Specificity for signaling by cAMP-dependent protein kinase (PKA) is achieved by both targeting and isoform diversity. The inactive PKA holoenzyme has two catalytic (C) subunits and a regulatory (R) subunit dimer (R ₂ :C ₂ ). Although the RIα, RIIα, and RIIβ isoforms are well studied, little is known about RIβ. We show here that RIβ is enriched selectively in mitochondria and hypothesized that its unique biological importance and functional nonredundancy will correlate with its structure. Small-angle X-ray scattering showed that the overall shape of RIβ ₂ :C ₂ is different from its closest homolog, RIα ₂ :C ₂ . The full-length RIβ ₂ :C ₂ crystal structure allows us to visualize all the domains of the PKA holoenzyme complex and shows how isoform-specific assembly of holoenzyme complexes can create distinct quaternary structures even though the R ₁ :C ₁ heterodimers are similar in all isoforms. The creation of discrete isoform-specific PKA holoenzyme signaling “foci” paves the way for exploring further biological roles of PKA RIβ and establishes a paradigm for PKA signaling.

Serine oligopeptidases of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. We report here the isolation and characterization of oligopeptidase B (OpdB) and its corresponding gene from Trypanosoma evansi, a pathogen of significant veterinary importance. The T. evansi opdB gene was present as a single copy per haploid genome containing an open reading frame of 2148 bp encoding a protein of 80.664 kDa. Purified OpdB hydrolyzed substrates with basic residues in P₁ (k_cat/K_m for carbobenzyloxy-l-arginyl-l-arginyl-7-amido-4-methylcoumarin, 337 s^–1·μm^–1) and exhibited potent arginyl carboxypeptidase activity (k_cat/K_m for Val-Lys-Arg↓Arg-OH, 231 s^–1·mm^–1). While not secreted, T. evansi released OpdB into the plasma of infected hosts where it retained catalytic activity. Plasma OpdB levels correlated with blood parasitemia. In vitro, OpdB cleaved the peptide hormone atrial natriuretic factor (ANF) at four sites: Arg³↓Arg⁴, Arg⁴↓Ser⁵, Arg¹¹↓Ile¹², and Arg²⁷↓Tyr²⁸, thereby abrogating smooth muscle relaxant and prohypotensive properties of ANF. Circulating plasma ANF levels in T. evansi-infected rats were depressed from 130 to 8 pg·ml^–1, and plasma ANF levels inversely correlated with plasma OpdB activity. The in vitro half-life of ANF in rat plasma was reduced 300-fold in plasma from T. evansi-infected rodents, which contains high levels of OpdB activity. Addition of OpdB inhibitors to cell-free plasma from infected rodents significantly abrogated this ANF hydrolysis. Furthermore the in vivo ANF half-life was reduced 5-fold in T. evansi-infected rats. Thus, we propose a role for OpdB in peptide hormone dysregulation in trypanosomiasis, specifically in generating the depressed plasma levels of ANF in mammals infected with T. evansi.

There is currently no “gold standard” test for the diagnosis of late-stage Chagas' disease. As a result, protection of the blood supply in areas where Chagas' disease is endemic remains problematic. A panel of 709 serum samples from subjects with confirmed Chagas' disease ( n = 195), healthy controls ( n = 400), and patients with other parasitic diseases ( n = 114) was used to assess enzyme-linked immunosorbent assays (ELISAs) based on a concentrated extract of excretory-secretory antigens from either Brazil or Tulahuen strain Trypanosoma cruzi trypomastigotes (total trypomastigote excretory-secretory antigens [TESAs]). The total TESA-based assays had excellent overall sensitivity (100%) and specificity (>94%), except for cross-reactivity with Leishmania -infected sera. In an attempt to increase the specificity of the assay, immunoaffinity chromatography was used to purify the TESA proteins (TESA _IA proteins). By Western blotting, a series of polypeptide bands with molecular masses ranging from 60 to 220 kDa were recognized by pooled sera positive for Chagas' disease. An ELISA based on TESA _IA proteins had a slightly lower sensitivity (98.6%) but an improved specificity (100%) compared to the sensitivity and specificity of the total TESA protein-based ELISAs. A 60-kDa polypeptide was identified as a major contributor to the cross-reactivity with Leishmania . These data suggest the need for field validation studies of TESA- and TESA _IA -based assays in regions where Chagas' disease is endemic.