Phospholipase A2 group XV activity during cardiopulmonary bypass surgery.

OBJECTIVES: All patients who undergo cardiopulmonary bypass (CPB) experience some degree of ischemia reperfusion injury (IRI). Severe IRI-induced acute kidney injury (AKI) occurs in approximately 1-2% of patients undergoing CPB. Previous studies using activity-based protein profiling of urine identified group XV phospholipase A2, PLA2G15/LPLA2, as potentially associated with patients who develop AKI post CPB. The present study examined urinary PLA2G15/LPLA2 activity during CPB and in the near postoperative period for associations with subsequent AKI development. DESIGN & METHODS: Samples were collected in a nested case controlled cohort of 21 patients per group who either did (AKI) or did not (non-AKI) develop AKI post-operatively. Serum and urine samples from each patient before, during and after CPB were assayed for PLA2G15/LPLA2 activity. RESULTS: Urine activity significantly increased during the intra operative period. In contrast the activities in paired sera were markedly decreased during CPB. There was no correlation between the serum and urine activity levels of patients. There were no significant differences in activity levels of PLA2G15/LPLA2 in the urine or sera from patients that did and did not develop AKI. CONCLUSIONS: The lack of correlation between serum and urine activity levels suggests that the rapid intraoperative increases in PLA2G15/LPLA2 activity may originate from the kidney and as such offer an intraoperative indicator of early renal response to CPB associated stressors.

Activity-based protein profiling guided identification of urine proteinase 3 activity in subclinical rejection after renal transplantation.

BACKGROUND: The pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection. METHODS: Serine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine. RESULTS: In-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group. CONCLUSIONS: There appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process.

Activity-based Protein Profiling Approaches for Transplantation.

Enzyme activity may be more pathophysiologically relevant than enzyme quantity and is regulated by changes in conformational status that are undetectable by traditional proteomic approaches. Further, enzyme activity may provide insights into rapid physiological responses to inflammation/injury that are not dependent on de novo protein transcription. Activity-based protein profiling (ABPP) is a chemical proteomic approach designed to characterize and identify active enzymes within complex biological samples. Activity probes have been developed to interrogate multiple enzyme families with broad applicability, including but not limited to serine hydrolases, cysteine proteases, matrix metalloproteases, nitrilases, caspases, and histone deacetylases. The goal of this overview is to describe the overall rationale, approach, methods, challenges, and potential applications of ABPP to transplantation research. To do so, we present a case example of urine serine hydrolase ABPP in kidney transplant rejection to illustrate the utility and workflow of this analytical approach. Ultimately, developing novel transplant therapeutics is critically dependent on understanding the pathophysiological processes that result in loss of transplant function. ABPP offers a new dimension for characterizing dynamic changes in clinical samples. The capacity to identify and measure relevant enzyme activities provides fresh opportunities for understanding these processes and may help identify markers of disease activity for the development of novel diagnostics and real-time monitoring of patients. Finally, these insights into enzyme activity may also help to identify new transplant therapeutics, such as enzyme-specific inhibitors.

Activity-Based Protein Profiling of Intraoperative Serine Hydrolase Activities during Cardiac Surgery.

The processes involved in the initiation of acute kidney injury (AKI) following cardiopulmonary bypass (CPB) are thought to occur during the intraoperative period. Such a rapid development might indicate that some of the inductive events are not dependent on de novo protein synthesis, raising the possibility that changes in activities of pre-existing enzymes could contribute to the development of AKI. Activity-based protein profiling (ABPP) was used to compare the serine hydrolase enzyme activities present in the urines of CPB patients who subsequently developed AKI versus those who did not (non-AKI) during the intra- and immediate postoperative periods. Sequential urines collected from a nested case-control cohort of AKI and non-AKI patients were reacted with a serine hydrolase activity probe, fluorophosphonate-TAMRA, and separated by SDS-PAGE. The patterns and levels of probe-labeled proteins in the two groups were initially comparable. However, within 1 h of CPB there were significant pattern changes in the AKI group. Affinity purification and mass spectrometry-based analysis of probe-labeled enzymes in AKI urines at 1 h CPB and arrival to the intensive care unit (ICU) identified 28 enzymes. Quantitative analysis of the activity of one of the identified enzymes, kallikrein-1, revealed some trends suggesting differences in the levels and temporal patterns of enzyme activity between a subset of patients who developed AKI and those who did not. A comparative analysis of affinity-purified probe reacted urinary proteins from these patient groups during the intraoperative period suggested the presence of both shared and unique enzyme patterns. These results indicate that there are intraoperative changes in the levels and types of serine hydrolase activities in patients who subsequently develop AKI. However, the role of these activity differences in the development of AKI remains to be determined.

