Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1

Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation.     

L-type Calcium Channel Cav1.2 Is Required for Maintenance of Auditory Brainstem Nuclei

Ca_v1.2 and Ca_v1.3 are the major L-type voltage-gated Ca²⁺ channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Ca_v1.3 is essential for proper maturation. Here, we investigated the role of Ca_v1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Ca_v1.3, loss of Ca_v1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Ca_v1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Ca_v1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Ca_v1.2 and Ca_v1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.     

Highly Virulent Non-O157 Enterohemorrhagic Escherichia coli (EHEC) Serotypes Reflect Similar Phylogenetic Lineages,Providing New Insights into the Evolution of EHEC

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene (stx), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx-negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.     

Expression of cGMP-dependent protein kinase type I in mature white adipocytes

The presence of cGMP-dependent protein kinase I (cGKI) in murine adipocytes has been questioned, although cGKI was implicated in the thermogenic program of fat cells (FCs) and to exert anti-hypertrophic/-inflammatory effects in white adipose tissue. Herein, cGKI was detected in adipocytes from control mice, whereas FCs from global cGKI knockouts (cGKI^−/−) and cGKIα rescue (αRM) mice remained cGKI-negative. cGKI mutants exhibit decreased adipocyte size, plasma leptin levels and reduced body-weights as compared to litter-matched controls. Low abundance of adiponectin in WAT and plasma of αRM animals together with previously confirmed high IL-6 levels indicate a low-grade inflammation. However, αRMs were protected from streptozotocin-induced hyperglycemia. Our results suggest that cGMP/cGKI affects both glucose and FC homeostasis in more complex mode than previously thought.     

Interaction between NOD2 and CARD9 involves the NOD2 NACHT and the linker region between the NOD2 CARDs and NACHT domain

NOD2 activation by muramyl dipeptide causes a proinflammatory immune response in which the adaptor protein CARD9 works synergistically with NOD2 to drive p38 and c-Jun N-terminal kinase (JNK) signalling. To date the nature of the interaction between NOD2 and CARD9 remains undetermined. Here we show that this interaction is not mediated by the CARDs of NOD2 and CARD9 as previously suggested, but that NOD2 possesses two interaction sites for CARD9; one in the CARD–NACHT linker and one in the NACHT itself.     

Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies

Neurodegenerative disorders such as Huntington’s disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin–proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington’s disease, and support the reported absence of proteasome impairment in mouse models of Huntington’s disease.     

A spider mating plug: origin and constraints of production

Males can increase their reproductive success by mechanically hindering females to mate with subsequent males. Research on mating plugs so far has focused on the fitness consequences and demonstrated that plug size can strongly determine its efficacy. Here, we explore: (1) the site of plug production in the erigonine spider Oedothorax retusus; and (2) whether males are limited in the production of plug material when mating with three females in succession. Micro‐computed tomography, histological and ultrastructural sections demonstrate that the plug material is produced in a massive gland inside the sperm transfer organs of the male, the pedipalps. The glandular lumen is connected with the tube‐like spermophore almost at its blind end. Probably, a reservoir of plug material is built up at the end of the spermophore and released after sperm transfer onto the female genital opening. Since not all males applied a large plug during their first mating, there was no significant decline in plug size over the course of the three successive matings. However, the size of the first plug significantly affected the size of the following plug. We discuss these findings in the light of plug limitation and mate choice. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 345–354.     

Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1^liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1^liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1^liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles.     

Retinoic acid as a survival factor in neuronal development of the grasshopper,Locusta migratoria

Based on experience with cell cultures of adult insect neurons, we develop a serum-free culture system for embryonic locust neurons. Influences of trophic substances on survival and neurite outgrowth of developing neurons are investigated. For the first time, a positive trophic effect of 9-cis retinoic acid (9-cis RA) was shown in vitro on embryonic neurons of an insect. We observed longer cell survival of 50 % developmental stage neurons in cultures supplemented with 0.3 nM 9-cis RA. Furthermore, an influence on neuron morphology was revealed, as the addition of 9-cis RA to cell culture medium led to an increase in the number of neurites per cell. Although an RA receptor gene, LmRXR (Locusta migratoria retinoid X receptor), was expressed in the central nervous system throughout development, the influence of 9-cis RA on neuronal survival and outgrowth was restricted to 50 % stage embryonic cells.