Equilibrium between monomers and dimers of the death domain of the p75 neurotrophin receptor in solution.

p75 neurotrophin receptor (p75NTR) contains a C-terminal globular protein module known as the death domain (DD), which plays a central role in apoptotic and inflammatory signaling through the formation of oligomeric protein complexes. A monomeric state of the p75NTR-DD also exists depending on its chemical environment in vitro. However, studies on the oligomeric states of the p75NTR-DD have produced conflicting findings and sparked great controversy. Here we present new evidence from biophysical and biochemical studies to demonstrate the coexistence of symmetric and asymmetric dimers of the p75NTR-DD, which may equilibrate with the monomeric form in solution and in the absence of any other protein. The reversible close-open solution behavior may be important for the p75NTR-DD to serve as an intracellular signaling hub. This result supports an intrinsic ability of the p75NTR-DD to self-associate, in congruence with the oligomerization properties of all members of the DD superfamily.

Bacterial death and TRADD-N domains help define novel apoptosis and immunity mechanisms shared by prokaryotes and metazoans.

Several homologous domains are shared by eukaryotic immunity and programmed cell-death systems and poorly understood bacterial proteins. Recent studies show these to be components of a network of highly regulated systems connecting apoptotic processes to counter-invader immunity, in prokaryotes with a multicellular habit. However, the provenance of key adaptor domains, namely those of the Death-like and TRADD-N superfamilies, a quintessential feature of metazoan apoptotic systems, remained murky. Here, we use sensitive sequence analysis and comparative genomics methods to identify unambiguous bacterial homologs of the Death-like and TRADD-N superfamilies. We show the former to have arisen as part of a radiation of effector-associated α-helical adaptor domains that likely mediate homotypic interactions bringing together diverse effector and signaling domains in predicted bacterial apoptosis- and counter-invader systems. Similarly, we show that the TRADD-N domain defines a key, widespread signaling bridge that links effector deployment to invader-sensing in multicellular bacterial and metazoan counter-invader systems. TRADD-N domains are expanded in aggregating marine invertebrates and point to distinctive diversifying immune strategies probably directed both at RNA and retroviruses and cellular pathogens that might infect such communities. These TRADD-N and Death-like domains helped identify several new bacterial and metazoan counter-invader systems featuring underappreciated, common functional principles: the use of intracellular invader-sensing lectin-like (NPCBM and FGS), transcription elongation GreA/B-C, glycosyltransferase-4 family, inactive NTPase (serving as nucleic acid receptors), and invader-sensing GTPase switch domains. Finally, these findings point to the possibility of multicellular bacteria-stem metazoan symbiosis in the emergence of the immune/apoptotic systems of the latter.

A comprehensive interaction study provides a potential domain interaction network of human death domain superfamily proteins.

Human death domain superfamily proteins (DDSPs) play important roles in many signaling pathways involved in cell death and inflammation. Disruption or constitutive activation of these DDSP interactions due to inherited gene mutations is closely related to immunodeficiency and/or autoinflammatory diseases; however, responsible gene mutations have not been found in phenotypical diagnosis of these diseases. In this study, we comprehensively investigated the interactions of death-fold domains to explore the signaling network mediated by human DDSPs. We obtained 116 domains of DDSPs and conducted a domain-domain interaction assay of 13,924 reactions in duplicate using amplified luminescent proximity homogeneous assay. The data were mostly consistent with previously reported interactions. We also found new possible interactions, including an interaction between the caspase recruitment domain (CARD) of CARD10 and the tandem CARD-CARD domain of NOD2, which was confirmed by reciprocal co-immunoprecipitation. This study enables prediction of the interaction network of human DDSPs, sheds light on pathogenic mechanisms, and will facilitate identification of drug targets for treatment of immunodeficiency and autoinflammatory diseases.

Progesterone induces apoptosis by activation of caspase-8 and calcitriol via activation of caspase-9 pathways in ovarian and endometrial cancer cells in vitro.