Interferon γ induced compositional changes in human bone marrow derived mesenchymal stem/stromal cells.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) display a range of immunoregulatory properties which can be enhanced by the exposure to cytokines such interferon γ (IFN-γ). However the compositional changes associated with the 'licensing' of these cells have not been clearly defined. The present study was undertaken to provide a detailed comparative proteomic analysis of the compositional changes that occur in human bone marrow derived MSC following 20 h treatment with IFN-γ. METHODS: 2D LC MSMS analysis of control and IFN-γ treated cells from 5 different healthy donors provided confident identification of more than 8400 proteins. RESULTS: In total 210 proteins were shown to be significantly altered in their expression levels (≥|2SD|) following IFN-γ treatment. The changes for several of these proteins were confirmed by flow cytometry. STRING analysis determined that approximately 30% of the altered proteins physically interacted in described interferon mediated processes. Comparison of the list of proteins that were identified as changed in the proteomic analysis with data for the same proteins in the Interferome DB indicated that ~35% of these proteins have not been reported to be IFN-γ responsive in a range of cell types. CONCLUSIONS: This data provides an in depth analysis of the proteome of basal and IFN-γ treated human mesenchymal stem cells and it identifies a number of novel proteins that may contribute to the immunoregulatory capacity if IFN-γ licensed cells.

The integrin PSI domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin ß subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine ß3, and human ß1 and ß2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of ß3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant ß3 subunit. We further developed mouse anti-mouse ß3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbß3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbß3 endogenous PDI-like activity reduced αIIbß3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

A proteomic evaluation of urinary changes associated with cardiopulmonary bypass.

BACKGROUND: The urinary proteome of patients undergoing cardiopulmonary bypass (CPB) may provide important insights into systemic and renal changes associated with the procedure. Such information may ultimately provide a basis to differentiate changes or properties associated with the development of acute kidney injury. While mass spectrometry (MS) analysis offers the potential for in-depth compositional analysis it is often limited in coverage and relative quantitation capacity. The aim of this study was to develop a process flow for the preparation and comparison of the intraoperative urinary proteome. METHODS: Urines were collected from patients at the start of CPB and 1-h into CPB. Pooled samples (n = 5) from each time point were processed using a modified Filter Assisted Sample Preparation protocol. The resulting peptides were analyzed by 2D-LC-MS/MS and by 1D-LC-MS/MS SWATH (Sequential Window acquisition of All Theoretical fragment ion spectra). RESULTS: The 2D-LC-MS/MS analysis identified 1324 proteins in the two pools, of which 744 were quantifiable. The SWATH approach provided quantitation for 730 proteins, 552 of which overlapped with the common population from the 2D-IDA results. Intensity correlation filtering between the two methods gave 475 proteins for biological interpretation. Proteins displaying greater than threefold changes (>log2 1.59) at 1-hour CPB relative to the initiation of CPB (26 down-regulated and 22 up-regulated) were selected for further analysis. Up-regulated proteins were enriched in GO terms related to humoral immune response, predominantly innate immunity (C4b, lactotransferrin, protein S100-A8, cathelicidin, myeloperoxidase) and extracellular matrix reorganization (e.g. MMP-9). CONCLUSIONS: This study describes a scheme for processing urine from patients undergoing CPB for mass spectrometry-based analysis. The introduction of SWATH into the workflow offers a sample and instrument sparing approach to obtaining consistent in-depth sample analysis. The design of the methodology is such that it can be readily applied to large numbers of clinical samples with the potential for automation. The results also suggest that activation of the innate immune responses occur during cardiac bypass surgery.

