Canonical G proteins are heterotrimeric, consisting of ␣, ␤, and ␥ subunits. Despite multiple G␣ subunits functioning in fungi, only a single G␤ subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the G␣ subunit Gpa1 that functions in cAMP signaling as bait in a twohybrid screen, we have identified a novel G␤-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed ␤ propeller structure characteristic of ␤ transducins. Gib2 is also shown to interact, respectively, with two G␥ subunit homologs, Gpg1 and Gpg2, similar to the conventional G␤ subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it is present in the plasma and at sites of inflammation. At physiological pH, native pentameric CRP exhibits calcium-dependent binding specificity for phosphocholine. In this study, we determined the binding specificities of CRP at acidic pH, a characteristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, IgG, amyloid ␤, and BSA immobilized on microtiter plates. At pH 7.0, CRP did not bind to any of these proteins, but, at pH ranging from 5.2 to 4.6, CRP bound to all six proteins. Acidic pH did not monomerize CRP but modified the pentameric structure, as determined by gel filtration, 1-anilinonaphthalene-8-sulfonic acid-binding fluorescence, and phosphocholine-binding assays. Some modifications in CRP were reversible at pH 7.0, for example, the phosphocholine-binding activity of CRP, which was reduced at acidic pH, was restored after pH neutralization. For efficient binding of acidic pHtreated CRP to immobilized proteins, it was necessary that the immobilized proteins, except factor H, were also exposed to acidic pH. Because immobilization of proteins on microtiter plates and exposure of immobilized proteins to acidic pH alter the conformation of immobilized proteins, our findings suggest that conformationally altered proteins form a CRP-ligand in acidic environment, regardless of the identity of the protein. This ligand binding specificity of CRP in its acidic pH-induced pentameric state has implications for toxic conditions involving protein misfolding in acidic environments and favors the conservation of CRP throughout evolution.

The severe combined immunodeficiency disorder (SCID)-beige/albumin (Alb)-urokinase plasminogen activator (uPA) mouse containing a human-mouse chimeric liver is currently the only small animal model capable of supporting hepatitis C virus (HCV) infection. This model was utilized to characterize the host transcriptional response to HCV infection. The purpose of these studies was to investigate the genetic component of the host response to HCV infection and also to distinguish virus-induced gene expression changes from adaptive HCV-specific immune-mediated effects. Gene expression profiles from HCV-infected mice were also compared to those from HCV-infected patients. Analyses of the gene expression data demonstrate that host factors regulate the response to HCV infection, including the nature of the innate antiviral immune response. They also indicate that HCV mediates gene expression changes, including regulation of lipid metabolism genes, which have the potential to be directly cytopathic, indicating that liver pathology may not be exclusively mediated by HCV-specific adaptive immune responses. This effect appears to be inversely related to the activation of the innate antiviral immune response. In summary, the nature of the initial interferon response to HCV infection may determine the extent of viral-mediated effects on host gene expression.

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Perturbation of pheromone signaling modulates not only mating but also virulence in Cryptococcus neoformans, an opportunistic human pathogen known to encode three G␣, one G␤, and two G␥ subunit proteins. We have found that G␣s Gpa2 and Gpa3 exhibit shared and distinct roles in regulating pheromone responses and mating. Gpa2 interacted with the pheromone receptor homolog Ste3␣, G␤ subunit Gpb1, and RGS protein Crg1. Crg1 also exhibited in vitro GAP activity toward Gpa2. These findings suggest that Gpa2 regulates mating through a conserved signaling mechanism. Moreover, we found that G␥s Gpg1 and Gpg2 both regulate pheromone responses and mating. gpg1 mutants were attenuated in mating, and gpg2 mutants were sterile. Finally, although gpa2, gpa3, gpg1, gpg2, and gpg1 gpg2 mutants were fully virulent, gpa2 gpa3 mutants were attenuated for virulence in a murine model. Our study reveals a conserved but distinct signaling mechanism by two G␣, one G␤, and two G␥ proteins for pheromone responses, mating, and virulence in Cryptococcus neoformans, and it also reiterates that the link between mating and virulence is not due to mating per se but rather to certain mating-pathway components that encode additional functions promoting virulence. INTRODUCTIONCryptococcus neoformans is an encapsulated yeast-like fungus capable of infecting both immunocompromised and healthy individuals to cause life-threatening meningoencephalitis (Mitchell and Perfect, 1995;Chayakulkeeree and Perfect, 2006). The organism belongs to basidiomycota taxonomically and has a defined life cycle and a bipolar mating system with MAT␣ being the predominant mating type in both environmental and clinical settings (Hull and Heitman, 2002;Wang and Fox, 2005;Nielsen and Heitman, 2007). Like other fungi and higher eukaryotes, heterotrimeric G protein-mediated signaling pathways are central for C. neoformans to sense environmental-and host-imposed cues and to respond through regulation of developmental processes such as mating and haploid differentiation, as well as the production of several virulence factors including melanin and capsule (Alspaugh et al., 1998;Wang and Fox, 2005).The lower eukaryotic organisms such as fungi also encode multiple G␣ subunits, similar to higher eukaryotic organisms such as animals and plants . For example, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe encode two G␣ subunits, the plant pathogen Ustilago maydis encodes four G␣ subunits, and many other fungal species contain three G␣ subunits. The S. cerevisiae G␣ Gpa1 is a negative regulator of the pheromone-responsive mating pathway (Whiteway et al., 1989;Whiteway et al., 1990;Clark et al., 1993;Kü bler et al., 1997;Colombo et al., 1998;Thevelein and Winde, 1999), whereas G␣ Gpa2 positively controls a glucose-sensing cAMP pathway for pseudohyphal differentiation and invasive growth (Kü bler et al., 1997;Lorenz and Heitman, 1997). Distinct, but largely similar, signaling pathways mediated by two G␣ subunits were also found in S. pombe (Sti...