Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase-or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a 'switch' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins. Mammalian cells can use a number of mechanisms to kill themselves. The best characterized depends on the Bcl-2 family members Bax and Bak that work via mitochondria to activate caspases. 1 Some caspases, notably caspase 8, can be activated independently of Bcl-2 family members, for example, after stimulation of members of the TNF receptor superfamily. 2 Recently, it has become apparent that some of these receptors, including TNFR1, can activate a third suicide mechanism that does not require caspases, and in which the morphology of the dying cell differs from classical apoptosis. This form of cell death, termed 'necroptosis', can often be blocked by necrostatin-1 (nec-1), an inhibitor of the kinase activity of receptor-interacting protein kinase 1 (RIPK1). 3,4 Accordingly, observations from several groups have shown that in some cell types, expression of RIPK1 can signal cell death by caspase-independent necroptosis. 5 It has previously been revealed that RIPK1 could function downstream of death receptors, but in those cases, cell death was usually blocked by coexpression of the viral inhibitor of caspases 1 and 8, CrmA, 6 and typically exhibited a classical 'apoptotic' morphology. It was revealed that RIPK1 engages FADD via homotypic binding of their death domains (DDs), and FADD in turn activates caspase 8. 6,7 RIPK3, like RIPK1, bears a kinase domain and RIP homology interaction motif (RHIM), but unlike RIPK1 does not have a DD. 8-11 RIPK3 is required for necroptosis. 12,13 Furthermore, RIPK1 appears to activate RIPK3 in this pathway, as cell death could be blocked by nec-1. 14 RIPK3 activates, by phosphorylation, MLKL, a pseudokinase essential for this death pathway. [15][16][17] Once activated, MLKL forms multimers that trigger breaches of the plasma membrane. [18][19][20] Although RIPK3 is necessary for necroptosis, it is unclear whether activation of RIPK3 is sufficient for cell death, because TNF activates signaling ...

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography. The protocol employs a rapid and simple strategy to isolate highly purified HRG that minimizes proteolytic cleavage of the protein. The purification of HRG was assessed at each stage by measuring the amount of HRG immunoreactive protein using a specific monoclonal antibody against total protein, and demonstrated $1,000-fold purification with an overall yield of $32%. Mass spectrometry analysis demonstrated that plasma-derived HRG was free of contaminating proteins and gel electrophoresis showed it to have minimal proteolytic degradation. Characterization of protein by physical method showed that the protein exists as a single, monodisperse species. In contrast to the previous studies of HRG purified by different methods, HRG purified using the new procedure demonstrated a reduced profile of functions. Although the HRG retained binding to heparin and phosphatidic acid, it did not interact with necrotic cells or other cellular lipids. These data demonstrate that HRG does not exhibit the broad interactive properties that have been reported previously, suggesting that copurification of HRGbinding partners or other impurities are responsible for some of the reported functional properties. The findings in this study demonstrate that the new purification procedure can provide a ready source of pure HRG to assess ligand specificity and biological function of this important plasma protein.