Evidence for a role of protein phosphorylation in the maintenance of the cnidarian–algal symbiosis

The endosymbiotic relationship between cnidarians and photosynthetic dinoflagellate algae provides the foundation of coral reef ecosystems. This essential interaction is globally threatened by anthropogenic disturbance. As such, it is important to understand the molecular mechanisms underpinning the cnidarian–algal association. Here we investigated phosphorylation‐mediated protein signalling as a mechanism of regulation of the cnidarian–algal interaction, and we report on the generation of the first phosphoproteome for the coral model system Aiptasia. Mass spectrometry‐based phosphoproteomics using data‐independent acquisition allowed consistent quantification of over 3,000 phosphopeptides totalling more than 1,600 phosphoproteins across aposymbiotic (symbiont‐free) and symbiotic anemones. Comparison of the symbiotic states showed distinct phosphoproteomic profiles attributable to the differential phosphorylation of 539 proteins that cover a broad range of functions, from receptors to structural and signal transduction proteins. A subsequent pathway enrichment analysis identified the processes of “protein digestion and absorption,” “carbohydrate metabolism,” and “protein folding, sorting and degradation,” and highlighted differential phosphorylation of the “phospholipase D signalling pathway” and “protein processing in the endoplasmic reticulum.” Targeted phosphorylation of the phospholipase D signalling pathway suggests control of glutamate vesicle trafficking across symbiotic compartments, and phosphorylation of the endoplasmic reticulum machinery suggests recycling of symbiosome‐associated proteins. Our study shows for the first time that changes in the phosphorylation status of proteins between aposymbiotic and symbiotic Aiptasia anemones may play a role in the regulation of the cnidarian–algal symbiosis. This is the first phosphoproteomic study of a cnidarian–algal symbiotic association as well as the first application of quantification by data‐independent acquisition in the coral field.     

Analysis of detergent-resistant membranes associated with apical and basolateral GPI-anchored proteins in polarized epithelial cells

Detergent-resistant membranes (DRMs) represent specialized membrane domains resistant to detergent extraction, which may serve to segregate proteins in a specific environment in order to improve their function. Segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in DRMs has been shown to be involved in their sorting to the apical membrane in polarized epithelial cells. Nonetheless, we have shown that both apical and basolateral GPI-APs associate with DRMs. In this report we investigated the lipid composition of DRMs associated with an apical and a basolateral GPI-AP. We found that apical and basolateral DRMs contain the same lipid species although in different ratios. This specific lipid ratio is maintained after mixing the cells before lysis indicating that DRMs maintain their identity after Triton extraction.     

Role of phospholipase C and D signalling pathways in vasopressin-dependent myogenic differentiation

Arg⁸-vasopressin (AVP) is a potent inducer of myogenic differentiation stimulating the expression of myogenic regulatory factors. To understand the mechanism of its effect on myogenesis, we investigated the early signals induced by AVP in myogenic target cells. In the rat skeletal muscle cell line L6, AVP selectively stimulates phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) breakdown, through the activation of phospholipases C and D (PLC, PLD), as shown by the generation of Ins(1,4,5)P₃ and phosphatidylethanol (PtdEtOH), respectively. AVP induces the biphasic increase of sn-1,2-diacylglycerol (DAG) consisting in a rapid peak followed by a sustained phase, and the monophasic generation of phosphatidic acid (PA). Propranolol (a PA phosphatase inhibitor) and Zn²⁺ (a PLD inhibitor), abolish the sustained phase of DAG generation. Our data indicate that PtdIns-PLC activity is mainly responsible for the rapid phase of AVP-dependent DAG generation, whereas the sustained phase is dependent upon PtdCho-PLD activity and PA dephosphorylation, ruling out any significant role of DAG kinase. Modifications of PA level correlate with parallel changes of PLC activity, indicating a possible cross-talk between the two signal transduction pathways in the intact cell. PLD activation is elicited at AVP concentrations two orders of magnitude lower than those required for PLC activation. The differentiation of L6 myoblasts into multinucleated fibers is stimulated significantly by AVP at concentrations at which PLD, but not PLC, is activated. These data provide the first evidence for an important role of PLD in the mechanism of AVP-induced muscle differentiation. J. Cell. Physiol. 171:34–42, 1997. © 1997 Wiley-Liss, Inc.     

SAR study and conformational analysis of a series of novel peptide G protein‐coupled receptor kinase 2 inhibitors

G protein‐coupled receptor kinase 2 (GRK2) plays a central role in the cellular transduction network. In particular, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. Thereby, its inhibition offers a potential therapeutic solution to several pathological conditions. In the present study, we performed a SAR study and a NMR conformational analysis of peptides derived from HJ loop of GRK2 and able to selectively inhibit GRK2. From Ala‐scan and d ‐Ala point replacement, we found that Arg residues don't affect the inhibitory properties, while a d ‐amino acid at position 5 is key to the activity. Conformational analysis identified two β‐turns that involve N‐terminal residues, followed by a short extended region. These information can help the design of peptides and peptido‐mimetics with enhanced GRK2 inhibition properties. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 121–128, 2014.     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.     

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

AKTIP interacts with ESCRT I and is needed for the recruitment of ESCRT III subunits to the midbody

To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission.     

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions.

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.