This protocol describes activation, isolation, and analysis of the CD95 (APO-1/Fas) death-inducing signaling complex (DISC) using affinity purification. Activation is achieved using a biotin-labeled ...anti-CD95 antibody and the native DISC complex is captured using streptavidin beads. This approach minimizes both the number of steps involved and any potential nonspecific interactions or cross-reactivity of antibodies commonly seen in immunoprecipitations using unlabeled antibodies and protein A/G beads. Composition of the isolated complex is analyzed via western blot to identify known DISC components, and dimerization-induced autocatalytic processing of procaspase-8 at the DISC can be confirmed by detection of caspase-8 cleavage products. The potential for DISC-associated caspase-8 to activate the caspase cascade can be determined by measuring caspase-8-dependent cleavage of the fluorigenic substrate Ac-IETD.AFC, or by performing a bioassay using exogenous protein substrates.
Regulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their ...mode of action is still not fully understood.
Here, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4-Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5'UTR of target mRNAs directly upstream of the AUG start codon.
Our data support a model whereby purine motifs towards the 3' end of the 5'UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding.
The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies ...have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,1). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling 2. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker 3,4. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes.
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An initial stage of many neurodegenerative processes is associated with compromised synaptic function and precedes synapse loss, neurite fragmentation, and neuronal death. We showed previously that ...deficiency of heme, regulating many proteins of pharmacological importance, causes neurodegeneration of primary cortical neurons via N-methyl-d-aspartate receptor (NMDAR)-dependent suppression of the extracellular signal-regulated kinase 1/2 pathway. Here, we asked whether the reduction of heme causes synaptic perturbation before neurite fragmentation in neuronal cultures and investigated molecular mechanisms of synaptic dysfunction in these cells. We showed the change in the NR2B subunit phosphorylation that correlates with compromised NMDAR function after the reduction of regulatory heme and a rapid rescue of NR2B phosphorylation and NMDAR function by exogenous heme. Electrophysiological recordings demonstrated diminished NMDAR currents and NMDAR-mediated calcium influx after 24 h of inhibition of heme synthesis. These effects were reversed by treatment with heme; however, inhibition of the Src family kinases abolished the rescue effect of heme on NMDA-evoked currents. Diminished NMDAR current and Ca(2+) influx resulted in suppressed cGMP production and impairment of spine formation. Exogenous heme exerted rescue effects on NR2B tyrosine phosphorylation and NMDA-evoked currents within minutes, suggesting direct interactions within the NMDAR complex. These synaptic changes after inhibition of heme synthesis occurred at this stage without apparent dysfunction of major hemoproteins. We conclude that regulatory heme is necessary in maintaining NR2B phosphorylation and NMDAR function. NMDAR failure occurs before neurite fragmentation and may be a causal factor in neurodegeneration; this could suggest a route for an early pharmacological intervention.
Mantle cell lymphoma (MCL) remains incurable with current therapeutic approaches. Proteins expressed aberrantly at the MCL plasma membrane are candidate molecules for the development of targeted ...therapy. In the current study, we characterized globally the expression of proteins in the MCL plasma membrane and in purified lipid raft fractions in comparison with normal B cells using proteomic and bioinformatic analyses. Plasma membrane fractions were prepared from MCL patients in leukemic phase of disease and were then separated on 1D SDS-PAGE gels; sequential gel slices were digested with trypsin and the extracted peptides identified by LC/MS-MS. This approach overcomes the limitations of 2D gels for membrane proteins and also provides information on protein localization and post-translational modifications. Bioinformatics identified 111 intrinsic transmembrane proteins, from which we profiled selected proteins across primary MCL cases, MCL derived cell lines and normal B cells by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Several transmembrane proteins, including CD27/CD70 and CD31 (PECAM) were abnormally expressed when compared to normal B cells. CD70 was significantly up-regulated (> 10 fold) in MCL patients along with its cognate receptor, CD27 which was also up-strongly up-regulated (4–9 fold), suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and PKC-βII were also detected in plasma membranes, suggesting constitutive activation of these proteins in MCL. Since activated PKC-βII has been reported to be recruited to lipid rafts during BCR signaling and to control IêB kinase lipid raft recruitment and activation, we undertook shotgun proteomics and protein profiling of MCL lipid rafts, purified using sucrose gradient centrifugation. This analysis revealed an abnormal composition of lipid rafts in MCL. Raftlin a myristoylated lipid raft B-cell specific protein, required for the integrity of lipid rafts and BCR signal transduction, was markedly down-regulated in MCL, as was the lymphoid transmembrane adaptor protein, Cbp/PAG (PAG1), which controls lymphocyte activation. These data were confirmed by RT-PCR which showed significant down-regulation of both genes in MCL. In comparison, other constitutive lipid raft proteins, such as Csk, Blk, Fyn and Lyn kinases and flotillin 1 were expressed within lipid rafts at levels similar to normal B lymphocytes. However, PKC-bII was not localized within lipid rafts indicating aberrant signaling via this molecule in MCL. Conversely, 5-lipoxygenase (5-LO/ALOX5) a key enzyme in leukotriene biosynthesis, which is normally expressed in either the nucleus or cytoplasm, was unexpectedly associated with lipid rafts isolated from MCL cells and was up-regulated ~7-fold in MCL as compared to normal B cells. Comparable results were obtained in chronic lymphocytic leukemia (CLL). Aberrant expression of 5-lipoxygenase has been associated with increased proliferation and suppression of apoptosis in other malignancies. To assess the possible functional activity of this pathway in malignant B-cells, the effects of inhibitors of 5-LO activity (AA861) and FLAP (MK886) its activating enzyme, were assessed on MCL cell lines and primary CLL cells. Both inhibitors induced high levels of apoptosis in a time-dependent and concentration-dependent manner in MCL cell lines and CLL cells, indicating an important role for this enzyme and the leukotriene biosynthetic pathway in MCL and other B-cell malignancies. Thus, using shotgun proteomics and protein expression profiling we have identified a subset of transmembrane proteins with aberrant expression and aberrant subcellular localization in MCL plasma membranes that may contribute to the pathology of the disease and are potential therapeutic targets in treating MCL.
MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to ...that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce approximately 1.4-MDa and approximately 700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This approximately 700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable approximately 200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable approximately 200-kDa complex with active caspase-7.
Identification of the processing/activation of multiple interleukin-1β converting enzyme (ICE)-like proteases and their target substrates in the intact cell is critical to our understanding of the ...apoptotic process. In this study we demonstrate processing/activation of at least four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by processing of Ich-1, CPP32, and Mch3α to their catalytically active subunits, and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3α but not of ICE. Fluorescence-activated cell sorting was used to obtain pure populations of normal and apoptotic cells. In apoptotic cells, extensive cleavage of Ich-1, CPP32, and Mch3α was observed together with proteolysis of the ICE-like protease substrates, poly (ADP-ribose) polymerase (PARP), the 70-kD protein component of U1 small nuclear ribonucleoprotein (U1-70K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3α or the substrates was observed in normal cells. In cells exposed to an apoptotic stimulus, some processing of Ich-1 was detected in morphologically normal cells, suggesting that cleavage of Ich-1 may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited apoptosis and cleavage of Ich-1, CPP32, Mch3α, Mch2α, PARP, U1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3α, and Mch2α. Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3α, and Mch2α accompanies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.
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