Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study.

Descriptive Proteome Analysis to Investigate Context-Dependent Treatment Responses to OXPHOS Inhibition in Colon Carcinoma Cells Grown as Monolayer and Multicellular Tumor Spheroids.

We have previously identified selective upregulation of the mevalonate pathway genes upon inhibition of oxidative phosphorylation (OXPHOS) in quiescent cancer cells. Using mass spectrometry-based proteomics, we here investigated whether these responses are corroborated on the protein level and whether proteomics could yield unique insights into context-dependent biology. HCT116 colon carcinoma cells were cultured as monolayer cultures, proliferative multicellular tumor spheroids (P-MCTS), or quiescent (Q-MCTS) multicellular tumor spheroids and exposed to OXPHOS inhibitors: nitazoxanide, FCCP, oligomycin, and salinomycin or the HMG-CoA-reductase inhibitor simvastatin at two different doses for 6 and 24 h.

Proteomics-Based Characterization of miR-574-5p Decoy to CUGBP1 Suggests Specificity for mPGES-1 Regulation in Human Lung Cancer Cells.

MicroRNAs (miRs) are one of the most important post-transcriptional repressors of gene expression. However, miR-574-5p has recently been shown to positively regulate the expression of microsomal prostaglandin E-synthase-1 (mPGES-1), a key enzyme in the prostaglandin E (PGE) biosynthesis, by acting as decoy to the RNA-binding protein CUG-RNA binding protein 1 (CUGBP1) in human lung cancer. miR-574-5p exhibits oncogenic properties and promotes lung tumor growth via induction of mPGES-1-derived PGE synthesis.

sRNA scr5239 Involved in Feedback Loop Regulation of Central Metabolism.

In contrast to transcriptional regulation, post-transcriptional regulation and the role of small non-coding RNAs (sRNAs) in streptomycetes are not well studied. Here, we focus on the highly conserved sRNA scr5239 in . A proteomics approach revealed that the sRNA regulates several metabolic enzymes, among them phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the central carbon metabolism.

Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts.

Objectives: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology.

Methods: Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA.

Mass Spectrometry-Based Proteomics Approach Characterizes the Dual Functionality of miR-328 in Monocytes.

MicroRNAs (miRs) are small noncoding RNAs which control the expression of target genes by either translational repression or RNA degradation, known as canonical miR functions. The recent discovery that miR-328 has a noncanonical function and can activate gene expression by antagonizing the activity of heterogeneous ribonuclear protein E2 (hnRNP E2) opens an unexplored and exciting field of gene expression regulation. The global importance of such noncanonical miR function is not yet known.

Inhibition of mPGES-1 or COX-2 Results in Different Proteomic and Lipidomic Profiles in A549 Lung Cancer Cells.

Pharmacological inhibition of microsomal prostaglandin E synthase (mPGES)-1 for selective reduction in prostaglandin E (PGE) biosynthesis is protective in experimental models of cancer and inflammation. Targeting mPGES-1 is envisioned as a safer alternative to traditional non-steroidal anti-inflammatory drugs (NSAIDs). Herein, we compared the effects of mPGES-1 inhibitor Compound III (CIII) with the cyclooxygenase (COX)-2 inhibitor NS-398 on protein and lipid profiles in interleukin (IL)-1β-induced A549 lung cancer cells using mass spectrometry.

Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification.

Mass spectrometry-based analysis of cerebrospinal fluid from arthritis patients-immune-related candidate proteins affected by TNF blocking treatment.

Background: Signs of inflammation in cerebrospinal fluid (CSF) of rheumatoid arthritis patients correlate positively with fatigue, a central nervous system (CNS)-related symptom that can be partially suppressed by TNF blockade. This suggests a possible role for CNS inflammation in arthritis that may be affected by TNF blockade. We therefore investigated the effects of TNF blockade on the arthritis CSF proteome and how candidate proteins related to clinical measures of disease activity and inflammation.

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACPA.

The human CSF pain proteome.

Chronic pain represents one of the major medical challenges in the 21st century, affecting >1.5 billion of the world population. Overlapping and heterogenous symptoms of various chronic pain conditions complicate their diagnosis, emphasizing the need for more specific biomarkers to improve the diagnosis and understand the disease mechanisms.

Light-activatable prodrugs based on hyaluronic acid biomaterials.

