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The function of viral protein 2 (VP2) of the immunosuppressive circovirus chicken anemia virus (CAV)has not yet been established. We show that the CAV VP2 amino acid sequence has some similarity to a number of eukaryotic, receptor, protein-tyrosine phosphatase (PTPase) ␣ proteins as well as to a cluster of human TT viruses within the Sanban group. To investigate if CAV VP2 functions as a PTPase, purified glutathione S-transferase (GST)-VP2 fusion protein was assayed for PTPase activity using the generalized peptide substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine), with free phosphate detected using the malachite green colorimetric assay. CAV GST-VP2 was shown to catalyze dephosphorylation of both substrates. CAV GST-VP2 PTPase activity for the ENDpYINASL substrate had a V max of 14,925 units/ mg⅐min and a K m of 18.88 M. Optimal activity was observed between pH 6 and 7, and activity was specifically inhibited by 0.01 mM orthovanadate. We also show that the ORF2 sequence of the CAV-related human virus TT-like minivirus (TLMV) possessed PTPase activity and steady state kinetics equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To establish whether these viral proteins were dual specificity protein phosphatases, the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were also assayed for serine/ threonine phosphatase (S/T PPase) activity using the generalized peptide substrate RRApTVA, with free phosphate detected using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV GST-ORF2 fusion proteins possessed S/T PPase activity, which was specifically inhibited by 50 mM sodium fluoride. CAV GST-VP2 exhibited S/T PPase activity with a V max of 28,600 units/mg⅐min and a K m of 76 M. Mutagenesis of residue Cys 95 to serine in CAV GST-VP2 abrogated both PTPase and S/T PPase activity, identifying it as the catalytic cysteine within the proposed signature motif. These studies thus show that the circoviruses CAV and TLMV encode dual specificity protein phosphatases (DSP) with an unusual signature motif that may play a role in intracellular signaling during viral replication. This is the first DSP gene to be identified in a small viral genome.

Abstract-Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling.In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT 1 ) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT 1A receptor (AT 1 R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA A receptor, as a binding partner for the AT 1 R. Interaction of GABARAP with the AT 1 R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT 1 R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT 1 R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT 1 R in PC-12 cells. Cotransfection of GABARAP with an AT 1 R fluorescent fusion protein increased PC-12 cell surface expression of the AT 1 R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT 1 R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT 1 R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT 1 R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT 1 R to the plasma membrane. receptors are 7-transmembrane G protein-coupled receptors (GPCRs) of the largest GPCR subfamily, family 1, or the rhodopsin-like family. The GPCR superfamily has more than 860 members 1 and more than 50 "GPCR-associated" proteins have now been discovered, the majority of which interact with GPCR cytoplasmic carboxyl termini. 2 Most of these are involved in trafficking, subcellular targeting, and intracellular signaling. Our preliminary studies were designed to identify proteins that bind to the cytoplasmic carboxyl terminus of the AT 1 receptor (AT 1 R), the most prevalent and best characterized of the Ang receptors. Such proteins are expected to be involved in trafficking of the AT 1 R through the secretory pathway and to the plasma membrane, as well as in ligand-mediated internalization and recycling. Moreover, our recent published studies suggest that the AT 1 R is cleaved in a ligand-dependent manner to liberate the cytoplasmic domain, a significant quantity of which traffics to the nucleus. 3 Presumably, this nuclear trafficking event also involves sequence-specific binding proteins. Using a yeast 2-hybrid (Y2H) approach to screen a mouse brain library, we have ide...

Background Rapid-response extracorporeal membrane oxygenation (RR-ECMO) has been implemented at select centers to expedite cannulation for patients placed on ECMO during cardiopulmonary resuscitation (ECPR). In 2008, we established such a program and used it for all pediatric veno-arterial ECMO initiations. This study was designed to compare outcomes before and after program implementation. Methods Between 2003 and 2011, 144 pediatric patients were placed on veno-arterial ECMO. Records of patients placed on ECMO before (17 ECPR and 62 non-ECPR) or after (14 ECPR and 51 non-ECPR) RR-ECMO program implementation were retrospectively compared. Results The peak performance of the ECMO team was assessed by measuring ECMO initiation times for the ECPR patient subgroup (n=31). There was a shift towards more ECPR initiations achieved in under 40 minutes (24% pre-RR-ECMO vs. 43% RR-ECMO, P=0.25) and fewer requiring more than 60 minutes (47% pre-RR-ECMO vs. 21% RR-ECMO, P=0.14) after program implementation, although these changes did not reach statistical significance. After multivariable risk-adjustment, RR-ECMO was associated with a 52% reduction in neurologic complications for all patients (adjusted odds ratio, 0.48; confidence interval, 0.23–0.98; P=0.04), but the risk of in-hospital death remained unchanged (adjusted odds ratio, 0.99; confidence interval, 0.50–1.99; P=0.99). Conclusion Implementation of a pediatric RR-ECMO program for veno-arterial ECMO initiation was associated with reduced neurologic complications but not improved survival during the first three years of program implementation. These data suggest that development of a coordinated system for rapid ECMO deployment may benefit both ECPR and non-ECPR patients, but further efforts are required to improve survival.

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