David M. Ferrari

CD137 ligand reverse signaling has multiple functions in human dendritic cells during an adaptive immune response.

T cell activation via dendritic cells (DC) is an important step in the adaptive immune response, which requires DC maturation, migration to lymph nodes and presentation of antigen to T cells. CD137 receptor expressed on activated T cells is a potent costimulatory molecule. Here, we investigated the functions of CD137 ligand (CD137L) in human monocyte‐derived DC during an immune response. Cross‐linking of CD137L on DC leads to cell maturation in an autocrine fashion, mostly via release of TNF‐α. Reverse signaling of CD137L also mediates migration of DC via up‐regulation of the CCR7 chemokine receptor, demonstrated by an in vivo MIP‐3β‐dependent SCID mouse migration model. Finally, CD137L‐activated DC induce differentiation of human T cells into potent Th1 effectors. Cocultivation of autologous T cells and CD137L‐activated DC in an antigen‐specific reaction leads to T cell proliferation and the release of IL‐12p70 and IFN‐γ. These findings deliver new insights into the multiple effects of reverse signaling of CD137L in human DC during the initiation of an adaptive immune response, including the key features of DC maturation, migration and, ultimately, antigen‐specific T cell differentiation.

Crystal structure and functional analysis of the protein disulfide isomerase-related protein ERp29.

The protein disulfide isomerase-related protein ERp29 is a putative chaperone involved in processing and secretion of secretory proteins. Until now, however, both the structure and the exact nature of interacting substrates remained unclear. We provide for the first time a crystal structure of human ERp29, refined to 2.9 A, and show that the protein has considerable structural homology to its Drosophila homolog Wind. We show that ERp29 binds directly not only to thyroglobulin and thyroglobulin-derived peptides in vitro but also to the Wind client protein Pipe and Pipe-derived peptides, although it fails to process Pipe in vivo. A monomeric mutant of ERp29 and a D domain mutant in which the second peptide binding site is inactivated also bind protein substrates, indicating that the monomeric thioredoxin domain is sufficient for client protein binding. Indeed, the b domains of ERp29 or Wind, expressed alone, are sufficient for binding proteins and peptides. Interacting peptides have in common two or more aromatic residues, with stronger binding for sequences with overall basic character. Thus, the data allow a view of the two putative peptide binding sites of ERp29 and indicate that the apparent, different processing activity of the human and Drosophila proteins in vivo does not stem from differences in peptide binding properties.

Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells

Mast cells are important players in innate immunity and mediate allergic responses. Upon stimulation, they release biologically active mediators including histamine, cytokines and lysosomal hydrolases. We used permeabilized rat basophilic leukaemia cells as model to identify R‐SNAREs (soluble NSF (N‐ethylmaleimide‐sensitive fusion protein)) mediating exocytosis of hexosaminidase from mast cells. Of a complete set of recombinant mammalian R‐SNAREs, only vesicle associated membrane protein (VAMP8)/endobrevin consistently blocked hexosaminidase release, which was also insensitive to treatment with clostridial neurotoxins. Thus, VAMP8, which also mediates fusion of late endosomes and lysosomes, plays a major role in hexosaminidase release, strengthening the view that mast cell granules share properties of both secretory granules and lysosomes.

Crystal structure and functional analysis of Drosophila Wind, a protein-disulfide isomerase-related protein

In the developing Drosophila melanogaster embryo, dorsal-ventral patterning displays an absolute requirement for the product of the essential windbeutel gene, Wind. In homozygous windbeutel mutant flies, dorsal-ventral patterning fails to initiate because of the failure of the Golgi-resident proteoglycan-modifying protein, Pipe, to exit the endoplasmic reticulum, and this leads to the death of the embryo. Here, we describe the three-dimensional structure of Wind at 1.9-Å resolution and identify a candidate surface for interaction with Pipe. This represents the first crystal structure of a eukaryotic protein-disulfide isomerase-related protein of the endoplasmic reticulum to be described. The dimeric protein is composed of an N-terminal thioredoxin domain and a C-terminal α-helical domain unique to protein-disulfide isomerase D proteins. Although Wind carries a CXXC motif that is partially surface accessible, this motif is redox inactive, and the cysteines are not required for the targeting of Pipe to the Golgi. However, both domains are required for targeting Pipe to the Golgi, and, although the mouse homologue ERp28 cannot replace the function of Wind, exchange of the Wind D-domain with that of ERp28 allows for efficient Golgi transport of Pipe.

A possible biochemical link between NADPH oxidase (Nox) 1 redox-signalling and ERp72.

Emerging evidence indicates that Nox (NADPH oxidase) 1-generated ROS (reactive oxygen species) play critical regulatory roles in various cellular processes, yet little is known of direct targets for the oxidase. In the present study we show that one of the proteins selectively oxidized in response to Nox1-generated ROS was ERp72 (endoplasmic reticulum protein 72 kDa) with TRX (thioredoxin) homology domains. Oxidation of ERp72 by Nox1 resulted in an inhibition of its reductase activity. EGF treatment of cells stimulated the Nox1 activity and the activated Nox1 subsequently mediated EGF-induced suppression of the ERp72 reductase activity. Co-immunoprecipitation, GST (glutathione transferase) pulldown assays and mutational analysis, indicated that Nox1 associates with ERp72, which involves its N-terminus encompassing a Ca(2+)-binding site and the first TRX-like motif. Furthermore, confocal microscopy showed co-localization between Nox1 and ERp72 at the plasma membrane. These results suggest that Nox1 functionally associates with ERp72, regulating redox-sensitive signalling pathways in a cellular context.

Conserved structural and functional properties of D-domain containing redox-active and -inactive protein disulfide isomerase-related protein chaperones

The structure and mode of binding of the endoplasmic reticulum protein disulfide isomerase-related proteins to their substrates is currently a focus of intensive research. We have recently determined the crystal structure of the Drosophila melanogaster protein disulfide isomerase-related protein Wind and have described two essential substrate binding sites within the protein, one within the thioredoxin b-domain and another within the C-terminal D-domain. Although a mammalian ortholog of Wind (ERp29/28) is known, conflicting interpretations of its structure and putative function have been postulated. Here, we have provided evidence indicating that ERp29 is indeed similar in both structure and function to its Drosophila ortholog. Using a site-directed mutagenesis approach, we have demonstrated that homodimerization of the b-domains is significantly reduced in vitro upon replacement of key residues at the predicted dimerization interface. Investigation of Wind-ERp29 fusion constructs showed that mutants of the D-domain of ERp29 prevent transport of a substrate protein (Pipe) in a manner consistent with the presence of a discrete, conserved peptide binding site in the D-domain. Finally, we have highlighted the general applicability of these findings by showing that the D-domain of a redox-active disulfide isomerase, from the slime mold Dictyostelium discoideum, can also functionally replace the Wind D-domain in vivo.

Structural Elucidation of the Pdi-Related Chaperone Wind with the Help of Mutants.

The structures of the PDI-related protein Wind (with a C-terminal His(6) tag) and the mutants Y53S, Y53F and Y55K have been determined and compared with the wild-type structure with the His(6) tag at the N-terminus. All five structures show the same mode of dimerization, showing that this was not an artefact introduced by the nearby N-terminal His(6) tag and suggesting that this dimer may also be the biologically active form. Although the mutants Y53S and Y55K completely abrogate transport of the protein Pipe (which appears to be the primary function of Wind in the cell), only subtle differences can be seen in the putative Pipe-binding region. The Pipe binding in the active forms appears to involve hydrophobic interactions between aromatic systems, whereas the inactive mutants may be able to bind more strongly with the help of hydrogen bonds, which could disturb the delicate equilibrium required for effective Pipe transport.

