Novel COMP Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases using Affinity Chromatography and Mass Spectrometry.

To identify patients at risk for progressive joint damage there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open for novel treatment strategies. Disease specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis (RA), osteoarthritis (OA) or acute trauma (AT). Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin and Asp-N for the digestions an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, S77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope S77 were released into the culture medium of cytokine (TNF-α and IL-6/sIL-6R) stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pin-point disease progression, evaluation methods for therapy and means to elucidate disease mechanisms will be provided

The glycosaminoglycan-binding domain of PRELP acts as a cell type–specific NF-κB inhibitor that impairs osteoclastogenesis

The PRELP heparin sulfate–binding protein translocates to the nucleus, where it impairs NF-κB transcriptional activity, which in turn regulates bone homeostasis

Team motivation - A model for understanding the motivational factors of a team -

The concept of team motivation is highly affecting the performance of the teams in organizations today. A team that is motivated is creative and has a high level of performance, which makes an investigation to understand the concept of motivation in a team relevant and interesting. This research concerns what factors affect team motivation and also how motivation is different between individuals and teams. To provide a basis for the thesis a deductive approach was used. Theories of teams and motivation were studied and out of these theories a model was created which contained five different types of motivational factors for a team. The empirical material was gathered through interviews with team members and team leaders at the research area. The material was then interpreted according to the created model and the conclusion that was drawn was that team motivation is affected from many different directions and that the different factors complement each other in creating team motivation

Interactions Between Small Heat Shock Proteins and Substrate Proteins Mass Spectrometric Detection of Crosslinked Peptides to Map Protein-Protein Interactions

The small heat shock proteins (sHsps) are a widespread class of molecular chaperones existing in all organisms. During heat stress the sHsps protect other proteins from unfolding and aggregation. In plants the sHsps play an especially prominent part of the stress response. In this thesis chemical crosslinking and mass spectrometric mapping of crosslinked peptides has been used to investigate the interaction between sHsps and substrate proteins. The focus has been on Hsp21, a chloroplast-localized sHsp which increases the stress resistance of Arabidopsis thaliana plants, and a thermosensitive model substrate protein, citrate synthase (CS), using the chemical crosslinker DTSSP (3,3?-dithiobis[sulfosuccinimidylpropionate]), for covalent crosslinking between lysine residues or other primary amines. In peptide array screening, binding of sHsps occurred to a peptide located in a stem-like structure protruding from the CS dimer, where the C-terminus from one CS monomer interacts with the N-terminal part of the other monomer. Mass spectrometric mapping of crosslinked Hsp21-CS peptides further confirmed interactions between Hsp21 and lysine residues (K16, K22, K432, K437) located in this stem-like structure of CS. We propose that sHsps bind to this region, which is absent in thermostable CS forms, to stabilize the Nand C-terminae and thereby prevent CS dimer dissociation and aggregation. The crosslinked Hsp21-CS peptides indicated that the substrate-binding region in the N-terminal arm in Hsp21 interacted with the thermosensitive part of CS. Similar mass spectrometric peptide mapping was also used to show how this N-terminal arm region of a mammalian sHsp, Hsp20, is involved in transglutaminase-catalyzed crosslinking of sHsps to various substrate proteins in cellular aggregates. This was studied using a peptide probe which could be crosslinked by transglutaminase to the conserved Q31 in the Nterminal arm of mammalian sHsps. Whereas the N-terminus of Hsp21 was detected in crosslinked Hsp21-Hsp21 and Hsp21-CS peptides under conditions where the Hsp21 dodecamer was intact, the C-terminal tail and a C-terminal binding groove was detected in crosslinked Hsp21-CS peptides only under conditions where the Hsp21 dodecamer dissociated. The crosslinked Hsp21-CS peptides mapped onto several sites on the CS surface, indicating that several weak and short-lived interactions between Hsp21 and CS occur already at normal temperatures. Single particle reconstruction EM of crosslinked Hsp21 in the presence of CS indicated that the crosslinker could capture the Hsp21 dodecamers in an ?activated? conformation induced by such weak and short-lived interactions with CS

Conserved methionines in chloroplasts

Heat shock proteins counteract heat and oxidative stress. In chloroplasts, a small heat shock protein (Hsp21) contains a set of conserved methionines, which date back to early in the emergence of terrestrial plants. Methionines M49, M52, M55, M59, M62, M67 are located on one side of an amphipathic helix, which may fold back over two other conserved methionines (M97 and M101), to form a binding groove lined with methionines, for sequence-independent recognition of peptides with an overall hydrophobic character. The sHsps protect other proteins from aggregation by binding to their hydrophobic surfaces, which become exposed under stress. Data are presented showing that keeping the conserved methionines in Hsp21 in a reduced form is a prerequisite to maintain such binding. The chloroplast generates reactive oxygen species under both stress and unstressed conditions, but this organelle is also a highly reducing cellular compartment. Chloroplasts contain a specialized isoform of the enzyme, peptide methionine sulfoxide reductase, the expression of which is light-induced. Recombinant proteins were used to measure that this reductase can restore Hsp21 methionines after sulfoxidation. This paper also describes how methionine sulfoxidation-reduction can be directly assessed by mass spectrometry, how methionine-to-leucine substitution affects Hsp21, and discusses the possible role for an Hsp21 methionine sulfoxidation-reduction cycle in quenching reactive oxygen species. (C) 2004 Elsevier B.V. All rights reserved

Quantitative mass spectrometry to study inflammatory cartilage degradation and resulting interactions with the complement system

