Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death.

The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex regulating key pathways in cell growth. Hyperactivation of mTORC1 is implicated in numerous cancers, thus making it a potential broad-spectrum chemotherapeutic target. Here, we characterized how mTORC1 responds to cell death induced by various anticancer drugs such rapamycin, etoposide, cisplatin, curcumin, staurosporine and Fas ligand. All treatments induced cleavage in the mTORC1 component, raptor, resulting in decreased raptor-mTOR interaction and subsequent inhibition of the mTORC1-mediated phosphorylation of downstream substrates (S6K and 4E-BP1). The cleavage was primarily mediated by caspase-6 and occurred at two sites. Mutagenesis at one of these sites, conferred resistance to cell death, indicating that raptor cleavage is important in chemotherapeutic apoptosis.

The death domain-containing protein Unc5CL is a novel MyD88-independent activator of the pro-inflammatory IRAK signaling cascade.

The family of death domain (DD)-containing proteins are involved in many cellular processes, including apoptosis, inflammation and development. One of these molecules, the adapter protein MyD88, is a key factor in innate and adaptive immunity that integrates signals from the Toll-like receptor/interleukin (IL)-1 receptor (TLR/IL-1R) superfamily by providing an activation platform for IL-1R-associated kinases (IRAKs). Here we show that the DD-containing protein Unc5CL (also known as ZUD) is involved in a novel MyD88-independent mode of IRAK signaling that culminates in the activation of the transcription factor nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase. Unc5CL required IRAK1, IRAK4 and TNF receptor-associated factor 6 but not MyD88 for its ability to activate these pathways. Interestingly, the protein is constitutively autoproteolytically processed, and is anchored by its N-terminus specifically to the apical face of mucosal epithelial cells. Transcriptional profiling identified mainly chemokines, including IL-8, CXCL1 and CCL20 as Unc5CL target genes. Its prominent expression in mucosal tissues, as well as its ability to induce a pro-inflammatory program in cells, suggests that Unc5CL is a factor in epithelial inflammation and immunity as well as a candidate gene involved in mucosal diseases such as inflammatory bowel disease.

PIDD orchestrates translesion DNA synthesis in response to UV irradiation.

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by γ-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polη). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polη in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-κB activation and cell death.

Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90.

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.

Metabolic labeling and protein linearization technology allow the study of proteins secreted by cultured cells in serum-containing media.

Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)-based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples.

Short- and long-term changes in proteome composition and kinetic properties in a culture of Escherichia coli during transition from glucose-excess to glucose-limited growth conditions in continuous culture and vice versa.

To investigate the ability of Escherichia coli K12 MG1655 to cope with excess and limitation of a carbon and energy source, we studied the changes in kinetic properties and two-dimensional (2D) gel protein patterns of an E. coli culture. The population was transferred from glucose-excess batch to glucose-limited continuous culture (D = 0.3 h(-1)), in which it was cultivated for 500 h (217 generations) and then transferred back to glucose-excess batch culture. Two different stages to glucose-limitation were recognized: a short-term physiological adaptation characterized by a general effort in enhancing the cell's substrate scavenging ability and mutations resulting in a population exhibiting increased glucose affinity. Physiological short-term adaptation to glucose-limitation was achieved by upregulation of 12 proteins, namely MglB, MalE, ArgT, DppA, RbsB, YdcS, LivJ (precursor), UgpB (precursor), AceA, AldA, AtpA and GatY. Eight of these proteins are periplasmic binding proteins of ABC transporters. Most of them are not involved in glucose transport regulons, but rather in chemotaxis and transport of other substrates, whereas MalE and MglB have previously been shown to belong to transport systems important in glucose transport under glucose-limited conditions. Evolution under low glucose concentration led to an up to 10-fold increase in glucose affinity (from a K(s) of 366 +/- 36 microg l(-1) at the beginning to 44 +/- 7 microg l(-1)). The protein pattern of a "500-h-old" continuous culture showed a highly increased expression of MglB and MalE as well as of the regulator protein MalI. When adapted cells taken from the "500-h-old" continuous culture were transferred to batch culture, an increased expression of MalE was observed, compared with cells from un-adapted batch-grown cells. Otherwise, no significant changes were observed in the protein pattern of batch-grown populations before and after 500 h of evolution in the glucose-limited continuous culture.

Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety.

We describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one- and two-dimensional polyacrylamide gel electrophoresis. The approach is based on the use of an isotopically labeled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. The method allows quantitation of the changes occurring in spots or bands that contain more than one protein and has a greater dynamic range than most staining methods. Since the reagent carries a fixed positive charge under acidic conditions and labels only the N-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. The sequences can easily be extracted for homology searches instead of using indirect mass spectral-based searches and are independent of posttranslational modifications.

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites.

Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of (32)P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904. (Blood. 2000;96:1550-1557)

Breakdown of cytoskeletal proteins during meiosis of starfish oocytes and proteolysis induced by calpain.

Meiosis reinitiation in starfish oocytes is characterized by Ca(2+) transients in the cytosol and in the nucleus and is accompanied by the disassembly of the nuclear envelope, a process which is likely to be mediated by the cleavage of selected proteins. We have used mass spectrometry analysis (mass profile fingerprinting) on 2D polyacrylamide gels of extracts of oocytes in which meiosis resumption was induced by 1-methyladenine and have identified five proteins that were specifically degraded: alpha-tubulin, lamin B, dynamin, and two kinds of actin. They are all components of the cytoskeleton or associated with it. We then investigated whether calpain, which is activated by the increase in cell Ca(2+), could cleave the same proteins that became degraded under the influence of 1-methyladenine and thus be involved in nuclear membrane breakdown. The investigation was prompted by the finding that microinjection of calpain into the nuclei of prophase arrested oocytes induced meiosis in the absence of 1-methyladenine. Incubation of prophase arrested (disrupted) oocytes with calpain produced a 2D gel protein pattern in which some of the degradation products coincided with those seen in oocytes challenged with 1-methyladenine.

Phosphopeptide analysis.

In this chapter we review the various methods available to the experimenter to analyse phosphorylated peptides. The initial steps in such an analysis involve the isolation of the phosphopeptides for analysis, and we outline the various current methods such as immobilised metal affinity chromatography, anti-phosphoamino acid antibodies as well as HPLC (High Pressure Liquid Chromatography) and TLC (Thin Layer Chromatography). The isolated peptides can be analysed by chemical modification followed by Edman degradation or by mass spectrometry (MS). We focus on MS methods and give examples illustrating the selective detection and sequencing of phosphopeptides.

A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification.

We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage. The mass differences between a series of N-terminally degraded peptides give short sequences of defined length. Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database. The sequence ladders are generated without the use of chain terminators or sample aliquoting and the degradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing. The entire procedure can be automated, and we describe a prototype device for the parallel analysis of multiple samples. We demonstrate the effectiveness of this chemical tagging method in a comparison with Edman sequencing, peptide mass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which consists of 57 proteins. We show that chemical tagging is a viable first-pass high-throughput identification method to be used prior to an in depth MS/MS analysis.

Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation-induced response in Pseudomonas aeruginosa PAO1.

A set of proteins induced in Pseudomonas aeruginosa PAO1 during growth in the absence of sulfate was characterized by differential two-dimensional electrophoresis and MS. Thirteen proteins were found to be induced de novo or upregulated in P. aeruginosa grown in a succinate/salts medium with sodium cyclohexylsulfamate as the sole sulfur source. Protein spots excised from the two-dimensional gels were analysed by N-terminal Edman sequencing and MS sequencing (MS/MS) of internal protein fragments. The coding sequences for 11 of these proteins were unambiguously identified in the P. aeruginosa genome sequence. Expression of these genes was investigated by reverse transcription-PCR, which confirmed that repression in the presence of sulfate was acting at a transcriptional level. Three classes of sulfur-regulated proteins were found. The first class (five proteins) were high-affinity periplasmic solute-binding proteins with apparent specificity for sulfate and sulfonates. A second class included enzymes involved in sulfonate and sulfate ester metabolism (three proteins). The remaining three proteins appeared to be part of a more general stress response, and included two antioxidant proteins and a putative lipoprotein. This study demonstrates the power of the proteomics approach for direct correlation of the responses of an organism to an environmental stimulus with the genetic structures responsible for that response, and the application of reverse transcription-PCR significantly increases the conclusions that can be drawn from the proteomic study.

Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.

Proteomics and automation.

Proteome analysis is concerned with the global changes in protein expression as visualized most commonly by two-dimensional gel electrophoresis and analyzed by mass spectrometry. A drastic increase in the rapidity and reproducibility of protein isolation and identification is needed for proteome analysis to become a useful complement to global mRNA analysis. Simplification and standardization, based on innovation in both hard- and software, are prerequisites to the creation of automated proteomics platforms that are both robust and user-friendly, and will allow many more laboratories access to this technique. In this review we highlight the weak points in the chain of analysis (such as sample handling, protein separation and digestion) and summarize recent trends toward automation in instrumentation and software and offer our own personal view of future developments in the field.

Molecular features underlying the sequential phosphorylation of HS1 protein and its association with c-Fgr protein-tyrosine kinase.

The hematopoietic lineage cell-specific protein HS1 was shown to undergo a process of sequential phosphorylation both in vitro and in vivo, which is synergistically mediated by Syk and Src family protein-tyrosine kinases and essential for B cell antigen receptor-mediated apoptosis. We have now identified tyrosine 222 as the HS1 residue phosphorylated by the Src family protein kinases c-Fgr and Lyn, and we show that a truncated form of HS1 (HS1-208-401) lacking the N-terminal putative DNA binding region and the C-terminal Src homology 3 (SH3) domain is still able to undergo all the steps of sequential phosphorylation as efficiently as full-length HS1. We also show that a stable association of phospho-HS1 with c-Fgr through its SH2 domain requires previous autophosphorylation of the kinase and is prevented by subsequent phosphorylation of Tyr-222. Kinetic studies with HS1 and its truncated forms previously phosphorylated by Syk and with a peptide substrate reproducing the sequence around tyrosine 222 support the view that efficient phosphorylation of HS1 by Src family protein kinases entirely relies on TyrP-SH2 domain interaction with negligible, if any, contribution of local specificity determinants. Our data indicate that the proline-rich region of HS1 bordered by tyrosyl residues affected by Syk and Src family kinases represents a functional domain designed to undergo a process of sequential phosphorylation.

Phosphorylation of calmodulin alters its potency as an activator of target enzymes.

Previous work has shown that calmodulin (CaM) is constitutively phosphorylated in rat liver, probably by casein kinase II [Quadroni, M., James, P., and Carafoli, E. (1994) J. Biol. Chem. 269, 16116-16122]. A procedure is now described for the isolation of the phosphorylated forms of calmodulin (PCaM) free from CaM, since in vitro phosphorylation experiments yield a 50:50 mixture of 3-4 times phosphorylated CaM and native CaM. The activation of six target enzymes by PCaM was tested: myosin light chain kinase, 3',5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, Ca2+-CaM-dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase, and CaM-kinase II. In general, the phosphorylation of CaM caused a decrease in enzyme binding affinity, increasing the Kact by 2-4-fold for MLCK, PDE, PM Ca2+-ATPase, and calcineurin. The Vmax at saturating concentrations of PCaM was less affected, with the exception of CaM-kinase II, which was only minimally activated by PCaM and NOS whose Vmax was increased 2.6 times by PCaM with respect to CaM. Phosphorylation of calmodulin had very little effect on the binding of calcium to the enzyme despite the fact that Ser 101 which is phosphorylated is located in the third calcium binding loop. CD measurements performed on CaM and PCaM indicated that phosphorylation causes a marked decrease in the alpha-helical content of the protein. Phosphorylated CaM is very prone to dephosphorylation and was thus tested as a substrate for several phosphatases. It was unaffected by calcineurin (PP2B), but was a reasonable substrate for the pleiotropic phosphatases PP1gamma and PP2A.

Concentration of, and SDS removal from proteins isolated from multiple two-dimensional electrophoresis gels.

