Augmented erythrocyte band-3 phosphorylation in septic mice

Infection-induced RBC dysfunction has been shown to play a role in the modulation of host response to injury and infection. The underlying biochemical mechanisms are not known. This study investigated alterations in RBC band-3 phosphorylation status and its relationship to anion exchange activity in vitro as well as under in vivo septic conditions induced by cecal ligation and puncture (CLP) in mice. Pervanadate treatment in vitro increased band-3 tyrosine phosphorylation that was accompanied by decreased RBC deformability and anion exchange activity. Following sepsis, band-3 tyrosine phosphorylation in whole RBC ghosts as well as in cytoskeleton-bound or soluble RBC protein fractions were elevated as compared to controls. Although anion exchange activity was similar in RBCs from septic and control animals, band-3 interaction with eosin-5-maleimide (EMA), which binds to band-3 lysine moieties, was increased in cells from septic animals as compared to controls, indicating that sepsis altered band 3 organization within the RBC membrane. Since glucose-6-phosphate dehydrogenase is a major antioxidant enzyme in RBC, in order to assess the potential role of oxidative stress in band-3 tyrosine phosphorylation, sepsis-induced RBC responses were also compared between WT and (G6PD) mutant animals (20% of normal G6PD activity). Band-3 membrane content and EMA staining were elevated in G6PD mutant mice compared to WT under control non-septic conditions. Following sepsis, G6PD mutant animals showed lessened responses in band-3 tyrosine phosphorylation and EMA staining compared to WT. RBC anion exchange activity was similar between mutant and WT animals under all tested conditions. In summary, these studies indicate that sepsis results in elevated band-3 tyrosine phosphorylation and alters band-3 membrane organization without grossly affecting RBC anion exchange activity. The observations also suggest that factors other than oxidative stress are responsible for the sepsis-induced increase in RBC band-3 tyrosine phosphorylation.     

Distribution of the glycoconjugate oligosaccharides in the human placenta from pregnancies complicated by altered glycemia: lectin histochemistry

The aim of this study was to investigate the distribution of the oligosaccharides of the glycoconjugates in placentas from pregnancies complicated by different degree of altered glycaemia. Placentas from women with physiological pregnancies (group 1), with pregnancies complicated by minor degree of glucose intolerance (group 2) and with pregnancies complicated by gestational diabetes mellitus (GDM) treated with insulin (group 3) were collected. Ten lectins were used (ConA, WGA, PNA, SBA, DBA, LTA, UEA I, GSL II, MAL II and SNA) in combination with chemical and enzymatic treatments. The data showed a decrease of sialic acid linked α(2–6) to galactose/N-acetyl-d-galactosamine and an increase of N-acetyl-d-glucosamine in the placentas of the pathological groups, in particular the group 3, comparing to the group 1. A decrease of l-fucose (LTA) and d-galactose-(β1–3)-N-acetyl-d-galactosamine, and an increase and/or appearance of l-fucose (UEA I) and N-acetyl-d-galactosamine were observed in both the pathological groups, particularly in the group 2, with respect to the group 1. In GDM, and even in pregnancies with a simple alteration of maternal glycaemia, the changes in the distribution of oligosaccharides could be related to alteration of the structure and functionality of the placenta.     

A novel detection strategy for odorant molecules based on controlled bioengineering of rat olfactory receptor I7

In this study, we report a dose-dependent detection of odorant molecules in solution by rat olfactory receptor I7 (OR I7) in its membrane fraction. The OR I7 is immobilized on a gold electrode by multilayer bioengineering based on a mixed self-assembled monolayer and biotin/avidin system, which allows for a well-controlled immobilization of the bioreceptor within its lipid environment. The odorant detection is electronically performed in a quantitative manner by electrochemical impedance spectroscopy (EIS) measurements on samples and controls.     

Interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α in asthenozoospermia

Impaired male fertility may have a variety of causes, among which asthenozoospermia. In its etiology, several bioactive substances, such as cytokines may be involved. In this context, our aim was to evaluate the expression of interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α, in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients. We evaluated twenty-eight infertile patients affected by idiopathic asthenozoospermia and twenty-three normospermic fertile donors, age-matched. Sperm parameters were evaluated; immunohistochemical analysis and enzyme-linked immunosorbent assay were then performed in isolated spermatozoa. Spermatozoa from the asthenozoospermic group presented an increased expression of IL-1β, COX-2, and HIF-1α compared with the normospermic fertile subjects. Our results can lead us to speculate that the increased expression of these substances may influence sperm motility. Nevertheless, further studies are needed in order to assess whether these bioactive mediators have a potential relevance as targets in future therapeutic strategies for the treatment of male infertility.     

The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero‐subunit peptidasome with enhanced peptide destruction properties

TET aminopeptidases assemble as large homo‐dodecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo‐oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero‐oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero‐oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo‐oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi‐enzymatic complex efficiency among exopeptidases.     

p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis

Introduction

Recent evidence suggests that tissue accumulation of senescent p16^INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16^INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16^INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).

Methods

We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16^INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16^INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.

Results

p16^INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16^INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16^INK4a. Accordingly, p16^INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.

Conclusions

We disclosed herein a new role of the senescence marker p16^INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.     

Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse

The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca²⁺ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity.Knockout (KO)¹ mouse models represent powerful methods for studying the physiological relevance of a protein. However, to elucidate the effects of KO-induced perturbations on the entire system, systems-wide molecular characterization is needed, as, for instance, provided by (phospho)proteomics. Recent technological and methodological advancements now allow the mapping of protein expression, at least in cell cultures, close to completion (1–3). More challenging, proteomics is also increasingly used to attempt systems-wide proteome characterizations in tissue. This has led to semi-quantitative (4–6) and quantitative (7) reasonably comprehensive proteome data on selected tissues, in both humans and animal models. More recently, proteomics has also been applied for the in-depth profiling of perturbations in the proteome occurring in KO models. For instance, de Graaf et al. (8) used an in-depth proteomic approach to identify the proteins changed by DNA-damage-induced premature aging, using a KO mouse model lacking the excision repair cross-complementing group 1 gene. Another recent study used a mouse model lacking apolipoprotein E in order to identify biomarker candidates for coronary artery disease (9).Adaptation and/or perturbations in the proteome caused by a KO can lead to changes in protein expression, but, at least equally likely, also to rewiring of signaling networks, through changes in post-translational modifications, such as protein phosphorylation. The application of (phospho)proteomics technology on KO or knock-in models is therefore also extremely relevant, albeit even more challenging. Hilger et al. (10) combined proteomics and phosphoproteomics on a cell line in which a phosphatase had been knocked out. To perform such experiments in a more (disease) relevant context, we should invest in functional, tissue-based phosphoproteomics approaches. A few examples of such approaches have very recently been reported. Lundby et al. (11) globally assessed phosphorylation events downstream of systemic adrenergic stimulation in mouse cardiac tissue. We recently reported on the use of a cardiac delimited CaMKII inhibited knock-in mouse to probe for substrates using a focused kinase-inhibition directed approach (12). Moreover, a mouse model lacking nitric oxide synthase (13), as a system of interest for Alzheimer disease, was recently studied via (phospho)proteomics.Here we explored how mature state-of-the-art (phospho)proteomics technology could be used to monitor the adaptation at the proteome level of the mouse cerebellum in a mouse line deficient for cGMP-dependent protein kinase type I (cGKI, also known as PKG-I), a kinase that plays an important role in synaptic plasticity, motor learning, and other brain functions (14). The cGMP-dependent protein kinases are serine/threonine kinases that act as key mediators of nitric oxide (NO) signaling, as well as of the natriuretic peptide pathway (15). In mammals, cGKs are encoded by two different genes: prkg1 coding for cGKI, and prkg2 coding for cGKII (16). The prkg1 gene encodes two cGKI isoforms, cGKIα and cGKIβ (17), which differ in their N-terminal leucine zipper and auto-inhibitory domains. cGKI regulates cardiovascular functions such as smooth muscle and cardiac contractility (16); in the nervous system it modulates synaptic plasticity in the hippocampus (18) and cerebellum (19).In the mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to contribute to learning and memory. In particular, cGKIα is highly expressed in cerebellar Purkinje cells (PCs) (20, 21). Long-term depression (LTD) is an activity-dependent reduction in the efficacy of synaptic transmission and occurs at the PC synapses. Both a pharmacological approach using enzyme inhibitors (22) and a conditional PC-specific cGKI-KO (23) showed that cGKI plays a role in cerebellar LTD. Several proteins have been identified in past years as cGKI substrates in vitro or in cultured cells (15), but only a small portion of these have been confirmed as cGKI substrates in vivo. Therefore, the understanding of cGKI signaling and function depends strongly on the identification of novel in vivo substrates and signaling partners. In this perspective, the currently described approach allows us to discover potentially novel cGKI signaling routes and substrates directly in relevant cerebellar tissue. Our study revealed that cGKI-KO led to differential expression in the cerebellum of a specific group of proteins, of which many were closely connected to cGMP-cGKI signaling. More changes were observed at the phosphoproteome level, with the regulation of phosphorylation of a few hundred proteins. In particular, we hypothesize that some of the down-regulated phosphoproteins, but certainly not all, may be putative substrates of cGKI.     

In pursuit of negative Fukui functions: molecules with very small band gaps

Journal of Molecular Modeling - A new 3D graphical representation of DNA sequences is introduced. This representation is called 3D-dynamic representation. It is a generalization of the 2D-dynamic...     

The proteolysis adaptor,NblA, initiates protein pigment degradation by interacting with the cyanobacterial light‐harvesting complexes

Degradation of the cyanobacterial protein pigment complexes, the phycobilisomes, is a central acclimation response that controls light energy capture. The small protein, NblA, is essential for proteolysis of these large complexes, which may reach a molecular mass of up to 4 MDa. Interactions of NblA in vitro supported the suggestion that NblA is a proteolysis adaptor that labels the pigment proteins for degradation. The mode of operation of NblA in situ, however, remained unresolved. Particularly, it was unclear whether NblA interacts with phycobilisome proteins while part of the large complex, or alternatively interaction with NblA, necessitates dissociation of pigment subunits from the assembly. Fluorescence intensity profiles demonstrated the preferential presence of NblA::GFP (green fluorescent protein) at the photosynthetic membranes, indicating co‐localization with phycobilisomes. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for interaction of NblA with phycobilisome protein pigments. Additionally, we demonstrated the role of NblA in vivo as a proteolysis tag based on the rapid degradation of the fusion protein NblA::GFP compared with free GFP. Taken together, these observations demonstrated in vivo the role of NblA as a proteolysis adaptor. Additionally, the interaction of NblA with phycobilisomes indicates that the dissociation of protein pigment subunits from the large complex is not a prerequisite for interaction with this adaptor and, furthermore, implicates NblA in the disassembly of the protein pigment complex. Thus, we suggest that, in the case of proteolysis of the phycobilisome, the adaptor serves a dual function: undermining the complex stability and designating the dissociated pigments for degradation.