Generation of locus-specific degradable tag knock-ins in mouse and human cell lines

Damhofer, Helene; Radzisheuskaya, Aliaksandra; Helin, Kristian

doi:10.1016/j.xpro.2021.100575

Cited by 6 publications

(4 citation statements)

References 9 publications

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“…Bioinformatic analysis revealed that HA-tagged both Z-WT and Z-G2A mutant proteins have five putative degrons [63].The functional degron region, which includes parts of the tripartite model and the tertiary structure necessary for E3 ligase engagement, is predicted to be located within 40 amino acids of the degron motif, whereas the HA tag was positioned more than 50 amino acids away from lysine 38 (K38) of the degron motif, supporting the exclusion of HA as an interfering factor. Moreover, experimental evidence supports that the HA tag does not interfere with C-terminal degrons in other proteins across different systems of eukaryotes [64][65][66][67], and we have shown that LCMV Z protein with a Cterminal HA tag is fully functional in cell-based assays virus [68]. Computational analysis of destabilizing N-terminal motifs indicated that in contrast to glycine, alanine at position 2 does not substantially contribute to protein destabilization [16].…”

Section: Discussionsupporting

confidence: 58%

Cellular N-myristoyl transferases Are Required for Mammarenavirus Multiplication

Witwit,

Betancourt,

Cubitt

et al. 2024

Preprint

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The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress, whereas heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect Z mediated virus budding and GP2 mediated fusion activity required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan NMT inhibitor DDD85464 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlated with reduced Z budding activity and GP2 mediated fusion activity, as well as proteasome mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic fever causing mammarenaviruses Junin (JUNV) and Lassa (LASV) viruses. Our results support exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.

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Section: Discussionsupporting

confidence: 58%

Cellular N-myristoyl transferases Are Required for Mammarenavirus Multiplication

Witwit,

Betancourt,

Cubitt

et al. 2024

Preprint

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“…The generation of the SMARCA5 knock‐in line involved methods as in Damhofer et al ( 2021 ). Briefly, the targeting construct was assembled from PCR products or synthetic DNA blocks (IDT) using the In‐Fusion cloning kit (Takara).…”

Section: Methodsmentioning

confidence: 99%

An alternative NURF complex sustains acute myeloid leukemia by regulating the accessibility of insulator regions

Radzisheuskaya,

Peña‐Rømer,

Lorenzini

et al. 2023

The EMBO Journal

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Efficient treatment of acute myeloid leukemia (AML) patients remains a challenge despite recent therapeutic advances. Here, using a CRISPRi screen targeting chromatin factors, we identified the nucleosome‐remodeling factor (NURF) subunit BPTF as an essential regulator of AML cell survival. We demonstrate that BPTF forms an alternative NURF chromatin remodeling complex with SMARCA5 and BAP18, which regulates the accessibility of a large set of insulator regions in leukemic cells. This ensures efficient CTCF binding and boundary formation between topologically associated domains that is essential for maintaining the leukemic transcriptional programs. We also demonstrate that the well‐studied PHD2‐BROMO chromatin reader domains of BPTF, while contributing to complex recruitment to chromatin, are dispensable for leukemic cell growth. Taken together, our results uncover how the alternative NURF complex contributes to leukemia and provide a rationale for its targeting in AML.

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“…The cell line was generated as previously described (Damhofer et al, 2021). For the donor vector, the BSD-P2A-2xHA-FKBP12 F36V and backbone pCRIS-PITCH cassettes were generated using PCR.…”

Section: Fkbp12 F36v Degradation Tag-nap1 Cell Line Generationmentioning

confidence: 99%

NAK-associated protein 1/NAP1 activates TBK1 to ensure accurate mitosis and cytokinesis

Paul,

Sarraf,

Nam

et al. 2023

Journal of Cell Biology

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Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1–TBK1 complex during cell division.

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Generation of locus-specific degradable tag knock-ins in mouse and human cell lines

Cited by 6 publications

References 9 publications

Cellular N-myristoyl transferases Are Required for Mammarenavirus Multiplication

Cellular N-myristoyl transferases Are Required for Mammarenavirus Multiplication

An alternative NURF complex sustains acute myeloid leukemia by regulating the accessibility of insulator regions

NAK-associated protein 1/NAP1 activates TBK1 to ensure accurate mitosis and cytokinesis

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