Structural and Functional Insights into Host Death Domains Inactivation by the Bacterial Arginine GlcNAcyltransferase Effector.

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.

Identification and analysis of dominant negative mutants of RIP1 DD that disrupt RIPoptosome core formation.

The RIPoptosome, composed of RIP1 and caspase-8, plays an important role in the regulation of apoptosis and necroptosis; however, the mechanism of complex formation by oligomerization and how the caspase-activating process and necroptosis are mediated by the formation of the RIPoptosome is not well-understood. This study revealed that the assembly mechanism of the RIPoptosome core is dependent on salt concentration and not on pH and time. In addition, we demonstrated that three RIP1 mutations, E626K, M637K, and S657K, have dominant negative effects. These dominant negative mutations in RIP1 may have potential applications in therapeutic intervention.

Enhanced UV Resistance Role of Death Domain-Associated Protein in Human MDA-MB-231 Breast Cancer Cells by Regulation of G2 DNA Damage Checkpoint.

PURPOSE: Death domain-associated protein (DAXX) is a multifunctional nuclear protein involved in apoptosis, transcription, deoxyribonucleic acid damage response, and tumorigenesis. However, the role of DAXX in breast cancer development and progression remains elusive. In this study, we examined the expression patterns and function of DAXX in human breast cancer samples and cell lines. METHODS: Immunohistochemistry was used to analyze the expression and localization patterns of DAXX. Additionally, we investigated whether DAXX played an intrinsic role in the cellular response to damage induced by ultraviolet (UV) irradiation in MDA-MB-231 breast cancer cells (isolated at M D Anderson from a pleural effusion of a patient with invasive ductal carcinoma). RESULTS: Our results showed that nucleus size, chromatin organization, and DAXX localization were altered in breast cancer tissues compared with those in control tissues. Compared with cytoplasmic and nuclear expression in benign breast tissues, DAXX was colocalized with promyelocytic leukemia in nuclei with a granular distribution. Endogenous DAXX messenger ribonucleic acid levels were upregulated upon UV radiation in MDA-MB-231 cells. DAXX-deficient cells tended to be more sensitive to irradiation than control cells. Conversely, DAXX-overexpressing cells exhibited reduced phosphorylated histone H2AX (γ-H2AX) accumulation, increased cell survival, and resistance to UV-induced damage. The protective effects of DAXX may be related to the activation of the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (ATM-CHK2)-cell division cycle 25c (CDC25c) signaling pathways in Gap2/Mitosis (G2/M) checkpoint and ultimately cell cycle arrest at G2/M phase. CONCLUSIONS: Taken together, these results suggested that DAXX may be an essential component in breast cancer initiation, malignant progression, and radioresistance.

Panel Sequencing Shows Recurrent Genetic FAS Alterations in Primary Cutaneous Marginal Zone Lymphoma.

Primary cutaneous marginal zone lymphoma (PCMZL) represents an indolent subtype of non-Hodgkin lymphoma that is clinically characterized by slowly growing skin tumors with a very low propensity for systemic dissemination. The underlying genetic basis of PCMZL has not been comprehensively elucidated. To gain deeper insight into the molecular pathogenesis of PCMZL, we performed hybridization-based panel sequencing of 38 patients with well-characterized PCMZL. In 32 of the 38 patients, we identified genetic alterations within 39 selected target genes. The most frequently detected alterations (24/38 patients, 63.2%) affected the FAS gene, of which 22 patients harbored alterations, which affect the functionally relevant death domain of the apoptosis-regulating FAS/CD95 protein in a dominant-negative manner. In addition, we identified highly recurrent mutations in three other genes, namely SLAMF1, SPEN, and NCOR2. Our molecular data suggest that apoptosis defects provide the molecular basis of the observed clinical features of PCMZL, which commonly presents with only slowly growing skin tumors, reflecting its invariably indolent behavior. From a diagnostic point of view, highly recurrent FAS mutations in PCMZL presumably separate this indolent lymphoma entity from pseudolymphoma, and this adds adjunctive discriminatory features at a molecular level.

PPARγ Ligand-induced Annexin A1 Expression Determines Chemotherapy Response via Deubiquitination of Death Domain Kinase RIP in Triple-negative Breast Cancers.

Metastatic breast cancer is still incurable so far; new specifically targeted and more effective therapies for triple-negative breast cancer (TNBC) are required in the clinic. In this study, our clinical data have established that basal and claudin-low subtypes of breast cancer (TNBC types) express significantly higher levels of Annexin A1 (ANXA1) with poor survival outcomes. Using human cancer cell lines that model the TNBC subtype, we observed a strong positive correlation between expression of ANXA1 and PPARγ. A similar correlation between these two markers was also established in our clinical breast cancer patients' specimens. To establish a link between these two markers in TNBC, we show de novo expression of ANXA1 is induced by activation of PPARγ both in vitro and in vivo and it has a predictive value in determining chemosensitivity to PPARγ ligands. Mechanistically, we show for the first time PPARγ-induced ANXA1 protein directly interacts with receptor interacting protein-1 (RIP1), promoting its deubiquitination and thereby activating the caspase-8-dependent death pathway. We further identified this underlying mechanism also involved a PPARγ-induced ANXA1-dependent autoubiquitination of cIAP1, the direct E3 ligase of RIP1, shifting cIAP1 toward proteosomal degradation. Collectively, our study provides first insight for the suitability of using drug-induced expression of ANXA1 as a new player in RIP1-induced death machinery in TNBCs, presenting itself both as an inclusion criterion for patient selection and surrogate marker for drug response in future PPARγ chemotherapy trials. Mol Cancer Ther; 16(11); 2528-42. ©2017 AACR.