3D HPLC-MS with Reversed-Phase Separation Functionality in All Three Dimensions for Large-Scale Bottom-Up Proteomics and Peptide Retention Data Collection.

The growing complexity of proteomics samples and the desire for deeper analysis drive the development of both better MS instrument and advanced multidimensional separation schemes. We applied 1D, 2D, and 3D LC-MS/MS separation protocols (all of reversed-phase C18 functionality) to a tryptic digest of whole Jurkat cell lysate to estimate the depth of proteome coverage and to collect high-quality peptide retention information. We varied pH of the eluent and hydrophobicity of ion-pairing modifier to achieve good separation orthogonality (utilization of MS instrument time). All separation modes employed identical LC settings with formic-acid-based eluents in the last dimension. The 2D protocol used a high pH-low pH scheme with 21 concatenated fractions. In the 3D protocol, six concatenated fractions from the first dimension (C18, heptafluorobutyric acid) were analyzed using the identical 2D LC-MS procedure. This approach permitted a detailed evaluation of the analysis output consuming 21× and 126× the analysis time and sample load compared to 1D. Acquisition over 189 h of instrument time in 3D mode resulted in the identification of ∼14 000 proteins and ∼250 000 unique peptides. We estimated the dynamic range via peak intensity at the MS(2) level as approximately 10(4.2), 10(5.6), and 10(6.2) for the 1D, 2D, and 3D protocols, respectively. The uniform distribution of the number of acquired MS/MS, protein, and peptide identifications across all 126 fractions and through the chromatographic time scale in the last LC-MS stage indicates good separation orthogonality. The protocol is scalable and is amenable to the use of peptide retention prediction in all dimensions. All these features make it a very good candidate for large-scale bottom-up proteomic runs, which target both protein identification as well as the collection of peptide retention data sets for targeted quantitative applications.

Phosphatidylinositol-3,4-Bisphosphate and Its Binding Protein Lamellipodin Regulate Chemotaxis of Malignant B Lymphocytes.

Cell migration is controlled by PI3Ks, which generate lipid messengers phosphatidylinositol-3,4,5-trisphosphate and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and consequently recruit pleckstrin homology (PH) domain-containing signaling proteins. PI3K inhibition impairs migration of normal and transformed B cells, an effect thought to partly underlie the therapeutic efficacy of PI3K inhibitors in treatment of B cell malignancies such as chronic lymphocytic leukemia. Although a number of studies have implicated phosphatidylinositol-3,4,5-trisphosphate in cell migration, it remains unknown whether PI(3,4)P2 plays a distinct role. Using the PI(3,4)P2-specific phosphatase inositol polyphosphate 4-phosphatase, we investigate the impact of depleting PI(3,4)P2 on migration behavior of malignant B cells. We find that cells expressing wild-type, but not phosphatase dead, inositol polyphosphate 4-phosphatase show impaired SDF-induced PI(3,4)P2 responses and reduced migration in Transwell chamber assays. Moreover, PI(3,4)P2 depletion in primary chronic lymphocytic leukemia cells significantly impaired their migration capacity. PI(3,4)P2 depletion reduced both overall motility and migration directionality in the presence of a stable chemokine gradient. Within chemotaxing B cells, the PI(3,4)P2-binding cytoskeletal regulator lamellipodin (Lpd) was found to colocalize with PI(3,4)P2 on the plasma membrane via its PH domain. Overexpression and knockdown studies indicated that Lpd levels significantly impact migration capacity. Moreover, the ability of Lpd to promote directional migration of B cells in an SDF-1 gradient was dependent on its PI(3,4)P2-binding PH domain. These results demonstrate that PI(3,4)P2 plays a significant role in cell migration via binding to specific cytoskeletal regulators such as Lpd, and they suggest that impairment of PI(3,4)P2-dependent processes may contribute to the therapeutic efficacy of PI3K inhibitors in B cell malignancies.

Development of an SRM method for absolute quantitation of MYDGF/C19orf10 protein.

