SUMMARY RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

Summary Receptor interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pro-necrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound; whereas, D161G, D143N, and K51A mutants only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3K51A/K51A) are viable and fertile, in stark contrast to the perinatal lethality of Rip3D161N/D161N mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Summary Apoptosis and necrosis are two major forms of cell death observed in normal and disease pathologies. Although there are many assays for detection of apoptosis, relatively few assays are available for measuring necrosis. A key signature for necrotic cells is the permeabilization of plasma membrane. This event can be quantified in tissue culture settings by measuring the release of the enzyme lactate dehydrogenase (LDH). When combined with other methods, measuring LDH release is a useful method for detection of necrosis. In this chapter, we describe the step-by-step procedure for detection of LDH release from necrotic cells using a microtiter plate based colorimetric absorbance assay.

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Abstract:The C57BL/6 mouse is the most well-known inbred mouse strain, and has been widely used as a genetic background for congenic and mutant mice. A number of C57BL/6 substrains have been derived from the C57BL/6 founder line and are reported to differ in several phenotypes. There are several major sources of C57BL/6 substrains for the biomedical research community. The importance of their genetic and phenotypic differences among substrains, however, has not yet been well recognized by biomedical researchers. Here, we report the result of screening of the functional deletion of the nicotinamide nucleotide transhydrogenase (Nnt) gene and 1,446 SNPs genotyping among seven C57BL/6 substrains from different sources, such as C57BL/6J, C57BL/6JJcl, C57BL/6JJmsSlc, C57BL/6NJcl, C57BL/6NCrlCrlj, C57BL/6NTac, and C57BL/6CrSlc. The deletion of exon 7-11 in the Nnt gene that was previously reported in C57BL/6J was also observed in other C57BL/6J substrains, indicating that this functional deletion probably occurred at an early stage in the establishment of C57BL/6J substrains. The genotyping of SNP loci clearly demonstrate genetic differences between C57BL/6J and C57BL/6N substrains at 11 loci. Besides, we found another SNP differing between C57BL/6J and other C57BL/6J substrains available from commercial breeders. No genetic difference was detected among C57BL/6N substrains. The C57BL/6CrSlc mouse, originally derived from the National Cancer Institute of the NIH was found to be the same as the C57BL/6N substrains by the SNP pattern. These data will be useful for accurate genetic monitoring of genetically engineered mice with the C57BL/6 background.

Cell proliferation and cell death are integral elements in maintaining homeostatic balance in metazoans. Disease pathologies ensue when these processes are disturbed. A plethora of evidence indicates that malfunction of cell death can lead to inflammation, autoimmunity or immuno-deficiency. Programmed necrosis or necroptosis is a form of non-apoptotic cell death driven by the receptor interacting protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL). RIPK3 partners with its upstream adaptors RIPK1, TRIF or DAI to signal for necroptosis in response to death receptor or toll-like receptor stimulation, pathogen infection, or sterile cell injury. Necroptosis promotes inflammation through leakage of cellular contents from damaged plasma membrane. Intriguingly, many of the signal adaptors of necroptosis have dual functions in innate immune signaling. This unique signature illustrates the cooperative nature of necroptosis and innate inflammatory signaling pathways in managing cell and organismal stresses from pathogen infection and sterile tissue injury.

The receptor-interacting protein kinase 3 (RIP3/RIPK3) has emerged as a critical regulator of programmed necrosis/necroptosis, an inflammatory form of cell death with important functions in pathogen-induced and sterile inflammation. RIP3 activation is tightly regulated by phosphorylation, ubiquitination, and caspase-mediated cleavage. These post-translational modifications coordinately regulate the assembly of a macromolecular signaling complex termed the necrosome. Recently, several reports indicate that RIP3 can promote inflammation independent of its pronecrotic activity. Here, we review our current understanding of the mechanisms that drive RIP3-dependent necrosis and its role in different inflammatory diseases.

Changes in oligosaccharide structures have been reported in certain types of malignant transformations and, thus, could be used for tumor markers in certain types of cancer. In the case of pancreatic cancer cell lines, a variety of fucosylated proteins are secreted into their conditioned media. To identify fucosylated proteins in the serum of patients with pancreatic cancer, we performed western blot analyses using Aleuria Aurantica Lectin (AAL), which is specific for fucosylated structures. An 40 kD protein was found to be highly fucosylated in pancreatic cancer and an N-terminal analysis revealed that it was the b chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer and colon cancer, the incidence was significantly higher in the case of pancreatic cancer. Fucosylated haptoglobin was observed more frequently at the advanced stage of pancreatic cancer and disappeared after an operation. A mass spectrometry analysis of haptoglobin purified from the serum of patients with pancreatic cancer and the medium from a pancreatic cancer cell line, PSN-1, showed that the a 1-3/a 1-4/a 1-6 fucosylation of haptoglobin was increased in pancreatic cancer. When a hepatoma cell line, Hep3B, was cultured with the conditioned media from pancreatic cancer cells, haptoglobin secretion was dramatically increased. These findings suggest that fucosylated haptoglobin could serve as a novel marker for pancreatic cancer. Two possibilities were considered in terms of the fucosylation of haptoglobin. One is that pancreatic cancer cells, themselves, produce fucosylated haptoglobin; the other is that pancreatic cancer produces a factor, which induces the production of fucosylated haptoglobin in the liver. ' 2005 Wiley-Liss, Inc.Key words: haptoglobin; pancreatic cancer; fucosylation; tumor marker; mass spectrometry; oligosaccharide; lectin; fucosyltransferase Pancreatic cancer is currently one of the leading causes of cancer-related deaths and the overall 5-year survival has been reported to be less than 5%.