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PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-B. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian twohybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1␤. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-B and cytokine processing.

The PYRIN domain is a recently identified proteinprotein interaction domain that is found at the N terminus of several proteins thought to function in apoptotic and inflammatory signaling pathways. We report here that PYPAF1 (

PYRIN-containing Apaf-1-like proteins (PYPAFs) are a recently identi¢ed family of proteins thought to function in apoptotic and in£ammatory signaling pathways. PYPAF1 and PYPAF7 proteins have been found to assemble with the PYRIN^CARD protein ASC and coordinate the activation of NF-U UB and pro-caspase-1. To determine if other PYPAF family members function in pro-in£ammatory signaling pathways, we screened ¢ve other PYPAF proteins (PYPAF2, PYPAF3, PY-PAF4, PYPAF5 and PYPAF6) for their ability to activate NF-U UB and pro-caspase-1. Co-expression of PYPAF5 with ASC results in a synergistic activation of NF-U UB and the recruitment of PYPAF5 to punctate structures in the cytoplasm. The expression of PYPAF5 is highly restricted to granulocytes and T-cells, indicating a role for this protein in in£ammatory signaling. In contrast, PYPAF2, PYPAF3, PYPAF4 and PYPAF6 failed to colocalize with ASC and activate NF-U UB. PYPAF5 also synergistically activated caspase-1-dependent cytokine processing when co-expressed with ASC. These ¢ndings suggest that PYPAF5 functions in immune cells to coordinate the transduction of pro-in£ammatory signals to the activation of NF-U UB and pro-caspase-1.

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Many structurally and functionally diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors, and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE; human testis isozyme) or one of four juxtamembrane (stalk) mutants containing either deletions of 17, 24, and 47 residues (ACE-JM delta 17, -JM delta 24, and -JM delta 47, respectively) or a substitution of 26 stalk residues with a 20-residue sequence from the stalk of the low-density lipoprotein receptor (ACE-JMLDL). The C termini of released, soluble WT-ACE and ACE-JM delta 17 and -JMLDL were determined by MALDI-TOF mass spectrometry analyses of C-terminal peptides generated by CNBr cleavage. Observed masses of 4264 (WT-ACE) and 4269 (ACE-JM delta 17) are in good agreement with an expected mass of 4262 for the C-terminal CNBr peptide ending at Arg-627, indicating cleavage at the Arg-627/Ser-628 bond in both WT-ACE and ACE-JM delta 17, at distances of 24 and 10 residues from the membrane, respectively. Data for ACE-JM delta 24 are also consistent with cleavage at or near Arg-627. For ACE-JMLDL, in which the native cleavage site is absent, observed masses of 4372 and 4542 are in close agreement with expected masses of 4371 and 4542 for peptides ending at Ala-628 and Gly-630, respectively, indicating cleavages at 17 or 15 residues from the membrane. These data indicate that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, cleavage at a distance of 10 residues from the membrane is more favorable, as ACE-JM delta 17 is cleaved 12-fold faster than WT-ACE. In contrast, ACE-JM delta 24 is released 17-fold slower, suggesting that a minimum distance from the membrane must be preserved. This is supported by results with the ACE-JM delta 47 mutant, which is membrane-bound but not cleaved, likely because the entire stalk has been deleted. Finally, soluble full-length (anchor-plus) WT-ACE is not cleaved when incubated with various CHO cell fractions or intact CHO cells. On the basis of these and other data, we propose that the CHO cell MPSP that solubilizes ACE (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain; and (4) may have a weak preference for cleavage at Arg/Lys-X bonds.

Targeted therapies and immunotherapy have changed the face of multiple solid malignancies, including metastatic melanoma and lung cancer, but no such therapies exist for pancreatic ductal adenocarcinoma (PDAC) despite the knowledge of key mutations and an increasing understanding of the tumor microenvironment. Until now, most clinical studies have not been biomarker driven in this highly immunosuppressive and heterogeneous cancer. Ongoing basic and translational studies are better classifying the disease in hopes of identifying critical pathways that distinguish the unique PDAC subtypes, which will lead to personalized therapies. In this review, we discuss the current treatment options for metastatic pancreatic cancer and highlight current ongoing clinical trials, which aim to target the stroma and the immune microenvironment either alone or in combination with standard chemotherapy. Identifying biomarkers and key resistance pathways and targeting these pathways in a personalized manner in combination with chemotherapy are likely to yield a more immediate and durable clinical benefit.