Bigenzahn, J. W. | bioRxiv Science

bioRxiv2025

Genetic identification of the RAS proteostatic machinery and its failure to regulate oncogenic variants

The regulation of cellular homeostasis, differentiation, and proliferation is safeguarded by proteostatic mechanisms, which are crucial for maintaining cellular function. We used endogenously affinity-tagged KRAS cells in a fluorescence-activated cell sorting (FACS)-based CRISPR knockout screen to map genome-wide genetic requirements for proteostatic KRAS regulation. Three regulatory modules emerged, specifically cullin E3 ligase activity (CUL3, NAE1, UBE2M, CAND1, and UBE2L3), LZTR1 protein function (LZTR1, NUDCD3, and ZRSR2), and RAS GTPase modulation and processing (NRAS, HRAS, FNTB, RCE1, ICMT, and GOLGA7), all critical for regulating KRAS abundance. This expands our knowledge on the machinery controlling ubiquitin-meditated RAS family member regulation. Combining endogenous affinity tagging with genetic variant introduction, we found that the oncogenic KRAS G12D mutant exhibits reduced regulation by CRL3LZTR1, potentially contributing to oncogenic transformation and proliferation. In summary, our study genetically defines the machinery regulating RAS GTPase protein abundance, providing a foundation for a deeper molecular understanding and potential therapeutic exploitation of CRL3LZTR1-RAS GTPase regulation in human disease.

cell biology↗

bioRxiv2018

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

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 TNF 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.

cell biology↗