Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking

Abstract

Regulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic technologies creates the opportunity to functionally map these essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss-and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor trafficking and ER homeostasis. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~ 400 SLC genes, for TNFR1-and FAS-but not TRAIL-R1-mediated responses. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9 synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1 (TNFAIP3-interacting protein 1), which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain-and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting investigation of their individual contribution and potential role in pathological conditions. not peer-reviewed)

Epistasis-driven identification of SLC25A51 as a regulator of human mitochondrial NAD import

“…Despite the high degree of functional redundancy displayed by transporters 46 , we show here that informative patterns of genetic interactions can be systematically derived for this family, allowing the generation of testable hypotheses for the de-orphanization of poorly characterized SLCs. Of all approaches that we have used in the past, including proteomics 47,48 , drug resistance 16,49 , and cell biological readouts 50,51 , genetic interactions among SLC has the potential to be one of the most powerful, as data points contribute to construct the evidence. As in a puzzle with a limited set of pieces, if all components are evaluated, individual genes may become implicated by exclusion principles.…”

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

“…ZIP7 has recently been reported to play a key role in zinc mobilization from the lumen of the ER (29)(30)(31), and the zinctransport activity of ZIP7 has further been reported to be facilitated by its phosphorylation by CK2 (37). To examine ZIP7 involvement in Tnap activation, we ectopically expressed WT ZIP7 or mutant ZIP7, either the phosphoablative form or the phosphomimetic active form (ZIP7S275A/S276A or ZIP7S275D/S276D) (37,43), in zip9 -/-zip13 -/cells ( Fig.…”

“…In comparison to the elucidation of the importance of ZNT proteins in zinc ectoenzyme activation, the involvement of another family of zinc transporter proteins, Zrt/Irt-like proteins (ZIPs), in the activation remains less well studied (20,21). However, certain ZIPs have recently been clearly shown to play several pivotal roles in early secretory pathway functions (28)(29)(30)(31). Specifically, ZIP7, ZIP9, and ZIP13 participate in zinc mobilization from the luminal side to the cytosol and contribute to the homeostatic maintenance of early secretory pathway functions, including those of the ER and Golgi apparatus (29,30,(32)(33)(34), in addition to performing crucial functions in zinc signaling by regulating signal transduction pathways in response to various stimuli (35)(36)(37)(38).…”

A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs

“…Finally, a set of 120 non-targeting sgRNAs was included by generating random 20-mers and selecting for sequences with at least three (for the strong PAM NGG) or two (for the PAM NAG) mismatches from any genomic sequence with E-CRISP Evaluation 67 . Adapter sequences were added to Of note, the library generated as described here, has been already successfully used in focused screens for phagocytosis 68 , necroptosis 69 and cell survival upon viral infection 70 .…”