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Zhao B Knepper MA Chou CL Pisitkun T 《American journal of physiology. Cell physiology》2012,302(1):C27-C45
Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination. 相似文献
13.
Xiaowen He Michael Knepper Cheng Ding Jun Li Suita Castro Maham Siddiqui Melitta Schachner 《PloS one》2012,7(9)
The L1 cell adhesion molecule promotes neurite outgrowth and neuronal survival in homophilic and heterophilic interactions and enhances neurite outgrowth and neuronal survival homophilically, i.e. by self binding. We investigated whether exploitation of homophilic and possibly also heterophilic mechanisms of neural stem cells overexpressing the full-length transmembrane L1 and a secreted trimer engineered to express its extracellular domain would be more beneficial for functional recovery of the compression injured spinal cord of adult mice than stem cells overexpressing only full-length L1 or the parental, non-engineered cells. Here we report that stem cells expressing trimeric and full-length L1 are indeed more efficient in promoting locomotor recovery when compared to stem cells overexpressing only full-length L1 or the parental stem cells. The trimer expressing stem cells were also more efficient in reducing glial scar volume and expression of chondroitin sulfates and the chondroitin sulfate proteoglycan NG2. They were also more efficient in enhancing regrowth/sprouting and/or preservation of serotonergic axons, and remyelination and/or myelin sparing. Moreover, degeneration/dying back of corticospinal cord axons was prevented more by the trimer expressing stem cells. These results encourage the view that stem cells engineered to drive the beneficial functions of L1 via homophilic and heterophilic interactions are functionally optimized and may thus be of therapeutic value. 相似文献
14.
The Human Kidney and Urine Proteome Project (HKUPP) was initiated in 2005 to promote proteomics research in the nephrology field, to better understand kidney functions as well as pathogenic mechanisms of kidney diseases, and to define novel biomarkers and therapeutic targets. This project was first approved in 2005 by the Human Proteome Organisation (HUPO) as a Kidney Disease Initiative under an umbrella of the HUPO Disease Biomarker Initiative (DBI), and more recently was approved as the HKUPP Initiative in 2007. Several sub-projects have been planned to achieve the ultimate goals. The most pressing is the establishment of "standard protocols and guidelines for urine proteome analysis". This sub-project had been extensively discussed during the first HKUPP symposium (during 6(th) HUPO Annual World Congress--October 2007, Seoul, Korea) and second workshop (during 40(th) American Society of Nephrology Renal Week--November 2007, San Francisco, CA, USA). Additional data and references have been collected after the symposium and workshop. An initial draft of standard protocols and guidelines for proteome analysis of non-proteinuric urine (urine protein excretion < or =150 mg/day) will soon be released as the first HKUPP product. 相似文献
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Correct phosphorylation site assignment is a critical aspect of phosphoproteomic analysis. Large-scale phosphopeptide data sets that are generated through liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis often contain hundreds or thousands of phosphorylation sites that require validation. To this end, we have created PhosphoScore, an open-source assignment program that is compatible with phosphopeptide data from multiple MS levels (MS(n)). The algorithm takes into account both the match quality and normalized intensity of observed spectral peaks compared to a theoretical spectrum. PhosphoScore produced >95% correct MS(2) assignments from known synthetic data, > 98% agreement with an established MS(2) assignment algorithm (Ascore), and >92% agreement with visual inspection of MS(3) and MS(4) spectra. 相似文献
17.
Arabidopsis (Arabidopsis thaliana) NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), a plasma membrane-localized protein, plays an essential role in resistance mediated by the coiled-coil-nucleotide-binding site-leucine-rich repeat class of resistance (R) proteins, which includes RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2), RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLA1, and RPS5. Infection with Pseudomonas syringae pv tomato DC3000 expressing the bacterial effector proteins AvrRpt2, AvrB, and AvrPphB activates resistance by the aforementioned R proteins. Whereas the genetic requirement for NDR1 in plant disease resistance signaling has been detailed, our study focuses on determining a global, physiological role for NDR1. Through the use of homology modeling and structure threading, NDR1 was predicted to have a high degree of structural similarity to Arabidopsis LATE EMBRYOGENESIS ABUNDANT14, a protein implicated in abiotic stress responses. Specific protein motifs also point to a degree of homology with mammalian integrins, well-characterized proteins involved in adhesion and signaling. This structural homology led us to examine a physiological role for NDR1 in preventing fluid loss and maintaining cell integrity through plasma membrane-cell wall adhesions. Our results show a substantial alteration in induced (i.e. pathogen-inoculated) electrolyte leakage and a compromised pathogen-associated molecular pattern-triggered immune response in ndr1-1 mutant plants. As an extension of these analyses, using a combination of genetic and cell biology-based approaches, we have identified a role for NDR1 in mediating plasma membrane-cell wall adhesions. Taken together, our data point to a broad role for NDR1 both in mediating primary cellular functions in Arabidopsis through maintaining the integrity of the cell wall-plasma membrane connection and as a key signaling component of these responses during pathogen infection. 相似文献
18.