Joint diseases are often characterized by inflammatory processes that result in pathological changes in joint tissues, including cartilage degradation and release of components into the synovial fluid. The complement system plays a central role in promoting the inflammation. Because several cartilage proteins are known to interact with complement, causing either activation or inhibition of the system, we aimed to investigate these interactions comprehensively. Bovine cartilage explants were cultured with IL-1α to induce cartilage degradation, followed by incubation with human serum. Label-free selected reaction monitoring mass spectrometry was used to specifically quantify complement proteins interacting with the cartilage explant. In parallel, the time-dependent degradation of cartilage was detected using mass spectrometry analysis (liquid chromatography-tandem mass spectrometry). Complement proteins resulting from activation of the classical, alternative, and terminal pathways were detected on IL-1α-stimulated cartilage at time points when clear alterations in extracellular matrix composition had occurred. Increased levels of the complement activation product C4d, as detected by ELISA in serum after incubation with IL-1α-stimulated cartilage, confirmed the selected reaction monitoring results indicating complement activation. Further, typical activated (cleaved) C3 fragments were detected by Western blotting in extracts of IL-1α-stimulated cartilage. No complement activation was triggered by cartilage cultured in the absence of IL-1α. Components released from IL-1α-stimulated cartilage during culture had an inhibitory effect on complement activation. These were released after a longer incubation period with IL-1α and may represent a feedback reaction to cartilage-triggered complement activation observed after a shorter incubation period

Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase

The molecular mechanism whereby the small heat-shock protein (sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp–substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase (CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,3′-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross-linker captured Hsp21 in dodecameric form, even at increased temperature (47°C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21–Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21–CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21

Quantification of heat shock proteins in the posterior interosseous nerve among subjects with type 1 and type 2 diabetes compared to healthy controls

Introduction: Diabetic peripheral neuropathy (DPN) is a common complication of both type 1 (T1D) and type 2 diabetes (T2D). No cure for DPN is available, but several potential targets have been proposed for treatment. Heat shock proteins (HSPs) are known to respond to both hyper- and hypoglycemia. DPN can be diagnosed using electrophysiology and studied using peripheral nerve biopsies.Aim: This study aimed to analyze the presence and patterns of HSPs in peripheral nerve biopsies from subjects with T1D, T2D, and healthy controls.Methods: Posterior interosseous nerves (PIN) from a total of 56 subjects with T1D (n = 9), with T2D (n = 24), and without diabetes (i.e., healthy controls, n = 23) were harvested under local anesthesia and prepared for quantitative mass spectrometry analysis. Protein intensities were associated with electrophysiology data of the ulnar nerve and morphometry of the same PIN, and differences in protein intensities between groups were analyzed.Results: In total, 32 different HSPs were identified and quantified in the nerve specimens. No statistically significant differences were observed regarding protein intensities between groups. Furthermore, protein intensities did not correlate with amplitude or conduction velocity in the ulnar nerve or with the myelinated nerve fiber density of PIN.Conclusion: Quantitative proteomics can be used to study HSPs in nerve biopsies, but no clear differences in protein quantities were observed between groups in this cohort

Effective protein extraction combined with data independent acquisition analysis reveals a comprehensive and quantifiable insight into the proteomes of articular cartilage and subchondral bone

OBJECTIVE: The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal.DESIGN: Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral library as reference database (FDR 1%).RESULTS: We identified to our knowledge the hitherto most comprehensive quantitative cartilage (1758 proteins) and subchondral bone (1482 proteins) proteomes in all species presented to date. Both extraction methods were sensitive and reproducible and the high consistency of the identified proteomes (>97% overlap) indicated that both methods preserved the diversity among the extracted proteins. Proteome mining revealed a substantial number of quantifiable cartilage and bone matrix proteins and proteins involved in osteogenesis and bone remodeling, including ACAN, BGN, PRELP, FMOD, COMP, ACP5, BMP3, BMP6, BGLAP, TGFB1, IGF1, ALP, MMP3, and collagens. A number of proteins, including COMP and TNN, were identified in different protein isoforms with potential unique biological roles.CONCLUSION: We have successfully developed two sensitive and reproducible non-species specific workflows enabling a comprehensive quantitative insight into the proteomes of cartilage and subchondral bone. This facilitates the prospect of investigating the molecular events at the osteochondral unit in the pathogenesis of OA in future projects

Quantitative proteomic analysis of human peripheral nerves from subjects with type 2 diabetes

AIMS: Diabetic peripheral neuropathy (DPN) is a common and severe complication to type 2 diabetes (T2D). The pathogenesis of DPN is not fully known, but several pathways and gene polymorphisms contributing to DPN are described. DPN can be studied using nerve biopsies, but studies on the proteome of the nerve itself, and its surrounding tissue as a whole, are lacking. Studies on the posterior interosseous nerve (PIN) have proposed PIN a useful indicator of DPN.METHODS: A quantitative mass spectrometry-based proteomics analysis was made of peripheral nerves from age- and gender-matched living human male tissue donors; nine T2D subjects, with decreased sural nerve action potentials indicating DPN, and six controls without T2D, with normal electrophysiology results.RESULTS: A total of 2617 proteins were identified. Linear regression was used to discover which proteins were differentially expressed between T2D and controls. Only soft signals were found. Therefore, clustering of the 500 most variable proteins were made in order to find clusters of similar proteins in T2D subjects and healthy controls.CONCLUSIONS: This feasibility study shows, for the first time, that the use of quantitative mass spectrometry enables quantification of proteins from nerve biopsies from subjects with and without T2D, which may aid in finding biomarkers of importance to DPN development