PURPOSE: To develop a MS-based selected reaction monitoring (SRM) assay for quantitation of myeloid-derived growth factor (MYDGF) formerly chromosome 19 open reading frame (C19orf10). EXPERIMENTAL DESIGN: Candidate reporter peptides were identified in digests of recombinant MYDGF. Isotopically labeled forms of these reporter peptides were employed as internal standards for assay development. Two reference peptides were selected SYLYFQTFFK and GAEIEYAMAYSK with respective LOQ of 42 and 380 attomole per injection. RESULTS: Application of the assay to human serum and synovial fluid determined that the assay sensitivity was reduced and quantitation was not achievable. However, the partial depletion of albumin and immunoglobulin from synovial fluids provided estimates of 300-650 femtomoles per injection (0.7-1.6 nanomolar (nM) fluid concentrations) in three of the six samples analyzed. CONCLUSIONS AND CLINICAL RELEVANCE: A validated sensitive assay for the quantitation of MYDGF in biological fluids was developed. However, the endogenous levels of MYDGF in such fluids are at or below the current levels of quantitation. The levels of MYDGF are lower than those previously reported using an ELISA. The current results suggest that additional steps may be required to remove high abundance proteins or to enrich MYDGF for SRM-based quantitation.

Identification of broadly conserved cross-species protective Leishmania antigen and its responding CD4+ T cells.

There is currently no clinically effective vaccine against leishmaniasis because of poor understanding of the antigens that elicit dominant T cell immunity. Using proteomics and cellular immunology, we identified a dominant naturally processed peptide (PEPCK335-351) derived from Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK). PEPCK was conserved in all pathogenic Leishmania, expressed in glycosomes of promastigotes and amastigotes, and elicited strong CD4(+) T cell responses in infected mice and humans. I-A(b)-PEPCK335-351 tetramer identified protective Leishmania-specific CD4(+) T cells at a clonal level, which comprised ~20% of all Leishmania-reactive CD4(+) T cells at the peak of infection. PEPCK335-351-specific CD4(+) T cells were oligoclonal in their T cell receptor usage, produced polyfunctional cytokines (interleukin-2, interferon-γ, and tumor necrosis factor), and underwent expansion, effector activities, contraction, and stable maintenance after lesion resolution. Vaccination with PEPCK peptide, DNA expressing full-length PEPCK, or rPEPCK induced strong durable cross-species protection in both resistant and susceptible mice. The effectiveness and durability of protection in vaccinated mice support the development of a broadly cross-species protective vaccine against different forms of leishmaniasis by targeting PEPCK.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy.

INTRODUCTION: Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. METHODS: We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. RESULTS: Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. CONCLUSIONS: We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.

N-capping motifs promote interaction of amphipathic helical peptides with hydrophobic surfaces and drastically alter hydrophobicity values of individual amino acids.

Capping rules, which govern interactions of helical peptides with hydrophobic surfaces, were never established before due to lack of methods for the direct measurement of polypeptide structure on the interphase boundary. We employed proteomic techniques and peptide retention modeling in reversed-phase chromatography to generate a data set sufficient for amino acid population analysis at helix ends. We found that interactions of amphipathic helical peptides with a hydrophobic C18 phase are induced by a unique motif featuring hydrophobic residues in the N1 and N2 positions adjacent to the N-cap (Asn, Asp, Ser, Thr, Gly), followed by Glu, Gln, or Asp in position N3 to complete a capping box. A favorable N-capping arrangement prior to amphipathic helix may result in the highest hydrophobicity (retention on C18 columns) of Asp/Asn (or Glu/Gln) peptide analogues among all naturally occurring amino acids when placed in N-cap or N3 position, respectively. These results contradict all previously reported hydrophobicity scales and provide new insights into our understanding of the phenomenon of hydrophobic interactions.

Quantification of the host response proteome after mammalian reovirus T1L infection.

All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular "transcriptome." We used SILAC (stable isotope labeling by amino acids in cell culture), combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L). 3,076 host proteins were detected at 6 hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24 hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.

Information-dependent LC-MS/MS acquisition with exclusion lists potentially generated on-the-fly: case study using a whole cell digest of Clostridium thermocellum.

