Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC-MS.

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20-30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.

Substrate determinants in the C99 juxtamembrane domains differentially affect γ-secretase cleavage specificity and modulator pharmacology.

The molecular mechanisms governing γ-secretase cleavage specificity are not fully understood. Herein, we demonstrate that extending the transmembrane domain of the amyloid precursor protein-derived C99 substrate in proximity to the cytosolic face strongly influences γ-secretase cleavage specificity. Sequential insertion of leucines or replacement of membrane-anchoring lysines by leucines elevated the production of Aß42, whilst lowering production of Aß40. A single insertion or replacement was sufficient to produce this phenotype, suggesting that the helical length distal to the ε-site is a critical determinant of γ-secretase cleavage specificity. Replacing the lysine at the luminal membrane border (K28) with glutamic acid (K28E) increased Aß37 and reduced Aß42 production. Maintaining a positive charge with an arginine replacement, however, did not alter cleavage specificity. Using two potent and structurally distinct γ-secretase modulators (GSMs), we elucidated the contribution of K28 to the modulatory mechanism. Surprisingly, whilst lowering the potency of the non-steroidal anti-inflammatory drug-type GSM, the K28E mutation converted a heteroaryl-type GSM to an inverse GSM. This result implies the proximal lysine is critical for the GSM mechanism and pharmacology. This region is likely a major determinant for substrate binding and we speculate that modulation of substrate binding is the fundamental mechanism by which GSMs exert their action.

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells.

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.

Leukocyte transmigration is modulated by chemokine-mediated PI3Kgamma-dependent phosphorylation of vimentin.

Phosphoinositide 3-kinase gamma (PI3Kgamma) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine-stimulated WT or PI3Kgamma-null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kgamma signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kgamma or the kinase activity of PI3Kgamma (PI3Kgamma(KD/KD)), phosphorylation of vimentin was reduced. PI3Kgamma-null macrophages displayed impaired chemokine-driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N-terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kgamma-null macrophages with a vimentin mutant mimicking serine phosphorylation of N-terminal residues rescued the transendothelial migration defect. These results define vimentin N-terminal phosphorylation and fiber reorganization as a target of chemokine-dependent PI3Kgamma signaling in leukocytes.

Detection and identification of plasma proteins that bind GlialCAM using ProteinChip arrays, SELDI-TOF MS, and nano-LC MS/MS.

In order to fully understand biological processes it is essential to identify interactions in protein complexes. There are several techniques available to study this type of interactions, such as yeast two-hybrid screens, affinity chromatography, and coimmunoprecipitation. We propose a novel strategy to identify protein-protein interactions, comprised of first detecting the interactions using ProteinChips and SELDI-TOF MS, followed by the isolation of the interacting proteins through affinity beads and RP-HPLC and finally identifying the proteins using nano-LC MS/MS. The advantages of this new strategy are that the primary high-throughput screening of samples can be performed with small amounts of sample, no specific antibody is needed and the proteins represented on the SELDI-TOF MS spectra can be identified with high confidence. Furthermore, the method is faster and less labor-intensive than other current approaches. Using this novel method, we isolated and identified the interactions of two mouse plasma proteins, mannose binding lectin C and properdin, with GlialCAM, a type 1 transmembrane glycoprotein that belongs to the Ig superfamily.

The redox sensor TXNL1 plays a regulatory role in fluid phase endocytosis.

BACKGROUND: Small GTPases of the Rab family can cycle between a GTP- and a GDP-bound state and also between membrane and cytosol. The latter cycle is mediated by the Guanine Nucleotide Dissociation Inhibitor GDI, which can selectively extract GDP-bound Rab proteins from donor membranes, and then reload them on target membranes. In previous studies, we found that capture of the small GTPase Rab5, a key regulator of endocytic membrane traffic, by GDI is stimulated by oxidative stress via p38MAPK, resulting in increased fluid phase endocytosis. METHODOLOGY/PRINCIPAL FINDINGS: When purifying the GDI stimulating activity we found that that it copurified with a high MW protein complex, which included p38MAPK. Here we report the identification and characterization of another component of this complex as the thioredoxin-like protein TXNL1. Our observations indicate that TXNL1 play a selective role in the regulation of fluid phase endocytosis, by controlling GDI capacity to capture Rab5. CONCLUSIONS/SIGNIFICANCE: Oxidants, which are known to cause cellular damage, can also trigger signaling pathways, in particular via members of the thioredoxin family. We propose that TXNL1 acts as an effector of oxidants or a redox sensor by converting redox changes into changes of GDI capacity to capture Rab5, which in turn modulates fluid phase endocytosis.

A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages.

Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.

Quantitative detection of therapeutic proteins and their metabolites in serum using antibody-coupled ProteinChip Arrays and SELDI-TOF-MS.

One of the important steps in developing protein therapeutics is the determination of their preliminary PK in vivo. These data are essential to design optimal dosing in animal models prior to progressing to clinical trials in man. The quantitative detection of protein therapeutics in serum is traditionally performed by ELISA, which has the prerequisite of the availability of the appropriate monoclonal antibodies. We have developed an alternative method using polyclonal antibodies immobilized on ProteinChip Arrays and SELDI-TOF mass spectrometry. This method has an advantage over ELISA since it provides simultaneously information on the clearance rate of the protein and it's in vivo processing. We compared these two methods using a RANTES variant, [(44)AANA(47)]-RANTES as the test protein in this study. Using SELDI-TOF mass spectrometry, we were able to establish that the protein is readily oxidized in serum, and moreover is processed in vivo to produce a truncated 3-68 protein, and undergoes a further cleavage to produce the 4-68 protein. These modifications are not identified by ELISA, whilst the serum exposure profiles determined by the two methods show essentially similar protein concentration values.

Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens.

Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a single-dominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a "minimal essential MHC" for chickens, in strong contrast to typical mammals.

PERIOD1-associated proteins modulate the negative limb of the mammalian circadian oscillator.

The clock proteins PERIOD1 (PER1) and PERIOD2 (PER2) play essential roles in a negative transcriptional feedback loop that generates circadian rhythms in mammalian cells. We identified two PER1-associated factors, NONO and WDR5, that modulate PER activity. The reduction of NONO expression by RNA interference (RNAi) attenuated circadian rhythms in mammalian cells, and fruit flies carrying a hypomorphic allele were nearly arrhythmic. WDR5, a subunit of histone methyltransferase complexes, augmented PER-mediated transcriptional repression, and its reduction by RNAi diminished circadian histone methylations at the promoter of a clock gene.

Extracellular lysosome-associated membrane protein-1 (LAMP-1) mediates autoimmune disease progression in the NOD model of type 1 diabetes.

Treatment (from 5 to 25 weeks of age) with a novel blocking monoclonal antibody, mAb I-10, directed against the plasma membrane (pm) form of LAMP-1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12 weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm-LAMP-1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4(+)CD25(+)) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic-infiltrating IFN-gamma-positive cells was significantly reduced in the mAb I-10-treated group. These results demonstrate an unexpected role for pm-LAMP-1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN-gamma-driven responses.

Role of LBPA and Alix in multivesicular liposome formation and endosome organization.

What are the components that control the assembly of subcellular organelles in eukaryotic cells? Although membranes can clearly be distorted by cytosolic factors, very little is known about the intrinsic mechanisms that control the biogenesis, shape, and organization of organellar membranes. Here, we found that the unconventional phospholipid lysobisphosphatidic acid (LBPA) could induce the formation of multivesicular liposomes that resembled the multivesicular endosomes that exist where this lipid is found in vivo. This process depended on the same pH gradient that exists across endosome membranes in vivo and was selectively controlled by Alix. In turn, Alix regulated the organization of LBPA-containing endosomes in vivo.

Proteomic analysis of the mouse liver mitochondrial inner membrane.

Mitochondria play a crucial role in cellular homeostasis, which justifies the increasing interest in mapping the different components of these organelles. Here we have focused our study on the identification of proteins of the mitochondrial inner membrane (MIM). This membrane is of particular interest because, besides the well known components of the respiratory chain complexes, it contains several ion channels and many carrier proteins that certainly play a key role in mitochondrial function and, therefore, deserve to be identified at the molecular level. To achieve this goal we have used a novel approach combining the use of highly purified mouse liver mitochondrial inner membranes, extraction of membrane proteins with organic acid, and two-dimensional liquid chromatography coupled to tandem mass spectrometry. This procedure allowed us to identify 182 proteins that are involved in several biochemical processes, such as the electron transport machinery, the protein import machinery, protein synthesis, lipid metabolism, and ion or substrate transport. The full range of isoelectric point (3.9-12.5), molecular mass (6-527 kDa), and hydrophobicity values (up to 16 transmembrane predicted domains) were represented. In addition, of the 182 proteins found, 20 were unknown or had never previously been associated with the MIM. Overexpression of some of these proteins in mammalian cells confirmed their mitochondrial localization and resulted in severe remodeling of the mitochondrial network. This study provides the first proteome of the MIM and provides a basis for a more detailed study of the newly characterized proteins of this membrane.

Pharmacological profiles of peptide drug candidates for the treatment of Alzheimer's disease.

Amyloid plaques in brain, composed of aggregates of amyloid-beta peptide, play a central role in the pathogenesis of Alzheimer's disease and represent a good target for treatment. We have shown previously that a 5-amino acid beta-sheet breaker peptide (iA beta 5p), end-protected, has the ability to induce a dramatic reduction in amyloid deposition in two different transgenic Alzheimer's models (Permanne, B., Adessi, C., Saborio, G. P., Fraga, S., Frossard, M.-J., Dewachter, I., Van Dorpe, J., Banks, W. A., Van Leuven, F., and Soto, C. (2002) FASEB J. 16, 860-862). The aim of this study was to evaluate the effect of chemical modifications of the peptide bonds at the metabolite cleavage sites on the pharmacological properties of iA beta 5p derivatives. Using a rational approach, peptide analogs were designed and tested for in vitro activity and enzymatic stability. One peptide analog containing a methyl group introduced at the nitrogen atom of one amide bond showed increased stability in vitro, a 10-fold higher in vivo half-life, and good brain uptake compared with iA beta 5p while maintaining a similar activity in vitro. Our results suggest that the pharmacological profile of beta-sheet breaker peptides can be improved to produce compounds with drug-like properties that might offer a new promise in the treatment of Alzheimer's disease.

Differential sorting and fate of endocytosed GPI-anchored proteins.

In this paper, we studied the fate of endocytosed glycosylphosphatidyl inositol anchored proteins (GPI- APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI-APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft-like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI-APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI-APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI-APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI-APs in different cell types depend on the residence time of GPI-APs in lipid rafts, and hence that raft partitioning regulates GPI-APs sorting in the endocytic pathway.