Efficient peptide recovery from secreted recombinant MHC-I molecules expressed via mRNA transfection.

Most current methods for identifying peptides that are bound to a distinct MHC-I product in a given cell sample utilize detergent solubilization of membrane proteins followed by immunoaffinity purification. Since detergent traces and cell debris hamper subsequent peptide analysis, exceedingly large cell samples are often required. To avoid the use of detergents, truncated MHC-I heavy chains have recently been expressed by stable DNA transfection or retroviral transduction, resulting in the secretion of soluble MHC-I complexes to the growth medium. The electroporation of in vitro-transcribed mRNA achieves remarkable efficacy and uniformity of gene expression in numerous cell types, exhibiting exceedingly fast kinetics. We reasoned that mRNA transfection offers a simple, fast and widely applicable alternative to current gene delivery protocols for expressing secreted MHC-I products in cells of interest. To test this assumption we used mRNA to express soluble derivatives of HLA-A2 in the human AF10 B cell myeloma and 624mel melanoma and H-2K(d) in the mouse SP2/0 B cell myeloma. The level of MHC-I complexes secreted by these cells peaked within less than 24h post-transfection and they could be affinity-purified directly from the culture medium in considerably greater yields when compared to nonionic detergent lysates on a cell-to-cell basis. Mass-spectrometry analysis of eluted peptides revealed larger pools in the secreted material than in lysates with substantial overlap in composition. Our results introduce mRNA transfection as a powerful tool for determining the cell's MHC-I peptidome, which can be potentially applied to a broad range of cell types.

Parkin promotes degradation of the mitochondrial pro-apoptotic ARTS protein.

Parkinson's disease (PD) is associated with excessive cell death causing selective loss of dopaminergic neurons. Dysfunction of the Ubiquitin Proteasome System (UPS) is associated with the pathophysiology of PD. Mutations in Parkin which impair its E3-ligase activity play a major role in the pathogenesis of inherited PD. ARTS (Sept4_i2) is a mitochondrial protein, which initiates caspase activation upstream of cytochrome c release in the mitochondrial apoptotic pathway. Here we show that Parkin serves as an E3-ubiquitin ligase to restrict the levels of ARTS through UPS-mediated degradation. Though Parkin binds equally to ARTS and Sept4_i1 (H5/PNUTL2), the non-apoptotic splice variant of Sept4, Parkin ubiquitinates and degrades only ARTS. Thus, the effect of Parkin on ARTS is specific and probably related to its pro-apoptotic function. High levels of ARTS are sufficient to promote apoptosis in cultured neuronal cells, and rat brains treated with 6-OHDA reveal high levels of ARTS. However, over-expression of Parkin can protect cells from ARTS-induced apoptosis. Furthermore, Parkin loss-of-function experiments reveal that reduction of Parkin causes increased levels of ARTS and apoptosis. We propose that in brain cells in which the E3-ligase activity of Parkin is compromised, ARTS levels increase and facilitate apoptosis. Thus, ARTS is a novel substrate of Parkin. These observations link Parkin directly to a pro-apoptotic protein and reveal a novel connection between Parkin, apoptosis, and PD.

Peptides mimicking the unique ARTS-XIAP binding site promote apoptotic cell death in cultured cancer cells.

PURPOSE: XIAP [X-linked inhibitor of apoptosis (IAP) protein] is the best characterized mammalian caspase inhibitor. XIAP is frequently overexpressed in a variety of human tumors, and genetic inactivation of XIAP in mice protects against lymphoma. Therefore, XIAP is an attractive target for anticancer therapy. IAP antagonists based on a conserved IAP-binding motif (IBM), often referred to as "Smac-mimetics," are currently being evaluated for cancer therapy in the clinic. ARTS (Sept4_i2) is a mitochondrial proapoptotic protein which promotes apoptosis by directly binding and inhibiting XIAP via a mechanism that is distinct from all other known IAP antagonists. Here, we investigated the ability of peptides derived from ARTS to antagonize XIAP and promote apoptosis in cancer cell lines. EXPERIMENTAL DESIGN: The ability of synthetic peptides, derived from the C-terminus of ARTS, to bind to XIAP, stimulate XIAP degradation, and induce apoptosis was examined. We compared the response of several cancer cell lines to different ARTS-derived peptides. Pull-down assays were used to examine binding to XIAP, and apoptosis was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, caspase activation, and Western blot analyses of caspase substrates. RESULTS: The C-terminus of ARTS contains a unique sequence, termed ARTS-IBM (AIBM), which is important for binding to XIAP and cell killing. AIBM peptides can bind to XIAP-BIR3, penetrate cancer cells, reduce XIAP levels, and promote apoptosis. CONCLUSIONS: Short synthetic peptides derived from the C-terminus of ARTS are sufficient for binding to XIAP and can induce apoptosis in cancer cells. These results provide proof-of-concept for the feasibility of developing ARTS-based anticancer therapeutics.