stress was almost time independent compared to normal microalgae cultivation times. The main shear stress effect was obtained within 1 min, with a secondary effect of up to 8 min

Cultivation of shear stress sensitive and tolerant microalgalspecies in a tubular photobioreactor equipped with a centrifugal pump

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Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway

The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes

Blood and brain biochemistry and behaviour in NTBC and dietary treated tyrosinemia type 1 mice

Tyrosinemia type 1 (TT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Neurocognitive deficiencies have been described in TT1 patients, that have, among others, been related to changes in plasma large neutral amino acids (LNAA) that could result in changes in brain LNAA and neurotransmitter concentrations. Therefore, this project aimed to investigate plasma and brain LNAA, brain neurotransmitter concentrations and behavior in C57 Bl/6 fumarylacetoacetate hydrolase deficient (FAH-/-) mice treated with 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and/or diet and wild-type mice. Plasma and brain tyrosine concentrations were clearly increased in all NTBC treated animals, even with diet (p <0.001). Plasma and brain phenylalanine concentrations tended to be lower in all FAH-/- mice. Other brain LNAA, were often slightly lower in NTBC treated FAH-/- mice. Brain neurotransmitter concentrations were usually within a normal range, although serotonin was negatively correlated with brain tyrosine concentrations (p <0.001). No clear behavioral differences between the different groups of mice could be found. To conclude, this is the first study measuring plasma and brain biochemistry in FAH-/- mice. Clear changes in plasma and brain LNAA have been shown. Further research should be done to relate the biochemical changes to neurocognitive impairments in TT1 patients

The SwissLipids knowledgebase for lipid biology.

MOTIVATION: Lipids are a large and diverse group of biological molecules with roles in membrane formation, energy storage and signaling. Cellular lipidomes may contain tens of thousands of structures, a staggering degree of complexity whose significance is not yet fully understood. High-throughput mass spectrometry-based platforms provide a means to study this complexity, but the interpretation of lipidomic data and its integration with prior knowledge of lipid biology suffers from a lack of appropriate tools to manage the data and extract knowledge from it. RESULTS: To facilitate the description and exploration of lipidomic data and its integration with prior biological knowledge, we have developed a knowledge resource for lipids and their biology-SwissLipids. SwissLipids provides curated knowledge of lipid structures and metabolism which is used to generate an in silico library of feasible lipid structures. These are arranged in a hierarchical classification that links mass spectrometry analytical outputs to all possible lipid structures, metabolic reactions and enzymes. SwissLipids provides a reference namespace for lipidomic data publication, data exploration and hypothesis generation. The current version of SwissLipids includes over 244 000 known and theoretically possible lipid structures, over 800 proteins, and curated links to published knowledge from over 620 peer-reviewed publications. We are continually updating the SwissLipids hierarchy with new lipid categories and new expert curated knowledge. AVAILABILITY: SwissLipids is freely available at http://www.swisslipids.org/. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Sinteza i farmakološko ispitivanje novih 4-(3-etilfenil)-1-supstituiranih 4H-[1,2,4]triazolo[4,3-a]kinazolin-5-ona kao nove klase H1-antihistaminika

A series of novel 4-(3-ethylphenyl)-1-substituted-4H-[1,2,4]triazolo[4,3-a]quinazolin-5-ones (4a-j) were synthesized by the cyclization of 3-(3-ethylphenyl)-2-hydrazino-3H-quinazolin-4-one (3) with various one-carbon donors. The starting material, compound 3, was synthesized from 3-ethyl aniline by a new innovative route with improved yield. When tested for their in vivo H1-antihistaminic activity on conscious guinea pigs, all test compounds protected the animals from histamine induced bronchospasm significantly. Compound 4-(3-ethylphenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a]quinazolin-5-one (4b) emerged as the most active compound of the series and it is more potent (74.6 % protection) compared to the reference standard chlorpheniramine maleate (71 % protection). Compound 4b shows negligible sedation (10 %) compared to chlorpheniramine maleate (30 %). Therefore compound 4b can serve as the leading compound for further development of a new class of H1-antihistamines.Ciklizacijom 3-(3-etilfenil)-2-hidrazino-3H-kinazolin-4-ona (3) s različitim donorima jednog C atoma sintetizirana je serija novih 4-(3-etilfenil)-1-supstituiranih 4H-[1,2,4]triazolo[4,3-a]kinazolin-5-ona (4a-j). Početni spoj 3 pripravljen je iz 3-etil anilina na novi, inovativni način, s poboljšanim iskorištenjem. U testovima in vivo na zamorcima, svi testirani spojevi pokazali su značajno zaštitno djelovanje protiv bronhospazma induciranog histaminom. Spoj 4-(3-etilfenil)-1-metil-4H-[1,2,4]triazolo[4,3-a]kinazolin-5-on (4b) najaktivniji je među testiranim spojevima (zaštita 74.6 %) i jači od referentnog standarda klorfeniramin maleata (zaštita 71 %). Spoj 4b pokazuje zanemarivu sedaciju (10 %) u usporedbi s klorfeniramin maleatom (30 %). Stoga spoj 4b može biti vodeći spoj za daljnji razvoj nove klase H1-antihistaminika

Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin