Hanno Langen

Orphanin FQ: A neuropeptide that activates an opioidlike G protein-coupled receptor

A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP- binding protein (G protein)- coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ, has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.

A Coat Protein on Phagosomes Involved in the Intracellular Survival of Mycobacteria

Mycobacteria are intracellular pathogens that can survive within macrophage phagosomes, thereby evading host defense strategies by largely unknown mechanisms. We have identified a WD repeat host protein that was recruited to and actively retained on phagosomes by living, but not dead, mycobacteria. This protein, termed TACO, represents a component of the phagosome coat that is normally released prior to phagosome fusion with or maturation into lysosomes. In macrophages lacking TACO, mycobacteria were readily transported to lysosomes followed by their degradation. Expression of TACO in nonmacrophages prevented lysosomal delivery of mycobacteria and prolonged their intracellular survival. Active retention of TACO on phagosomes by living mycobacteria thus represents a mechanism preventing cargo delivery to lysosomes, allowing mycobacteria to survive within macrophages.

Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB

We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150–200 species, corresponding to 15–25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (≥90 kDa) but only 18% of the small (≤30 kDa) cytosolic proteins and include essential proteins. The DnaK system in addition acts with ClpB to form a bi‐chaperone system that quantitatively solubilizes aggregates of most of these proteins. Efficient solubilization also occurred in an in vivo order‐of‐addition experiment in which aggregates were formed prior to induction of synthesis of the bi‐chaperone system. Our data indicate that large‐sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central, dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation.

A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNAMet (tRNAi Met). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNAi Met with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNAi Met by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.

Guidelines for the next 10 years of proteomics

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol

We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol. In ΔrpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5–10% and 20–30% of total protein aggregated at 30°C and 42°C respectively. The aggregates contained 350–400 protein species, of which 93 were identified by mass spectrometry. The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30°C, and showed strong overlap with previously identified DnaK substrates. Overproduction of the DnaK system, or low‐level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to ΔrpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival. In rpoH+ cells, DnaK depletion did not lead to protein aggregation at 30°C, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins. Lon was the most efficient protease in degrading misfolded proteins in DnaK‐depleted cells. At 42°C, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42°C.

Activating interactions in human NK cell recognition: the role of 2B4‐CD48

2B4 is a cell surface glycoprotein of the immunoglobulin superfamily structurally related to CD2‐like molecules. It was originally identified in the mouse as a receptor that mediates non‐MHC‐restricted cytotoxicity by NK cells and CD8+ T cells. Recently, 2B4 was shown to bind CD48 by molecular binding assays and surface plasmon resonance. Here, we have investigated the cell surface expression, biochemical characteristics and function of human 2B4. Our results show that 2B4 is expressed not only on NK cells and CD8+ T cells, but also on monocytes and basophils, indicating a broader role for 2B4 in leukocyte activation. In NK cells, engagement of 2B4 with a specific monoclonal antibody or with CD48 can trigger NK cell‐mediated cytotoxicity. The contribution of 2B4‐CD48 interaction to target cell lysis by different NK cell clones varies, probably dependent on the relative contribution of other receptor‐ligand interactions. In T cells and monocytes, ligation of 2B4 does not lead to T cell or monocyte activation. Thus, it appears that the primary function of 2B4 is to modulate other receptor‐ligand interactions to enhance leukocyte activation.

Mass spectrometry : A tool for the identification of proteins separated by gels

Mass spectrometry (MS) has become the technique of choice to identify proteins. This has been largely accomplished by the combination of high‐resolution two‐dimensional (2‐D) gel separation with robotic sample preparation, automated MS measurement, data analysis, and database query. Developments during the last five years in MS associated with protein gel separation are reviewed.

Reliable automatic protein identification from matrix-assisted laser desorption/ionization mass spectrometric peptide fingerprints.

Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry of protein samples from two‐dimensional (2‐D) gels in conjunction with protein sequence database searches is frequently used to identify proteins. Moreover, the automatic analysis of complete 2‐D gels with hundreds and even thousands of protein spots (“proteome analysis”) is possible, without human intervention, with the availability of highly accurate mass spectrometry instruments, and high‐throughput facilities for preparation and handling of protein samples from 2‐D gels. However, the lack of software for precise automatic analysis and annotation of mass spectra, as well as software for in‐batch sequence database queries, is increasingly becoming a significant bottleneck for the proteomics work flow. In the present paper we outline an algorithm for reliable, accurate, and automatic evaluation of mass spectrometric data and database searches. We show here that simply selecting from the sequence database the protein that has the most matching fragment masses often leads to false‐positive results. Reliable protein identification is dependent on several parameters: the accuracy of fragment mass determination, the number of masses submitted for query, the mass distribution of query masses, the number of masses matching between sample and database protein, the size of the sequence database, and the kind and number of modifications considered. Using these parameters, we derive a simple statistical estimation that can be used to calculate the probability of true‐positive protein identification.

Tetraspan microdomains distinct from lipid rafts enrich select peptide-MHC class II complexes.

Complexes of peptide and major histocompatibility complex (MHC) class II are expressed on the surface of antigen-presenting cells but their molecular organization is unknown. Here we show that subsets of MHC class II molecules localize to membrane microdomains together with tetraspan proteins, the peptide editor HLA-DM and the costimulator CD86. Tetraspan microdomains differ from other membrane areas such as lipid rafts, as they enrich MHC class II molecules carrying a selected set of peptide antigens. Antigen-presenting cells deficient in tetraspan microdomains have a reduced capacity to activate CD4+ T cells. Thus, the organization of uniformly loaded peptide–MHC class II complexes in tetraspan domains may be a very early event that determines both the composition of the immunological synapse and the quality of the subsequent T helper cell response.