miR-155-3p: processing by-product or rising star in immunity and cancer?

MicroRNAs (miRNAs) are key players in gene regulation that target specific mRNAs for degradation or translational repression. Each miRNA is synthesized as a miRNA duplex comprising two strands (5p and 3p). However, only one of the two strands becomes active and is selectively incorporated into the RNA-induced silencing complex in a process known as miRNA strand selection. Recently, significant progress has been made in understanding the factors and processes involved in strand selection. Here, we explore the selection and functionality of the miRNA star strand (either 5p or 3p), which is generally present in the cell at low levels compared to its partner strand and, historically, has been thought to possess no biological activity. We also highlight the concepts of miRNA arm switching and miRNA isomerism. Finally, we offer insights into the impact of aberrant strand selection on immunity and cancer. Leading us through this journey is miR-155, a well-established regulator of immunity and cancer, and the increasing evidence that its 3p strand plays a role in these arenas. Interestingly, the miR-155-5p/-3p ratio appears to vary dependent on the timing of the immune response, and the 3p strand seems to play a regulatory role upon its partner 5p strand.

Interfering with the CCL2-glycosaminoglycan axis as a potential approach to modulate neuroinflammation.

Multiple Sclerosis, a chronic inflammatory demyelinating disease of the central nervous system, involves an increased expression of monocyte chemotactic protein 1 MCP1-/CCL2. For exerting its chemotactic effects, chemokine binding to glycosaminoglycans (GAGs) is required and therefore this interaction represents a potential target for therapeutic intervention. We have designed an anti-inflammatory decoy variant, Met-CCL2 (Y13A S21K Q23R), embodying increased affinity for GAGs as well as knocked-out GPCR activation properties. This non-signalling dominant-negative mutant is shown here to be able to displace wild type CCL2 from GAGs by which it is supposed to interfere with the chemokine-related inflammatory response. In vivo, the anti-inflammatory properties were successfully demonstrated in a murine model of zymosan-induced peritonitis as well as in an experimental autoimmune encephalomyelitis, a model relevant for multiple sclerosis, where the compound lead to significantly reduced clinical scores due to reduction of cellular infiltrates and demyelination in spinal cord and cerebellum. These findings indicate a promising potential for future therapeutic development.

Structure-based design of decoy chemokines as a way to explore the pharmacological potential of glycosaminoglycans.

Glycosaminoglycans (GAGs) are a class of highly negatively charged, unbranched, O-linked polysaccharides that are involved in many diseases. Their role as a protein-binding matrix on cell surfaces has long been recognized, but therapeutic approaches to interfere with protein-GAG interactions have been limited due to the complex chemistry of GAGs, on one hand, and due to the lack of specific antibodies against GAGs, on the other hand. We have developed a protein engineering platform (the so-called CellJammer(®) technology), which enables us to introduce higher GAG-binding affinity into wild-type GAG-binding proteins and to combine this with impaired biological, receptor-binding function. Chemokines are among the prototypic GAG-binding proteins and here we present selected results of our CellJammer technology applied to several of these proinflammatory proteins. An overview is given of our lead decoy protein, PA401, which is a CXCL8-based mutant protein with increased GAG-binding affinity and decreased CXCR1/2 binding and activation. Major results from our CCL2 and CCL5 programmes are also summarized and the potential for clinical application of these decoy proteins is presented.

A new monocyte chemotactic protein-1/chemokine CC motif ligand-2 competitor limiting neointima formation and myocardial ischemia/reperfusion injury in mice.

OBJECTIVES: A nonagonist monocyte chemotactic protein-1 (MCP-1/CCL2) mutant (PA508) with increased affinity for glycosaminoglycans and thus competing with CCL2 was evaluated as a candidate for preventing neointima formation or myocardial ischemia/reperfusion injury. BACKGROUND: Myocardial infarction (MI) remains a major cause of death worldwide despite improved interventional and therapeutic options. Therefore, the discovery of drugs that limit restenosis after intervention and post-MI damage remains an important challenge. METHODS: The function of PA508 was assessed in functional assays in vitro and in mouse models of wire-induced neointima formation and experimental MI. RESULTS: PA508 was functionally inactive in CC chemokine receptor 2 (CCR2) binding and calcium influx but inhibited monocyte chemotaxis or transendothelial migration toward CCL2, suggesting that it interferes with CCL2 presentation. In wild-type but not CCR2-deficient mice, PA508 reduced inflammatory leukocyte recruitment without affecting differential leukocyte counts, CCL2 levels, organ function, or morphology, indicating that it specifically attenuates the CCL2-CCR2 axis. Compared with vehicle, daily intraperitoneal injection of PA508 significantly (p < 0.05, n = 5) reduced neointimal plaque area and mononuclear cell infiltration in carotid arteries of hyperlipidemic apolipoprotein E-deficient mice while increasing smooth muscle cell content. In C57Bl/6J mice that underwent myocardial ischemia/reperfusion, treatment with PA508 significantly reduced infarction size, monocyte infiltration, and collagen and myofibroblast content in the infarction area and preserved heart function compared with vehicle (p < 0.05, n = 4 to 8). CONCLUSIONS: Here we demonstrate that administration of a rationally designed CCL2 competitor reduced inflammatory monocyte recruitment, limited neointimal hyperplasia, and attenuated myocardial ischemia/reperfusion injury in mice and could therefore be envisioned as a combined therapeutic approach for restenosis and MI.

Rationally evolving MCP-1/CCL2 into a decoy protein with potent anti-inflammatory activity in vivo.

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.

A proteomic snapshot of the human heat shock protein 90 interactome.

Heat shock protein 90 (Hsp90) is a molecular chaperone which modulates several signalling pathways within a cell. By applying co-immunoprecipitation with endogeneous Hsp90, we were able to identify 39 novel protein interaction partners of this chaperone in human embryonic kidney cells (HEK293). Interestingly, levels of DNA-activated protein kinase catalytic subunit, an Hsp90 interaction partner found in this study, were found to be sensitive to Hsp90 inhibitor treatment only in HeLa cells but not in HEK293 cells referring to the tumorgenicity of this chaperone.

Chiral separation of natural and unnatural amino acid derivatives by micro-HPLC on a Ristocetin A stationary phase.

This paper deals with the chiral separation of Fmoc- and Z-derivatives of natural and unnatural sulfur containing amino acids by micro-HPLC. The separations were carried out in microbore columns packed with a new material based on Ristocetin A bonded to 3.5 microm silica gel. The columns were run in the normal phase, polar organic mode as well as in the reversed phase mode, whereby best results were obtained with the reversed-phase mode using mixtures of triethylamine acetate (TEAA) buffer and methanol as mobile phases.