Forward into terra incognita: Proteome and Protein Analysis edited by R.M Kamp, D. Kyriakidis and T. Choli-Papadopoulou, Springer, 2000. £76.00 (372 pages) ISBN 3 540 65891 2

This book review was commission by Trends in Cell Biology and published in the November 2000 issue of Trends in Cell Biology.There are those occasions when, with ones experiments left to their own devices for a while, one ponders over a cup of steaming coffee new avenues down which the pursuit of the ultimate truth can be envisioned. At times, it is not merely methods well established in ones own laboratory that come to mind, rather one might simply wish to find new ideas and play with the possibilities. Proteome and Protein Analysis is a book to this end. Although it contains many useful protocols, it is primarily suited as a resource for investigators from other fields of research who are attempting to gain an insight into some of the new techniques of proteome (i.e. the entire protein complement expressed in a given cell, tissue type or organism 1xFrom proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Wilkins, M.R. et al. Biotechnology. 1996; 14: 61–65Crossref | PubMed | Scopus (563)See all References1) and/or protein analysis.This book comprises a selection of papers presented by active researchers at the Twelfth International Conference on Methods in Protein Structure Analysis (MPSA), 1998, and is divided into four parts: proteomics and mass spectrometry (MS), protein structure analysis, protein–protein and protein–DNA interactions and posttranslational modifications. This is a well-balanced division, ensuring the greatest probability that it will contain a variety of flavours to please any reader. At 360 pages, more than half of the chapters include some mention of MS, attesting to the great potential of these methods in proteome and protein analysis.The methods described throughout are accurate, up to date and, by avoiding much of the specialist jargon, written in reader-friendly mode. Practical examples abound, as do many informative figures enlightening the experimental procedure.In the first part of the book, the reader is taken on a guided tour of proteomics and MS, encompassing all stages from initial protein separation of, for instance, total cell extracts by isoelectric focusing, PAGE and two-dimensional electrophoresis, through reversible staining down to femtomolar concentrations of protein in polyacrylamide gels using imidazole-zinc salts, to sample preparation for MS (for peptide separation and determination of peptide mass and sequence) and the analysis of MS-derived data by automated database searches. The advantages, importance and limitations of MS in protein identification and protein/ proteome characterization are presented.A lot of ground is covered in the second and larger part of the book. Here, a brief but accessible introduction to the analysis of protein structure by NMR is given, followed by various matrix-assisted laser desorption/ionization (MALDI)-based methods such as MALDI-TOF and the new post-source decay (PSD) method for the analysis of protein structure or protein modifications (such as oligosaccharide side chains and disulfide bonding pattern). An interesting example is given in the application of MALDI-TOF in the detection of immune-induced protein in the body fluid from a single fruit-fly.Among the methods described that do not involve MS are HPLC and capillary zone electrophoresis (CZE) in the isolation and identification of cis/trans isomers of proline-containing peptides, the use of a hydrophobic probe, bis-ANS [bis(4-anilino-5-naphthalenesulfonate)], in determining stages in the unfolding of a protein as measured by circular dichroism or spectrofluorimetry, and proteolytic (trypsin) digestion in protein domain analysis. A new method for the identification of proteins by isolation of C-terminal peptides after N-terminal blocking of proteolytic fragments is also described.The penultimate section, which could have benefited from a little more detail, deals with the study of protein–protein and protein–DNA interaction. However, the most useful information to be gleaned here comes from a quick view into the heterologous expression of a protein in a bacterial host, its extraction, purification and analysis (atomic absorption measurement of a zinc-binding protein). A glimpse of protein interaction studies performed using the powerful BIAcore system (uses surface plasmon resonance for detection of, for example, binding of analyte to a ligand coupled to a sensor chip) is given.Many proteins undergo some form of modification either during or after their synthesis. The analysis of such modifications is the focus of the last part of the book. Some topics mentioned here are methods for deblocking N-terminally modified (e.g. acetylated or containing pyroglutamic acid or myristoyl groups) proteins, a procedure required for later N-terminal sequencing. A nice point is the proposal of a scheme for the sequential deblocking of substrates with uncharacterized blocking groups. Other questions tackled here are the isolation of proteoglycans and characterization of the glycan moiety by methods including HPLC and capillary electrophoresis.Although this book contains a variety of excellent protocols, it is not a protocols book – rather, it adds up well to the modest intentions of the editors in being ‘beneficial because of the new and interesting ideas it introduces’ to receptive researchers willing to push forward into terra (slightly) incognita.