Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias)

We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.     

Novel Function and Intracellular Localization of Methionine Adenosyltransferase 2β Splicing Variants

Human methionine adenosyltransferase 2β (MAT2β) encodes for two major splicing variants, V1 and V2, which are differentially expressed in normal tissues. Both variants are induced in human liver cancer and positively regulate growth. The aim of this work was to identify interacting proteins of V1 and V2. His-tagged V1 and V2 were overexpressed in Rosetta pLysS cells, purified, and used in a pulldown assay to identify interacting proteins from human colon cancer cell line RKO cell lysates. The eluted lysates were subjected to Western blot and in solution proteomic analyses. HuR, an mRNA-binding protein known to stabilize the mRNA of several cyclins, was identified to interact with V1 and V2. Immunoprecipitation and Western blotting confirmed their interaction in both liver and colon cancer cells. These variant proteins are located in both nucleus and cytoplasm in liver and colon cancer cells and, when overexpressed, increased the cytoplasmic HuR content. This led to increased expression of cyclin D1 and cyclin A, known targets of HuR. When endogenous expression of V1 or V2 is reduced by small interference RNA, cytoplasmic HuR content fell and the expression of these HuR target genes also decreased. Knockdown of cyclin D1 or cyclin A blunted, whereas knockdown of HuR largely prevented, the ability of V1 or V2 overexpression to induce growth. In conclusion, MAT2β variants reside mostly in the nucleus and regulate HuR subcellular content to affect cell proliferation.     

p38 MAP Kinase and MAPKAP Kinases MK2/3 Cooperatively Phosphorylate Epithelial Keratins

The MAPK-activated protein kinases (MAPKAP kinases) MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation, and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phosphoproteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. In addition to differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8). The phosphorylation of K8-Ser⁷³ is catalyzed directly by p38, which in turn shows MK2-dependent expression. Notably, analysis of small molecule p38 inhibitors on K8-Ser⁷³ phosphorylation also demonstrated reduced phosphorylations of keratins K18-Ser⁵² and K20-Ser¹³ but not of K8-Ser⁴³¹ or K18-Ser³³. Interestingly, K18-Ser⁵² and K20-Ser¹³ are not directly phosphorylated by p38 in vitro, but by MK2. Furthermore, anisomycin-stimulated phosphorylations of K20-Ser¹³ and K18-Ser⁵² are inhibited by small molecule inhibitors of both p38 and MK2. MK2 knockdown in HT29 cells leads to reduced K20-Ser¹³ phosphorylation, which further supports the notion that MK2 is responsible for K20 phosphorylation in vivo. Physiologic relevance of these findings was confirmed by differences of K20-Ser¹³ phosphorylation between the ileum of wild-type and MK2/3-deficient mice and by demonstrating p38- and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18, and K20 in intestinal epithelia.     

A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.     

Comprehensive Mapping of the Human Kinome to Epidermal Growth Factor Receptor Signaling

Disregulation of epidermal growth factor receptor (EGFR) signaling directly promotes bypass of proliferation and survival restraints in a high frequency of epithelia-derived cancer. As such, much effort is currently focused on decoding the molecular architecture supporting EGFR activation and function. Here, we have leveraged high throughput reverse phase protein lysate arrays, with a sensitive fluorescent nanocrystal-based phosphoprotein detection assay, together with large scale siRNA-mediated loss of function to execute a quantitative interrogation of all elements of the human kinome supporting EGF-dependent signaling. This screening platform has captured multiple novel contributions of diverse protein kinases to modulation of EGFR signal generation, signal amplitude, and signal duration. As examples, the prometastatic SNF1/AMPK-related kinase hormonally upregulated Neu kinase was found to support EGFR activation in response to ligand binding, whereas the enigmatic kinase MGC16169 selectively supports coupling of active EGFR to ERK1/2 regulation. Of note, the receptor tyrosine kinase MERTK and the pyrimidine kinase UCK1 were both found to be required for surface accumulation of EGFR and subsequent pathway activation in multiple cancer cell backgrounds and may represent new targets for therapeutic intervention.     

Autocrine effects of VEGF-D on endothelial cells after transduction with AD-VEGF-D

Endothelial cells in tumor vessels display unusual characteristics in terms of survival and angiogenic properties which result from the increased expression of VEGF-D and its autocrine effect. To evaluate mechanisms by which VEGF-D leads to such abnormal phenotype, we searched for proteins with modified expression in HUVECs enriched in the recombinant mature VEGF-D (VEGFD^ΔNΔC) delivered by adenovirus. Expression of membrane proteins in endothelial cells was characterized by FACS using anti-human IT-Box-135 antibodies. HUVECs transduced with Ad-VEGF-D^ΔNΔC revealed markedly increased expression of proteins involved in adhesion and migration such as (a) integrins (αVβ5, α2β1, α5β1, αMβ2, αLβ2), (b) matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), (c) components of fibrinolytic system (PAI-1, u-PAR), and (d) CD45, CD98, CD147. Interestingly, there also were numerous proteins with significantly reduced expression, particularly among surface exposed membrane proteins. Thus, it can be concluded that to induce proangiogenic phenotype and facilitate migration of HUVECs, VEGF-D^ΔNΔC not only upregulates expression of proteins known to participate in the cell-matrix interactions but also silences some membrane proteins which could interfere with this process.     

PTK6 Inhibits Down-regulation of EGF Receptor through Phosphorylation of ARAP1

PTK6 (also known as Brk) is a non-receptor-tyrosine kinase containing SH3, SH2, and catalytic domains, that is expressed in more than 60% of breast carcinomas but not in normal mammary tissues. To analyze PTK6-interacting proteins, we have expressed Flag-tagged PTK6 in HEK293 cells and performed co-immunoprecipitation assays with Flag antibody-conjugated agarose. A 164-kDa protein in the precipitated fraction was identified as ARAP1 (also known as centaurin δ-2) by MALDI-TOF mass analysis. ARAP1 associated with PTK6 in an EGF/EGF receptor (EGFR)-dependent manner. In addition, the SH2 domain of PTK6, particularly the Arg¹⁰⁵ residue that contacts the phosphate group of the tyrosine residue, was essential for the association. Moreover, PTK6 phosphorylated residue Tyr²³¹ in the N-terminal domain of ARAP1. Expression of ARAP1, but not of the Y231F mutant, inhibited the down-regulation of EGFR in HEK293 cells expressing PTK6. Silencing of endogenous PTK6 expression in breast carcinoma cells decreased EGFR levels. These results demonstrate that PTK6 enhances EGFR signaling by inhibition of EGFR down-regulation through phosphorylation of ARAP1 in breast cancer cells.     

Eye lens proteomics

Summary. The eye lens is a fascinating organ as it is in essence living transparent matter. Lenticular transparency is achieved through the peculiarities of lens morphology, a semi-apoptotic process where cells elongate and loose their organelles and the precise molecular arrangement of the bulk of soluble lenticular proteins, the crystallins. The 16 crystallins ubiquitous in mammals and their modifications have been extensively characterized by 2-DE, liquid chromatography, mass spectrometry and other protein analysis techniques. The various solubility dependant fractions as well as subproteomes of lenticular morphological sections have also been explored in detail. Extensive post translational modification of the crystallins is encountered throughout the lens as a result of ageing and disease resulting in a vast number of protein species. Proteomics methodology is therefore ideal to further comprehensive understanding of this organ and the factors involved in cataractogenesis.     

Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria’s proteome was achieved using Beckman Coulter’s Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.