Significance Understanding loci nominated by genome-wide association studies (GWASs) is challenging. Here, we show, using the specific example of Parkinson disease, that identification of protein–protein interactions can help determine the most likely candidate for several GWAS loci. This result illustrates a significant general principle that will likely apply across multiple diseases.

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization.

Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNF␣ stimulation. In cells from TRADD-deficient mice, TNF␣-mediated apoptosis and TNF␣-stimulated NF-B, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNF␣-mediated inflammatory responses in vivo. TRADD deficiency does not enhance IFN␥-induced signaling. Importantly, TRADD has a novel role in TLR3 and TLR4 signaling. TRADD participates in the TLR4 complex formed upon LPS stimulation, and TRADD-deficient macrophages show impaired cytokine production in response to TLR ligands in vitro. Thus, TRADD is a multifunctional protein crucial both for TNFR1 signaling and other signaling pathways relevant to immune responses. TNF ͉ innate immunityT umor necrosis factor alpha (TNF␣) is a pleiotropic cytokine involved in a broad range of biological activities, including inflammation and cell differentiation, survival, and death (1). TNF␣ mediates these activities by engaging two distinct cell surface receptors: TNFR1 and TNFR2. Activation of TNFR1 leads to the recruitment of the intracellular death domain (DD)-containing adaptor TNFR-associated DD protein (TRADD) through a homotypic interaction with DD of TNFR1. On one hand, the recruitment of TRADD can promote the association of the TNFR1 complex with Fas-associated DD (FADD), which induces caspase activation and cell death. On the other hand, TRADD can also recruit receptor-interacting protein kinase 1 (RIP1) and TNFR-associated factor-2 (TRAF2), which trigger NF-B activation, leading to cell survival and proinflammatory responses (1).TRADD was originally identified in a yeast two-hybrid screen performed to identify TNFR1-interacting proteins (2). Intriguingly, TRADD is the first adaptor protein identified that binds directly to the DD of TNFR1 but transduces signals resulting in either apoptosis or NF-B activation (2-4). Indeed, TRADD overexpression in 293T cells activates both apoptotic and cellsurvival signaling pathways (2). In addition to TNFR1, TRADD may mediate signaling downstream of the death receptors (and TNFR superfamily members) DR3 and DR6 (5, 6). As well, recent TRADD knockdown studies have indicated that TRADD may be involved in signaling mediated by receptors unrelated to the TNFR superfamily, such as in IFN␥ receptor (7-9). These findings give tantalizing hints of the potential breadth of TRADD functions.To date, a knockout animal model has yet to be reported for TRADD, even though this important adaptor was identified more than ten years ago. In this study, we generate TRADD knockout mice by using conditional gene targeting and show that not only is TRADD indispensable for TNF␣-induced NF-B activation and apoptosis in vitro and TNF␣-induced inflammatory responses in vivo, but also that this molecule is involved in germinal center (GC) formation, T cell costimulation, and TLR signaling. Our TRADD knockout mice represent a very useful t...