A Proteome-wide Domain-centric Perspective on Protein Phosphorylation

Phosphorylation is a widespread post-translational modification that modulates the function of a large number of proteins. Here we show that a significant proportion of all the domains in the human proteome is significantly enriched or depleted in phosphorylation events. A substantial improvement in phosphosites prediction is achieved by leveraging this observation, which has not been tapped by existing methods. Phosphorylation sites are often not shared between multiple occurrences of the same domain in the proteome, even when the phosphoacceptor residue is conserved. This is partly because of different functional constraints acting on the same domain in different protein contexts. Moreover, by augmenting domain alignments with structural information, we were able to provide direct evidence that phosphosites in protein-protein interfaces need not be positionally conserved, likely because they can modulate interactions simply by sitting in the same general surface area.Phosphorylation, the most widespread protein post-translational modification, is an important regulator of protein function. The addition of phosphate groups on serine, threonine, and tyrosine residues can modulate the activity of the target protein by inducing complex conformational changes, by modifying protein electrostatics, and by regulating domain-peptide interactions, as in 14-3-3 or SH2 domains, that specifically recognize phosphorylated residues. The standard experimental technique for the high-throughput identification of phosphorylation sites is mass spectrometry (1).Phosphorylation is catalyzed by protein kinases, a family that in humans comprises ∼540 members (2, 3). It is well understood that these enzymes recognize specific sequence motifs in their substrates (4, 5). Accordingly the sequence around the phosphorylation site is undisputedly the most important feature for phosphosite prediction (6, 7). However the “context,” in a broad sense, where these motifs occur is also important as sequence alone is not enough to achieve the observed specificity of phosphorylation. Therefore, several studies have characterized multiple aspects of phosphosites such as their preference for loops and disordered regions (reviewed in (8)), or the tendency of phosphoserines and phosphothreonines to occur in clusters (9), and these features have been used to improve the performance of phosphosite predictors (6, 7, 10–12). Moreover placing kinases and substrates in the context of protein interaction networks has been shown to improve the prediction of phosphorylation by specific kinases (13).Perhaps one of the most puzzling observations when looking at the phosphoproteome as a whole, is the fact that a large proportion of phosphorylation sites is poorly conserved. This has led to various hypotheses. First some sites may represent nonfunctional, possibly low-stoichiometry, phosphorylation events that are picked up because of the sensitivity of mass-spectrometry (14, 15). Indeed functionally characterized sites and those matching known kinase motifs are more conserved on average (15–17). However, although in biology function often equates with conservation, there could be genuinely functional fast-evolving phosphosites, that are responsible for species-specific differences in signaling and regulation. Moreover in some cases, especially in the regulation of protein-protein interactions, the exact position of the phosphosites may be unimportant (18, 19).Here we explore the issues of “context” and “conservation” of phosphorylation sites from the perspective of protein domains. To this end, we assembled a comprehensive database of phosphosites from publicly available sources and studied their proteome distribution with respect to the location and identity of protein domains. We focus on the human phosphoproteome because it has been very well characterized in a multitude of low- and high-throughput experiments, thus providing the opportunity for a comprehensive, proteome-wide, study. In particular, the issues we want to address are the following:

1. Are specific domain types preferentially phosphorylated? Or conversely are some domains specifically depleted of phosphorylation sites?
2. Can the domain context be used to improve the prediction of phosphorylation sites?
3. What is the conservation pattern of phosphosites when looking at multiple instances of the same domain in the proteome?

     

天童常绿阔叶树种栲树生殖个体大小及其生殖构件特征

对浙江天童木荷-栲树林内的常绿阔叶树种栲树(Castanopsis fargesii Franch.)的生殖个体大小、生殖构件的分布及其动态变化特征进行了研究。结果表明, 该地区栲树生殖个体的胸径在17~50 cm 间, 平均胸径为31. 2±8.0 cm, 平均年龄约36.3±6.6 年;林缘附近的生殖个体小于木荷-林内。相对稳定的群落和比较丰富的土壤养分条件有利于生殖枝数量和花序数量的增多。栲树生殖个体的数量在两年中变化较大, 部分栲树个体可以在连续年份中生殖。从枝系水平分析:在持续生殖的栲树个体上, 生殖枝数量有明显变化, 并非所有的生殖枝在两年中都可开花或结果, 保持连续生殖的枝系约占48.2%。栲树果序枝数量在连续年份有明显差异(p < 0.01), 而且果序枝上的幼蕾数、果实数量及结实率等都有明显差异(p < 0.05)。     

Lymphoproliferative and cytotoxic responses of human peripheral blood mononuclear cells to mannoprotein constituents of Candida albicans.