Renal tubular actions of ANF. 总被引:1,自引:0,他引:1
M A Knepper S P Lankford Y Terada 《Canadian journal of physiology and pharmacology》1991,69(10):1537-1545
Many of the earliest investigations of the renal effects of atrial natriuretic factor (ANF) pointed to the glomerulus as a major site of the peptide's action. More recently, there have been many reports showing various effects of ANF on renal tubular epithelia, including collecting ducts, thick ascending limbs of Henle's loop, thin limbs of Henle's loops, and proximal tubules. The purpose of this review is to summarize the evidence for renal tubular actions of ANF and analyze it from the perspective of the specialized functions of the individual nephron segments, addressing the question: can renal tubule effects of ANF play a significant role in the precise day-to-day regulation of renal NaCl and water excretion? Based on these considerations, we propose that long-term renal tubular action of ANF may be distinct from its short-term natriuretic effect. The short-term action of ANF to accelerate salt and water excretion may play a role in the overall response to acute volume overload. This action of ANF appears to be largely due to an ANF-mediated increase in glomerular filtration rate accompanied by a blunting of the tubuloglomerular feedback mechanism, perhaps with some contribution from ANF-mediated inhibition of fluid absorption in the proximal tubule. In contrast, contributions of ANF to the precise day-to-day regulation of salt and water excretion are likely to be chiefly due to ANF-mediated inhibition of NaCl and water absorption in collecting ducts, but may also involve actions of ANF on the loop of Henle. 相似文献
19.
Mitogenic effects of the proto-oncogene and oncogene forms of c-H-ras DNA in human diploid fibroblasts. 总被引:6,自引:3,他引:3 下载免费PDF全文
C K Lumpkin J E Knepper J S Butel J R Smith O M Pereira-Smith 《Molecular and cellular biology》1986,6(8):2990-2993
Nuclear microinjection of c-H-ras DNA induced DNA synthesis in reversibly nonproliferating quiescent human cells. The proto-oncogene and oncogene forms were equally effective inducers. In contrast, c-H-ras DNA either alone or in combination with the adenovirus E1A gene did not cause terminally nondividing senescent cells to synthesize DNA. 相似文献
20.
Reduction in sample complexity enables more thorough proteomic analysis using mass spectrometry (MS). A solution-based two-dimensional (2D) protein fractionation system, ProteomeLab PF 2D, has recently become available for sample fractionation and complexity reduction. PF 2D resolves proteins by isoelectric point (pI) and hydrophobicity in the first and second dimensions, respectively. It offers distinctive advantages over 2D gel electrophoresis with respects to automation of the fractionation processes and characterization of proteins having extreme pIs. Besides fractionation, PF 2D is equipped with built-in UV detectors intended for relative quantification of proteins in contrasting samples using its software tools. In this study, we utilized PF 2D for the identification of basic and acidic proteins in mammalian cells, which are generally under-characterized. In addition, mass spectrometric methods (label-free and 18O-labeling) were employed to complement protein quantification based on UV absorbance. Our studies indicate that the selection of chromatographic fractions could impact protein identification and that the UV-based quantification for contrasting complex proteomes is constrained by coelution or partial coelution of proteins. In contrast, the quantification post PF 2D chromatography based on label-free or 18O-labeling mass spectrometry provides an alternative platform for basic/acidic protein identification and quantification. With the use of HCT116 colon carcinoma cells, a total of 305 basic and 183 acidic proteins was identified. Quantitative proteomics revealed that 17 of these proteins were differentially expressed in HCT116 p53-/- cells. 相似文献