Human ribonuclease 4 (RNase 4) is the most evolutionarily conserved member of the 8 canonical human pancreatic-like RNases showing more than 90% identity with bovine and porcine homologues. which have been found in the pancreas (Weickmann et al. 1981 in urine and kidney (Cranston et al. 1980 Iwama et al. 1981 Mizuta et al. 1990 in blood serum (Rabin and Tattrie 1982 in the placenta (Zhang et al. 2002 in the liver (Zhang et al. 2003 and in seminal plasma (De Prisco et al. 1984 to name a few. While all pancreatic-like human members conserve varying degrees of ribonucleolytic activities and a high structural similarity each member appears to have also acquired additional biological functions (Sorrentino 2010 Boix et al. 2013 These alternative roles include antibacterial antipathogenic cytotoxic and neurotoxic activities (Durack et al. 1981 angiogenesis (Strydom et al. 1985 and immunosuppressive activity (Bystrom et al. 2011 It is believed that all members are secretory proteins although it was recently suggested that RNase 8 might act through a different mechanism in light of the hydrophilic nature of its amino terminal extension (Chan et al. 2012 RNase 4 is the shortest member among human RNase A representatives with a primary structure of 119 residues. It is also the most evolutionarily conserved Danoprevir (RG7227) orthologue among mammalian species showing ~90% identity with bovine porcine and rat RNases (Fig. 1) (Zhou and Strydom 1993 Because of the high degree of conservation among mammalian species it is hypothesized that RNase 4 plays a crucial biological function. Although this role remains elusive RNase 4 has been linked to cytotoxicity in carcinoma cell lines together with the induction of cellular migration and the proliferation of human umbilical vein endothelial cells (Di Liddo et al. 2010 Fig. 1 Sequence alignment of RNase 4 members found among vertebrates. Alignment was performed with T-Coffee (Notredame et al. 2000 using the following orthologues: (human)(chimpanzee)(cattle)(wild boar) … RNase 4 retains all the important catalytic residues found in members of the RNase A superfamily (His12 His116 and Lys40 Fig. 2) in addition to 8 strictly conserved cysteine residues involved in the formation of 4 disulfide bridges. It Danoprevir (RG7227) contains a unique deletion site Danoprevir (RG7227) of two residues (residues 77-78 RNase 1 numbering) and no motif for expression and subcloned into a BL21(DE3) and protein expression was induced with 1 mM IPTG for 4 hours in 1H/13C/15N-labeled M9 minimal medium supplemented with non-essential Danoprevir (RG7227) amino acids (Invitrogen Grand Island NY US) metals and ammonium acetate. Cells were lysed by sonication and inclusion bodies were recovered by centrifugation. Proteins were refolded using a combination of oxidized/reduced glutathione for 72 hours as described (Doucet et al. 2009 RNase 4 was purified on HiTrapQ HP and MonoS ion exchange columns using an ?KTA Purifier (GE Healthcare Piscataway NJ US). The final purity was estimated by SDS-PAGE and a total of 12.8 mg of >98% pure RNase 4 was obtained from 2 L of cell culture. Solution NMR spectroscopy NMR samples were prepared in 15 mM sodium acetate and pH 5.0. The protein was quantified by UV-Vis spectrophotometry using a theoretical extinction coefficient of 7082 M?1cm?1. All NMR experiments were recorded at 298 K on samples containing 0.5-0.7 mM 1H/13C/15N-labeled RNase 4 in 15 mM sodium acetate at pH 5.0 with 10% 2H2O in 5-mm Shigemi NMR tubes filled with 350 μL of Danoprevir (RG7227) protein solution. NMR experiments were carried out on Agilent 500 CETP MHz and 800 MHz NMR spectrometers equipped with triple-resonance cold probes and pulsed-field gradients. For backbone resonance assignments of wild-type human RNase 4 2 HSQC 3 TOCSY-HSQC and 3D NOESY-HSQC were collected and sequence-specific assignments of the backbone atoms were achieved by running a series of independent CBCA(CO)NH HNCACB and HNCO 3D experiments. All heteronuclear NMR experiments were processed by NMRPipe and analyzed using Sparky and NMRView. Evolutionary conservation of amino acid positions in RNase 4 The evolutionary conservation of amino acid positions based on the phylogenetic relations between homologous sequences was performed using the.