No abstract

Human Monocyte Chemotactic Protein (MCP)-2 has originally been isolated from stimulated osteosarcoma cells as a chemokine coproduced with MCP-1 and MCP-3. Here, a 5'-end extended MCP-2 cDNA was cloned from a human testis cDNA library. It encoded a 76 residue MCP-2 protein, but differed from the reported bone marrow-derived MCP-2 cDNA sequence in codon 46, which coded for a Lys instead of a Gln. This MCP-2Lys46 variant, caused by a single nucleotide polymorphism (SNP), was biologically compared with MCP-2Gln46. The coding regions were subcloned into the bacterial expression vector pHEN1, and after transformation of Escherichia coli, the two MCP-2 protein variants were recovered from the periplasm. The recombinant proteins were purified to homogeneity by heparin-Sepharose affinity chromatography and reversed-phase HPLC. Edman degradation revealed a Gln residue at the NH2 terminus instead of a pGlu. To evaluate the influence of the cyclization, this Gln was chemically converted into pGlu in both MCP-2 variants. The conversion was confirmed by electrospray mass spectrometry. rMCP-2Gln46 and rMCP-2Lys46 and the NH2-terminal cyclic counterparts were tested on monocytic cells in calcium mobilization and chemotaxis assays. No significant difference in biological activity was observed between the rMCP-2Gln46 and rMCP-2Lys46 isoforms. However, for both MCP-2 variants the NH2-terminal pyroglutamate was shown to be essential for chemotaxis, but not for calcium mobilization. NH2-terminal truncation of rMCP-2Lys46 by the serine protease CD26/dipeptidyl peptidase IV (CD26/DPP IV) resulted in the cleavage of the NH2-terminal Gln-Pro dipeptide, whereas synthetic MCP-2 with an amino-terminal pGlu remained unaffected. CD26/DPP IV-clipped rMCP-2Lys46(3-76) was almost completely inactive in both chemotaxis and signaling assays. These observations indicate that the NH2-terminal pGlu in MCP-2 is necessary for chemotactic activity but also that it protects the protein against degradation by CD26/DPP IV.

It was previously shown that CD26

Populations often face changes in environmental conditions in a relatively short timescale, which may lead to microevolution of traits to cope with these changing selective pressures. Here, we demonstrate microevolution of a physiological trait in a natural population of the water flea Daphnia magna. Levels of the stress protein Hsp60 showed genetic variation, indicating in situ evolutionary potential, and the levels increased through time. The observed microevolutionary increase did not fit the historically documented changes in fish predation pressure in this pond, but it paralleled an increase in the load of infective stages of epibionts through time. In line with this, the locally most abundant epibiont caused an induction of Hsp60. Because stress proteins show evolutionary potential and protect organisms against a wide array of environmental factors, microevolution of stress proteins in natural populations is likely to be common.

SummaryLoss-of-function of ADAR1 causes the severe autoinflammatory disease Aicardi-Goutières Syndrome (AGS). ADAR1 converts adenosines into inosines within double-stranded (ds) RNA. This process called A-to-I editing masks self-dsRNA from detection by the antiviral dsRNA sensor MDA5. ADAR1 binds to dsRNA in both the canonical A-form and in the poorly defined Z-conformation (Z-RNA). Mutations in the Z-RNA binding Zα-domain of ADAR1 are common in AGS patients. How loss of ADAR1/Z-RNA interaction contributes to disease development is unknown. Using ADAR1 Zα-domain mutant human cells and knock-in mice, we demonstrate that abrogated binding of ADAR1 to Z-RNA leads to reduced A-to-I editing of dsRNA structures formed by pairing of inversely oriented SINEs. As a result, ADAR1 Zα-domain mutant human cells and transgenic mice develop a spontaneous MDA5-dependent immune response. This shows that the interaction between ADAR1 and Z-RNA restricts sensing of self-dsRNA and prevents AGS development.