Karim Rezaul

Annexin I is an endogenous ligand that mediates apoptotic cell engulfment

Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.

Quantitative Phosphoproteomic Analysis of T Cell Receptor Signaling Reveals System-Wide Modulation of Protein-Protein Interactions

Serine-threonine phosphorylation plays a role in regulating the interactions among proteins involved in T cell responses. Further Interactions The binding of antigen to the T cell receptor (TCR) complex triggers a cascade of responses that culminate in T cell activation. Key to the initial stages of this cascade is the phosphorylation of tyrosine residues in proteins proximal to the TCR, which enables the recruitment of other proteins that contain phosphotyrosine-binding domains. Given its importance to TCR signaling, tyrosine phosphorylation of target proteins has received considerable attention. To view protein phosphorylation from a larger perspective, Mayya et al. performed a system-level phosphoproteomics analysis of the events triggered by TCR activation in the human Jurkat T cell line. They found that the status of hundreds of phosphorylation sites was modulated in response to stimulation of the TCR. In addition to identifying previously unknown TCR-responsive phosphorylation events, this analysis also suggests a role for phosphorylated serine and threonine residues in modulating protein-protein interactions between many proteins involved in T cell responses. Protein phosphorylation events during T cell receptor (TCR) signaling control the formation of complexes among proteins proximal to the TCR, the activation of kinase cascades, and the activation of transcription factors; however, the mode and extent of the influence of phosphorylation in coordinating the diverse phenomena associated with T cell activation are unclear. Therefore, we used the human Jurkat T cell leukemia cell line as a model system and performed large-scale quantitative phosphoproteomic analyses of TCR signaling. We identified 10,665 unique phosphorylation sites, of which 696 showed TCR-responsive changes. In addition, we analyzed broad trends in phosphorylation data sets to uncover underlying mechanisms associated with T cell activation. We found that, upon stimulation of the TCR, phosphorylation events extensively targeted protein modules involved in all of the salient phenomena associated with T cell activation: patterning of surface proteins, endocytosis of the TCR, formation of the F-actin cup, inside-out activation of integrins, polarization of microtubules, production of cytokines, and alternative splicing of messenger RNA. Further, case-by-case analysis of TCR-responsive phosphorylation sites on proteins belonging to relevant functional modules together with network analysis allowed us to deduce that serine-threonine (S-T) phosphorylation modulated protein-protein interactions (PPIs) in a system-wide fashion. We also provide experimental support for this inference by showing that phosphorylation of tubulin on six distinct serine residues abrogated PPIs during the assembly of microtubules. We propose that modulation of PPIs by stimulus-dependent changes in S-T phosphorylation state is a widespread phenomenon applicable to many other signaling systems.

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P₁) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.

A Systematic Characterization of Mitochondrial Proteome from Human T Leukemia Cells

Global understanding of tissue-specific differences in mitochondrial signal transduction requires comprehensive mitochondrial protein identification from multiple cell and tissue types. Here, we explore the feasibility and efficiency of protein identification using the one-dimensional gel electrophoresis in combination with the nano liquid-chromatography tandem mass spectrometry (GeLC-MS/MS). The use of only 40 μg of purified mitochondrial proteins and data analysis using stringent scoring criteria and the molecular mass validation of the gel slices enables the identification of 227 known mitochondrial proteins (membrane and soluble) and 453 additional proteins likely to be associated with mitochondria. Replicate analyses of 60 μg of mitochondrial proteins on the faster scanning LTQ mass spectrometer validate all the previously identified proteins and most of the single hit proteins except the 81 single hit proteins. Among the identified proteins, 466 proteins are known to functionally participate in various processes such as respiration, tricarboxylic acid cycle (TCA cycle), amino acid and nucleotide metabolism, glycolysis, protection against oxidative stress, mitochondrial assembly, molecular transport, protein biosynthesis, cell cycle control, and many known cellular processes. The distribution of identified proteins in terms of size, pI, and hydrophobicity reveal that the present analytical strategy is largely unbiased and very efficient. Thus, we conclude that this approach is suitable for characterizing subcellular proteomes form multiple cells and tissues.