Previously we have shown inhibition of endometrial cancer cell growth with progesterone and calcitriol. However, the mechanisms by which the two agents attenuate proliferation have not been well characterized yet. Herein, we investigated how progesterone and calcitriol induce apoptosis in cancer cells. DNA fragmentation was upregulated by progesterone and calcitriol in ovarian and endometrial cancer cells. Time-dependent treatment of ovarian cancer cells, ES-2, and TOV-21G with progesterone enhanced caspase -8 activity after 12 h, whereas OV-90, TOV-112D, HEC-1A, and HEC-59 cells showed increased activity after 24 h. Caspase 9 activity was increased in all cell lines after 24 h treatment with calcitriol. Pretreatment of cancer cells with a caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (Z-LEHD-fmk) significantly attenuated progesterone and calcitriol induced caspase-8 and caspase-9 expression, respectively. The expression of FasL, Fas, FAD, and pro-caspase-8, which constitute the death-inducing signaling complex (DISC), was upregulated in progesterone treated cancer cells. Knockdown of FAS or FADD with specific siRNAs significantly blocked progesterone-induced caspase-8. Cleavage of the BID was not affected by caspase-8 activation suggesting the absence of cross-talk between caspase-8 and caspase-9 pathways. Calcitriol treatment decreased mitochondrial membrane potential and increased the release of cancer cytochrome C. These findings indicate that progesterone induces apoptosis through activation of caspase-8 and calcitriol through caspase-9 activation in cancer cells. A combination of progesterone-calcitriol activates both extrinsic and intrinsic apoptotic pathways in cancer cells.

Domain swapping of death domain superfamily: Alternative strategy for dimerization.

The role of death domain (DD) protein-mediated inter-protein interactions in cell death and immune cell signaling have been extensively investigated as they are tentative targets for therapeutic interventions and are involved in signal transduction. Structural studies, especially those involving the recent advanced cryo-electron microscopy, indicated that the DD superfamily proteins can assemble into different forms of oligomers, including homo and heterodimer, honey comb-like circular oligomer, and helical filament via three types of interactions, namely type I, type II, and type III. Recently, several structural reports indicated that domain swapping-mediated dimerization of the DD superfamily proteins might be an alternative oligomerization strategy in this family of protein interacting domains. In this review, all the binding strategies associated with the DD superfamily are summarized with a special focus on the novel domain swapping mechanism.

High-Level Prokaryotic Expression and Purification of Death Domain Superfamily with MBP Tag.

BACKGROUND: Death domain superfamily are compact structural motifs that play critical roles in many biological processes including inflammation and apoptosis. Due to mediating protein-protein interactions, these death domains have a high tendency to form oligomers and are notoriously difficult to deal with when overexpressed in vitro. In this study, we found that maltose binding protein (MBP) is very effective in improving the behavior of many death domain superfamily members. METHODS: In order to achieve high-level expression of death domain superfamily in E. coli, we designed two MBPtagged expression vectors based on a pET30a backbone: one with a short flexible noncleavable linker, the other with a TEV cleavage site. Soluble protein was purified from cell lysate by HisTrapTM IMAC column and Superdex-200 gel filtration column. RESULTS: We tested seven targets of death domain superfamily, including two PYDs, three CARDs, and two DDs; for all of these, it is challenging to obtain recombinant protein without a tag. We expressed and purified these death domain proteins successfully as soluble and highly purified protein with both vectors. Among them, two proteins were crystallized successfully. CONCLUSIONS: Our study demonstrated that both recombinant MBP expression vectors significantly enhanced production. In addition, MBP tagged recombinant proteins can sometimes produce crystals. This strategy may be applied to other challenging proteins.

Structure of the C-terminal domain of TRADD reveals a novel fold in the death domain superfamily.

The TNFR1-associated death domain protein (TRADD) is an intracellular adaptor protein involved in various signaling pathways, such as antiapoptosis. Its C-terminal death domain (DD) is responsible for binding other DD-containing proteins including the p75 neurotrophin receptor (p75NTR). Here we present a solution structure of TRADD DD derived from high-resolution NMR spectroscopy. The TRADD DD comprises two super-secondary structures, an all-helix Greek key motif and a ß-hairpin motif flanked by two α helices, which make it unique among all known DD structures. The ß-hairpin motif is essential for TRADD DD to fold into a functional globular domain. The highly-charged surface suggests a critical role of electrostatic interactions in TRADD DD-mediated signaling. This novel structure represents a new class within the DD superfamily and provides a structural basis for studying homotypic DD interactions. NMR titration revealed a direct weak interaction between TRADD DD and p75NTR DD monomers. A binding site next to the p75NTR DD homodimerization interface indicates that TRADD DD recruitment to p75NTR requires separation of the p75NTR DD homodimer, explaining the mechanism of NGF-dependent activation of p75NTR-TRADD-mediated antiapoptotic pathway in breast cancer cell.