We have developed a real-time graphic-processor-unit-based search engine capable of high-quality peptide identifications in <500 µs per spectrum. The steps of peptide/protein identification, in-silico prediction of all possible tryptic peptides from these proteins, and the prediction of their expected retention times and m/z values take less than 5 s per cycle over ∼3000 MS/MS spectra. This lays the foundation for information-dependent acquisition with exclusion lists generated on-the-fly, as the instrument continues to acquire data. While a complete evaluation of the dynamic exclusion system requires the participation from instrument vendors, we conducted a series of model experiments using a whole cell tryptic digestion of the bacterium Clostridium thermocellum. We ran a series of five iterative LC-MS/MS runs, adding a new exclusion list at each of four chromatographic "tripping points" - the elution times of the four standard peptides spiked into the sample. Retention times of these standard peptides were also used for real-time "chromatographic calibration." The dynamic exclusion approach gave a ≈ 5% increase in confident protein identification (for typical 2 h LC-MS/MS run), and reduced the average number of identified peptides per protein from 4.7 to 2.9. Its application to a two-times shorter gradient gave a ≈ 17% increase in proteins identified. Further improvements are possible for instruments with better mass accuracy, by employing a more accurate retention prediction algorithm and by developing better understanding of the possible chemical modifications and fragmentations produced during electrospray ionization.

Glia maturation factor gamma regulates the migration and adherence of human T lymphocytes.

BACKGROUND: Lymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG) as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration. RESULTS: Microscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of ß1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the ß2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1. CONCLUSIONS: The present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated adhesion.

Functional interaction of CD154 protein with α5ß1 integrin is totally independent from its binding to αIIbß3 integrin and CD40 molecules.

In addition to its classical CD40 receptor, CD154 also binds to αIIbß3, α5ß1, and αMß2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbß3, and α5ß1 receptors. We found that the binding affinity of CD154 for αIIbß3 is ∼4-fold higher than for α5ß1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbß3 and show that CD154 residues involved in its binding to CD40 or αIIbß3 are distinct from those implicated in its interaction to α5ß1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5ß1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5ß1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.

Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3.

Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD(1408)S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.

IQGAP1 involvement in MTOC and granule polarization in NK-cell cytotoxicity.

Natural killer (NK) cells form a region of tight contact called the NK immunological synapse (NKIS) with their target cells. This is a dynamic region serving as a platform for targeted signaling and exocytotic events. We previously identified IQGAP1 as a cytoskeletal component of the NK-like cell line YTS. The present study was undertaken to determine the role of IQGAP1 in the function of NK cells. Silencing of IQGAP1 expression resulted in almost complete loss of the cytotoxic activity of YTS cells. Loss of IQGAP1 did not prevent conjugate formation with target cells but it did result in a failure to reorient the microtubule organizing centre to the immune synapse. Significantly, IQGAP1 expression was required for the perigranular accumulation of an F-actin network. IQGAP1 was shown to undergo marked rearrangements during synapse maturation in effector target conjugates of YTS or primary NK cells. These results suggest previously undescribed role(s) for IQGAP1 in regulating multiple aspects of cytoskeletal organization and granule polarization in NK cells.

Requirement of podocalyxin in TGF-beta induced epithelial mesenchymal transition.

Epithelial mesenchymal transition (EMT) is characterized by the development of mesenchymal properties such as a fibroblast-like morphology with altered cytoskeletal organization and enhanced migratory potential. We report that the expression of podocalyxin (PODXL), a member of the CD34 family, is markedly increased during TGF-ß induced EMT. PODXL is enriched on the leading edges of migrating A549 cells. Silencing of podocalyxin expression reduced cell ruffle formation, spreading, migration and affected the expression patterns of several proteins that normally change during EMT (e.g., vimentin, E-cadherin). Cytoskeleton assembly in EMT was also found to be dependent on the production of podocalyin. Compositional analysis of podocalyxin containing immunoprecipitates revealed that collagen type 1 was consistently associated with these isolates. Collagen type 1 was also found to co-localize with podocalyxin on the leading edges of migrating cells. The interactions with collagen may be a critical aspect of podocalyxin function. Podocalyxin is an important regulator of the EMT like process as it regulates the loss of epithelial features and the acquisition of a motile phenotype.