Systematic Characterization of Nuclear Proteome during Apoptosis A Quantitative Proteomic Study by Differential Extraction and Stable Isotope Labeling

Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis.

Systematic Comparison of a Two-dimensional Ion Trap and a Three-dimensional Ion Trap Mass Spectrometer in Proteomics

The utility and advantages of the recently introduced two-dimensional quadrupole ion trap mass spectrometer in proteomics over the traditional three-dimensional ion trap mass spectrometer have not been systematically characterized. Here we rigorously compared the performance of these two platforms by using over 100,000 tandem mass spectra acquired with identical complex peptide mixtures and acquisition parameters. Specifically we compared four factors that are critical for a successful proteomic study: 1) the number of proteins identified, 2) sequence coverage or the number of peptides identified for every protein, 3) the data base matching SEQUEST Xcorr and Sp score, and 4) the quality of the fragment ion series of peptides. We found a 4–6-fold increase in the number of peptides and proteins identified on the two-dimensional ion trap mass spectrometer as a direct result of improvement in all the other parameters examined. Interestingly more than 70% of the doubly and triply charged peptides, but not the singly charged peptides, showed better quality of fragmentation spectra on the two-dimensional ion trap. These results highlight specific advantages of the two-dimensional ion trap over the conventional three-dimensional ion traps for protein identification in proteomic experiments.

Global Survey of Human T Leukemic Cells by Integrating Proteomics and Transcriptomics Profiling

A global protein survey is needed to gain systems-level insights into mammalian cell signaling and information flow. Human Jurkat T leukemic cells are one of the most important model systems for T cell signaling study, but no comprehensive proteomics survey has been carried out in this cell type. In the present study we combined subcellular fractionation, multiple protein enrichment methods, and replicate tandem mass spectrometry analyses to determine the protein expression pattern in a single Jurkat cell type. The proteome dataset was evaluated by comparison with the genome-wide mRNA expression pattern in the same cell type. A total of 5381 proteins were identified by mass spectrometry with high confidence. Rigorous comparison of RNA and protein expression afforded removal of the false positive identifications and redundant entries but rescued the proteins identified by a single high scoring peptide, resulting in the final identification of 6471 unique gene products among which 98% of the corresponding transcripts were detected with high probability. Using hierarchical clustering of the protein expression patterns in five subcellular fractions (cytosol, light membrane, heavy membrane, mitochondria, and nuclei), the primary subcellular localization of 2241 proteins was assigned with high confidence including 792 previously uncharacterized proteins. This proteome landscape can serve as a useful platform for systems-level understanding of organelle composition and cellular functions in human T cells.

Sphingosine Interaction with Acidic Leucine-rich Nuclear Phosphoprotein-32A (ANP32A) Regulates PP2A Activity and Cyclooxygenase (COX)-2 Expression in Human Endothelial Cells

Sphingolipid metabolites regulate cell fate by acting on specific cellular targets. Although the influence of sphingolipids in cellular signaling has been well recognized, the exact molecular targets and how these targets influence cellular signaling mechanisms remain poorly understood. Toward this goal, we used affinity chromatography coupled with proteomics technology and identified acidic leucine-rich nuclear phosphoprotein-32A (ANP32A), an inhibitor of protein phosphatase 2A (PP2A) as a direct target of sphingosine, N,N′-dimethyl sphingosine (DMS) and phytosphingosine but not dihydrosphingosine or sphingosine 1-phosphate. Treatment of human umbilical vein endothelial cells (HUVEC) with DMS, which is not phosphorylated by sphingosine kinases, led to the activation of PP2A activity. Suppression of ANP32A with siRNA enhanced basal and DMS-activated PP2A activity suggesting that the sphingoid base binds to and relieves the inhibitory action of ANP32A on the PP2A complex. Indeed, DMS relieved the ANP32A-mediated inhibition of PP2A enzyme complex in vitro. Interestingly, DMS treatment induced the p38 stress-activated protein kinase (SAPK) and expression of cyclooxygenase (COX)-2 transcript and protein. Knockdown of ANP32A expression further induced p38 SAPK and COX-2. These data identify ANP32A as a novel molecular target of sphingoid bases that regulates cellular signaling events and inflammatory gene expression.