Two major proteoglycan constituents (designated F1 and F2) of the cell wall of Candida albicans were separated by ion-exchange chromatography from a crude carbohydrate-rich extract (GMP), and investigated for their chemical and molecular composition, antigenicity and immunomodulatory properties in cultures of human peripheral blood mononuclear cells (PBMC). Both fractions consisted predominantly of Periodic acid-Schiff (PAS) and concanavalin A (Con A)-reactive material consisting of greater than 90% mannose, 3-5% protein and small amounts of phosphorus; each was recognized by an anti-Candida rabbit serum as well as by a monoclonal antibody (mAb AF1) directed against an oligosaccharide epitope present on the fungal cell surface. When F1 and F2 were subjected to SDS-PAGE, transblotted and stained with enzyme-conjugated mAb AF1 or Con A, most of the antibody or lectin bound to high molecular mass (greater than 200 kDa) polydisperse material, some of which was present in F2 (as in the starting GMP extract) but absent in F1. This difference was also observed in PAS-stained gels of the two fractions. The F2, but not the F1, constituent was as active as the unfractionated GMP extract in inducing lymphoproliferation, production of the cytokines interleukin-2 and interferon-gamma, and generation of cytotoxicity against a natural-killer-sensitive target cell line (K562). These immunomodulatory properties were, like those possessed by GMP, protease-sensitive and heat-stable. Treatment of PMBC cultures with a modulatory anti-T-cell receptor antibody abolished the lymphoproliferation induced by GMP and F2 but not that induced by phytohaemagglutinin, showing that the mannoprotein materials of C. albicans acted through interaction with the antigen receptor complex.     

Polarization of gelsolin and actin binding protein in kidney epithelial cells

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.     

Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.     

Indoor and outdoor winter activity of Culicoides biting midges,vectors of bluetongue virus,in Italy

Indoor and outdoor winter activity of Culicoides spp. (Diptera: Ceratopogonidae) in central Italy was investigated in order to evaluate whether indoor activity might account for the overwintering of bluetongue virus, as has been hypothesized by some authors. Weekly Culicoides collections were performed at three farms over three consecutive winter seasons. At each farm, two black‐light traps were operated simultaneously, indoors and outdoors. Culicoides were identified using both morphological and molecular means. The Culicoides obsoletus group accounted for 98.2% of sampled specimens. Within this group, C. obsoletus s.s. accounted for 56.8% and Culicoides scoticus for 43.2% of samples. Nulliparous, parous and engorged females were caught throughout the entire winter, both indoors and outdoors. At times, indoor catch sizes outnumbered outdoor collections. A significant inverse correlation was found between minimum temperature and the proportion of indoor Culicoides of the total midge catch, thus indicating that lower outdoor temperatures drive Culicoides midges indoors. High rates of engorged females were recorded indoors, possibly as the result of the propensity of C. obsoletus females to feed indoors. Higher proportions of parous females were found in indoor than in outdoor catches, indicating higher survival rates indoors and, consequently, higher vectorial capacities of midges sheltering indoors compared with those remaining outdoors.     

Short-Term In Vitro Culture and Molecular Analysis of the Microsporidian, Enterocytozoon bieneusi

ABSTRACT. The microsporidium, Enterocytozoon bieneusi , causes a severe, debilitating, chronic diarrhea in patients with the acquired immunodeficiency syndrome. Specific diagnosis of intestinal microsporidiosis, especially due to Enterocytozoon , is difficult and there is no known therapy that can completely eradicate this parasite. Preliminary studies indicate that a short term (about 6 months) in vitro culture of this parasite yielding low numbers of spores, may be established by inoculating human lung fibroblasts and/or monkey kidney cell cultures with duodenal aspirates and or biopsy from infected patients. The cultures may subsequently be used for the isolation and molecular analysis of parasite DNA.     

Gram-Chromotrope: a New Technique that Enhances Detection of Microsporidial Spores in Clinical Samples

We have developed a new staining procedure that combines the traditional Gram staining for bacteria and the Weber's chromotrope staining method, the standard technique for the detection of microsporidia spores in clinical Specimens. This “Gram-chromotrope” staining technique enhances the staining characteristics of microsporidia spores and facilitates the easy detection and differentiation of spores from other microorganisms that are found in clinical specimens, especially stool samples. This new technique is fast, reliable, and simple to perform, and can be easily adapted for use in clinical laboratories.