Photosensitive in situ cross-linked hyaluronan (HA) hydrogels are prepared by modular chemoselective assembly from the biopolymer precursors and novel heterobifunctional linkers with middle photo-labile ortho-nitrobenzyl group and orthogonally reactive terminals. Starting from the thiol-modified HA and a linker with activated disulfide and hydrazide terminals, a photo-degradable HA hydrogel was prepared by the hydrazone cross-linking reaction. Moreover, a light-triggered drug-releasing hydrogel prodrug was constructed by an orthogonal conjugation of dopamine (DA) via a photo-labile linker to HA dually modified with thiol and hydrazide groups (hy-HA-SH) and a subsequent cross-linking with aldehyde-derivatized HA (HA-al).

Endurance Exercise Improves Molecular Pathways of Aerobic Metabolism in Patients With Myositis.

Objective: Endurance exercise demonstrates beneficial effects in polymyositis/dermatomyositis (PM/DM); however, the molecular effects of exercise on skeletal muscle are incompletely understood. We undertook this controlled pilot study to investigate the effects of a 12-week endurance exercise training program on the molecular profile of skeletal muscle in patients with established PM/DM compared to a nonexercised control group of patients with established PM/DM.

Methods: Fifteen patients (7 in the exercise group and 8 in the control group) with paired baseline and 12-week follow-up muscle biopsy samples were included.

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens.

Introduction: We have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA).

Methods: The autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system.

IgG antibodies to cyclic citrullinated peptides exhibit profiles specific in terms of IgG subclasses, Fc-glycans and a fab-Peptide sequence.

The Fc-glycan profile of IgG1 anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG1, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns.

Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint.

Introduction: A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response.

Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis: identification and validation.

Objectives: Immunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs of patients with RA.

Patients And Methods: Proteins extracted from bronchial (n=6) and synovial (n=7) biopsy specimens from patients with RA were investigated by mass spectrometry-based proteomics.

Mass spectrometry-based proteomics identifies UPF1 as a critical gene expression regulator in MonoMac 6 cells.

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Recently, we have demonstrated that 5-LO mRNA expression is regulated by alternative splicing and nonsense-mediated mRNA decay (NMD). In addition to this, 5-LO protein expression was reduced on translational level in UPF1 knockdown cells, suggesting that UPF1 has a positive influence on 5-LO translation.

Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin.

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism.

MS analysis of rheumatoid arthritic synovial tissue identifies specific citrullination sites on fibrinogen.

Purpose: Citrullination is a post-translational modification of arginine residues to citrulline catalyzed by peptidyl arginine deiminases. Induced expression of citrullinated proteins are frequently detected in various inflammatory states including arthritis; however, direct detection of citrullination in arthritic samples has not been successfully performed in the past.

Experimental Design: Citrullination of human fibrinogen, a candidate autoantigen in arthritis, was studied.

Development of T-cell acute lymphoblastic leukemia in a patient in very long lasting complete remission of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) occurs with an incidence of 1.2 per million children a year, and represents 18% to 30% of all myelodysplastic (MDS) and myeloproliferative (MPS) disorders in the age group below 15, being by far the most common MDS/MPS in children younger than 4 years. The only therapeutic approach which results in a definitive cure of patients with JMML is myeloablative chemo-therapy/radio-therapy, followed by allogeneic hematopoietic cell transplantation.

Optimizing search conditions for the mass fingerprint-based identification of proteins.

The two central problems in protein identification by searching a protein sequence collection with MS data are the optimal use of experimental information to allow for identification of low abundance proteins and the accurate assignment of the probability that a result is false. For comprehensive MS-based protein identification, it is necessary to choose an appropriate algorithm and optimal search conditions. We report a systematic study of the quality of PMF-based protein identifications under different sequence collection search conditions using the Probability algorithm, which assigns the statistical significance to each result.

Method for differential detection and identification of components in protein mixtures analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We demonstrate that the semi-quantitative information in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of tryptically digested protein mixtures can, via a systematic statistical approach, be utilized for the identification of a protein present in different concentrations in two samples. Multiple mass spectra were acquired from a series of tryptically digested test samples in which the concentration of one protein was varied and the concentrations of three other proteins were held constant. The mass spectra were subjected to soft independent modeling of class analogy (SIMCA) analysis assuming that spectra originating from two different samples belonged to different data classes.