Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

AbstractAquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca2+-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg2+-dependent, Ca2+-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1

Plant Aquaporins: A study of expression, localization, specificity, and regulation

Aquaporins, or Major Intrinsic Proteins (MIPs), are integral proteins that facilitate transport of water and other small neutral solutes across biological membranes. They belong to a well conserved and ancient family of proteins, present in all organisms ranging from bacteria to plants and humans. The aquaporin family in plants is large, indicating complex and regulated water transport within the plant in order to adapt to different environmental conditions. All aquaporin isoforms probably work together in an orchestrated manner, where each individual aquaporin isoform displays a specific localization pattern, substrate specificity, and regulatory mechanism. When analyzing the whole genome of the model plant Arabidopsis thaliana, 35 aquaporin-encoded genes were identified. Based on sequence similarities and by phylogenetic analyses they were divided into four subfamilies; Plasma membrane Intrinsic Proteins (PIPs), Tonoplast Intrinsic Proteins (TIPs), NOD26-like Intrinsic Proteins (NIPs), and Small basic Intrinsic Proteins (SIPs). These subfamilies are conserved in many plant species. Based on the four subfamilies a new uniform nomenclature for all plant aquaporins was proposed, which is now widely accepted and used. A gene expression study, using a DNA microarray and quantitative real-time reverse transcriptase PCR, of all the 35 aquaporin genes in Arabidopsis was performed. The relative amounts of each isoform in leaves, roots, and flowers were analyzed, as well as their individual responses to drought stress. Focusing on four of the nine isoforms in the Arabidopsis NIP subfamily, i.e. AtNIP1;2, AtNIP2;1, AtNIP4;2, and AtNIP6;1, the gene expression on tissue and cell level was studied with promoter::GUS constructs, the protein localization was studied on the subcellular level in different organs with immunoblots, and the permeability to water and glycerol was examined by heterologous expression in Xenopus oocytes. The spinach leaf plasma membrane aquaporin SoPIP2;1 was heterologously overexpressed in the yeast Pichia pastoris, purified, and functionally characterized by reconstitution into proteoliposomes. The water channel activity of SoPIP2;1 has previously been shown to be regulated by phosphorylation of Ser115 and Ser274. Two protein kinases, acting on these two phosphorylation sites in SoPIP2;1, were partly purified and characterized

In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V(max). The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product

Initiation of biphasic insulin aspart 30/70 in subjects with type 2 diabetes mellitus in a largely primary care-based setting in Sweden.

AIMS: Despite a wealth of clinical trial data supporting use of the premixed insulin analogue, biphasic insulin aspart 30 (BIAsp 30) in the treatment of type 2 diabetes mellitus (T2DM), there is limited documentation of its use in primary care-based clinical practice. METHODS: An observational study investigating the safety and efficacy of BIAsp 30 in routine clinical practice was conducted. Patients were followed up 3 and 6 months after initiating insulin treatment. Safety and efficacy measures were documented. RESULTS: During the course of the study, 1154 patients were included (age range 20-95years), of whom 89% completed the 6-month follow-up period. Mean HbA(1c) at baseline was 8.8% (73mmol/mol), and had improved to 7.2% (55mmol/mol) after 6 months of treatment. The rate of total hypoglycaemia at completion of the study was 4.1 events per patient year. Major hypoglycaemic events were rare (two in total). CONCLUSIONS: BIAsp 30 was initiated safely and effectively in insulin-naïve patients with T2DM. The safety and efficacy profile observed in clinical trials was confirmed in this largely primary care-based setting in Sweden

The Complete Set of Genes Encoding Major Intrinsic Proteins in Arabidopsis Provides a Framework for a New Nomenclature for Major Intrinsic Proteins in Plants

Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes. MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants. In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes. Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns. Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons. Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies. The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species. Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed. The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species