We have developed a gel electrophoresis system that can concentrate proteins from spots cut out of up to 50 two-dimensional electrophoresis gels. During protein concentration, SDS is substituted with a non-ionic detergent (octyl beta-glucopyranoside) which allows digestion and MS analysis of the protein directly extracted from the gel without fixation or staining. The system avoids the problems associated with the digestion of dilute protein in multiple bands by (a) greatly reducing the gel volume for digestion and thus the amount of protease required, hence lowering contamination by autodigestion products, (b) reducing the volume of solvent required for extraction of protein from the gel, thus minimising loss of material to container surfaces, and (c) removing SDS which interferes with subsequent MS or HPLC analysis. The efficiency of protein recovery ranges between an average of 80% for proteins from silver stained two-dimensional gels to 90% for fluorescence and Coomassie-blue-stained gels. The method is compatible with MS analysis of very low amounts of protein from any staining system, but appears not to be useful for Edman sequencing of silver-stained or fluorescent-stained proteins since the amount of N-terminal blockage appears to increase as the amount of protein isolated from the two-dimensional gel decreases.

Probing protein function using a combination of gene knockout and proteome analysis by mass spectrometry.

Recently the determination of the genome sequences of three procaryotes (Haemophilus influenzae, Methanococcus jannaschii and Mycoplasma genitalium) as well as the first eucaryotic genome (Saccharomyces cerevisiae) were completed. Between 40-60% of the genes were found to code for proteins to which no function could be assigned. We describe an approach which combines proteome analysis (mapping of expressed proteins isolated by two-dimensional polyacrylamide gel electrophoresis to the genome) with genetic manipulations to study the complex pattern of protein regulation occurring in Escherichia coli in response to sulfate starvation. We have previously described the upregulation of eight spots on two-dimensional (2-D) gels in response to sulfate starvation and the assignment of six of these to entries in the E. coli genome sequence (Quadroni et al., Eur. J. Biochem. 1996, 239, 773-781). Here we describe the identification of the remaining two proteins which are encoded in a sulfate-controlled operon in the 21.5' region of the E. coli genome. Upregulated protein spots were cut from multiple 2-D gels collected and run on a modified funnel gel to concentrate the proteins and remove the sodium dodecyl sulfate before digestion. The peptide masses obtained from the digests were used to search the SwissProt database or a six-frame translation of the EMBL DNA database using a peptide mass fingerprinting algorithm. A digest can be reanalyzed after deuterium exchange to obtain a second, orthogonal data set to increase the confidence level of protein identification. The digests of the remaining unidentified proteins were used for peptide fragment generation using either post-source decay in a matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometer or collision-induced dissociation (CID) coupled mass spectrometry (MS/MS) with triple stage quadrupole or ion trap mass spectrometers. The spectra were used as peptide fragment fingerprints to search the SwissProt and EMBL databases.

Analysis of global responses by protein and peptide fingerprinting of proteins isolated by two-dimensional gel electrophoresis. Application to the sulfate-starvation response of Escherichia coli.

A set of 8 proteins (SSI, sulfate-starvation-induced proteins) was observed by comparative two-dimensional electrophoresis to be induced when Escherichia coli were grown using compounds other than sulfate or cysteine as the sole sulfur source. These proteins were isolated after two-dimensional gel electrophoresis, digested with trypsin and the masses of the resulting peptides determined by mass spectrometry. The list of peptide masses served as a protein fingerprint which was used to search the databases, allowing four of the SSI proteins (SSI2, 5, 7, 8) to be identified with a high degree of confidence. To identify the other SSI proteins, and to obtain sequence information for as many of the proteins as possible, automated on-line HPLC MS/MS (fragmentation analysis using coupled mass scanning devices) data collection was performed. The uninterpreted MS/MS spectra were used as peptide fingerprints to search the databases. Genes encoding two further proteins (SSI 1 and 3) were identified in the 8.5' region of the Escherichia coli genome. N-terminal sequencing of all of the proteins confirmed the results of protein and peptide fingerprinting and in addition showed that SSI 6 shows 50% similarity to the Bacillus subtilis orfM gene product. SSI 4 was not found in the databases by any of these methods. The methods described are of general use for the rapid analysis of complex cell responses. MS data accumulation takes about 5 min/protein for protein fingerprinting and 30 min for peptide fingerprinting and requires approximately 100 fmol of material. N-terminal sequencing however, takes about 5 h/protein and approximately 1 pmol to obtain a 10 amino acid sequence for a search.