Differential Protein Expression Profiles in Estrogen Receptor–Positive and –Negative Breast Cancer Tissues Using Label-Free Quantitative Proteomics

Identification of the proteins that are associated with estrogen receptor (ER) status is a first step towards better understanding of the hormone-dependent nature of breast carcinogenesis. Although a number of gene expression analyses have been conducted, protein complement has not been systematically investigated to date. Because proteins are primary targets of therapeutic drugs, in this study, we have attempted to identify proteomic signatures that demarcate ER-positive and -negative breast cancers. Using highly enriched breast tumor cells, replicate analyses from 3 ERα+ and 3 ERα- human breast tumors resulted in the identification of 2,995 unique proteins with ≥2 peptides. Among these, a number of receptor tyrosine kinases and intracellular kinases that are abundantly expressed in ERα+ and ERα- breast cancer tissues were identified. Further, label-free quantitative proteome analysis revealed that 236 proteins were differentially expressed in ERα+ and ERα- breast tumors. Among these, 141 proteins were selectively up-regulated in ERα+, and 95 proteins were selectively up-regulated in ERα- breast tumors. Comparison of differentially expressed proteins with a breast cancer database revealed 98 among these have been previously reported to be involved in breast cancer. By Gene Ontology molecular function, dehydrogenase, reductase, cytoskeletal proteins, extracellular matrix, hydrolase, and lyase categories were significantly enriched in ERα+, whereas selected calcium-binding protein, membrane traffic protein, and cytoskeletal protein were enriched in ERα- breast tumors. Biological process and pathway analysis revealed that up-regulated proteins of ERα+ were overrepresented by proteins involved in amino acid metabolism, proteasome, and fatty acid metabolism, while up-regulated proteins of ERα- were overrepresented by proteins involved in glycolysis pathway. The presence and relative abundance of 4 selected differentially abundant proteins (liprin-α1, fascin, DAP5, and β-arrestin-1) were quantified and validated by immunohistochemistry. In conclusion, unlike in vitro cell culture models, the in vivo signaling proteins and pathways that we have identified directly from human breast cancer tissues may serve as relevant therapeutic targets for the pharmacological intervention of breast cancer.

Cyp1b1 Protein in the Mouse Eye during Development: An Immunohistochemical Study

We show, for the first time, the spatiotemporal appearance of Cyp1b1 protein during mouse eye ontogeny. The protein was unambiguously identified in the adult mouse eye and newborn (P0) whole mouse microsomes and was shown to be localized in inner ciliary epithelium, corneal epithelium, retinal inner nuclear cells, and ganglion cells. The enzyme protein was present in the lens epithelium adjacent to the developing ciliary body at 15.5 days postconception (E15.5) and was most strongly expressed during E17.5 to 7 days postnatally (P07). Subsequently, it declined to very low levels. The protein was also expressed in the corneal endothelial cells adjacent to the ciliary body at P07. Cyp1b1 was barely detectable in the inner ciliary epithelium before E17.5 but increased rapidly postnatally, reaching adult levels by P28. Levels of the enzyme protein in the corneal epithelium were seen from E15.5 onward, increasing sharply, and after a decrease at P07, were highest in the adult animal eye. The presence of Cyp1b1 protein in the inner nuclear layer of the retina was very low in the prenatal eye, increasing rapidly postnatally, and was highest in the adult animal eye. In the ganglion cell layer of the retina, it increased slowly from E15.5 to P07 and then rapidly reached adult levels. Interestingly, Cyp1b1 was not detected in the trabecular meshwork at any stage of development or in the adult eye. We conclude that the enzyme may play important roles in normal eye development and function in mice as in humans, and that the mouse may prove to be an excellent model for determination of the roles of CYP1B1 in human eye development and function.