Whole gene family expression and drought stress regulation of aquaporins

Since many aquaporins (AQPs) act as water channels, they are thought to play an important role in plant water relations. It is therefore of interest to study the expression patterns of AQP isoforms in order to further elucidate their involvement in plant water transport. We have monitored the expression patterns of all 35 Arabidopsis AQPs in leaves, roots and flowers by cDNA microarrays, specially designed for AQPs, and by quantitative real-time reverse transcriptase PCR (Q-RT-PCR). This showed that many AQPs are pre-dominantly expressed in either root or flower organs, whereas no AQP isoform seem to be leaf specific. Looking at the AQP subfamilies, most plasma membrane intrinsic proteins (PIPs) and some tonoplast intrinsic proteins (TIPs) have a high level of expression, while NOD26-like proteins (NIPs) are present at a much lower level. In addition, we show that PIP transcripts are generally down-regulated upon gradual drought stress in leaves, with the exception of AtPIP1;4 and AtPIP2;5, which are up-regulated. AtPIP2;6 and AtSIP1;1 are constitutively expressed and not significantly affected by the drought stress. The transcriptional down-regulation of PIP genes upon drought stress could also be observed on the protein level

Dynamic changes in mucus thickness and ion secretion during Citrobacter rodentium infection and clearance.

Citrobacter rodentium is an attaching and effacing pathogen used as a murine model for enteropathogenic Escherichia coli. The mucus layers are a complex matrix of molecules, and mucus swelling, hydration and permeability are affected by many factors, including ion composition. Here, we used the C. rodentium model to investigate mucus dynamics during infection. By measuring the mucus layer thickness in tissue explants during infection, we demonstrated that the thickness changes dynamically during the course of infection and that its thickest stage coincides with the start of a decrease of bacterial density at day 14 after infection. Although quantitative PCR analysis demonstrated that mucin mRNA increases during early infection, the increased mucus layer thickness late in infection was not explained by increased mRNA levels. Proteomic analysis of mucus did not demonstrate the appearance of additional mucins, but revealed an increased number of proteins involved in defense responses. Ussing chamber-based electrical measurements demonstrated that ion secretion was dynamically altered during the infection phases. Furthermore, the bicarbonate ion channel Bestrophin-2 mRNA nominally increased, whereas the Cftr mRNA decreased during the late infection clearance phase. Microscopy of Muc2 immunostained tissues suggested that the inner striated mucus layer present in the healthy colon was scarce during the time point of most severe infection (10 days post infection), but then expanded, albeit with a less structured appearance, during the expulsion phase. Together with previously published literature, the data implies a model for clearance where a change in secretion allows reformation of the mucus layer, displacing the pathogen to the outer mucus layer, where it is then outcompeted by the returning commensal flora. In conclusion, mucus and ion secretion are dynamically altered during the C. rodentium infection cycle

List of primers for SyberGreen based RT-PCR.

<p>List of primers for SyberGreen based RT-PCR.</p

Ussing chamber responses are larger in a goblet cell-like <i>in vitro</i> model than in an enterocyte-like model after infection with <i>C. rodentium</i>.

<p>A: Membrane current (Im) response to forskolin of the goblet cell containing model (LS513) <i>vs</i> enterocyte like model (Caco-2) co-cultured with <i>C. rodentium</i> for 24 h (black bars) and cells without bacteria (white bars). B: Membrane current (Im) response of LS513 vs Caco-2 cells treated with <i>C. rodentium</i> for 30 min after insertion into the Ussing chamber and then stimulated with carbachol and forskolin. C: Transepithelial resistance (Rp) response of LS513 vs Caco-2 cells treated with <i>C. rodentium</i> for 30 min after insertion into the Ussing chamber and then stimulated with carbachol and forskolin. (2-tailed T-test, **p<0.01, ***p<0.001).</p

Mucus and bacteria during bacterial expulsion (day 14).

<p>A–C: The anti-Muc2C3 immunostained Muc2 mucin (green) formed an inner mucus layer (marked with a yellow bar in panel C) that is less well organized than in uninfected colon. Bacteria (FISH, eubacterial probe, hybridizing with both <i>C. rodentium</i> and other eubacteria) are mainly present in the outer mucus layer. Photos were taken with a Nikon Eclipse 90 i microscope, white bar = 50 µm. D–F: The mucus stained as in A–C showing the rod-shaped bacteria within the green mucus (white arrows), white bar = 50 µm. Photos taken with a LSM 510 META (Zeiss) confocal microscope with a 40x objective.</p