Construction of cloning-friendly minigenes for mammalian expression of full-length human NF1 isoforms

Cui, Yan; Morrison, Helen

doi:10.1002/humu.23681

Cited by 10 publications

(6 citation statements)

References 21 publications

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“…Assay Performance: We see similar dynamic ranges between the GTP-Ras and pERK/ERK experiments, with WT cDNA able to repress both GTP-Ras and pERK/ERK ratios by about half that seen with EV control. The Morrison lab reported that inactive variants (R1276P) served as better controls than empty vectors (Cui & Morrison, 2019). While our R1276Q and R1276X both displayed similar GTP-Ras levels as our EV, both showed insignificantly higher pERK/ERK ratios.…”

Section: Assay Validation With Cdna Titrationsmentioning

confidence: 54%

“…This is partially due to the size of the gene and toxicity of the human cDNA construct. We are aware of three potentially availableNF1 cDNAs: mouse Nf1 (Wallis et al, 2018) (isoform 2 with 2839 amino acids), codon optimized human NF1 (Bonneau, Lenherr, Pena, Hart, & Scheffzek, 2009) (isoform 1 with 2818 aa), and a humanNF1 with mini-intron 35-36 (Cui & Morrison, 2019) (isoforms 1 and 2). As the mNf1 cDNA is highly homologous to endogenous humanNF1 , we have developed and validated the mouse Nf1 cDNA expression system that allows us to examine the biochemical effects of any Nf1 genetic variant.…”

Section: Assay Validation With Cdna Titrationsmentioning

confidence: 99%

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Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine

Long

Liu

et al. 2021

Preprint

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We have created a panel of twenty-nine NF1 variant cDNAs representing benign missense (MS) variants, pathogenic MS variants, many with clinically relevant phenotypes, in-frame deletions, splice variants, and nonsense (NS) variants. We have determined the functional consequences of the variants, assessing their ability to produce mature neurofibromin and restore Ras signaling activity in NF1 null (-/-) cells. cDNAs demonstrate variant-specific differences in neurofibromin protein levels, suggesting that some variants lead to protein instability or enhanced degradation. When expressed at high levels, some variant proteins are still able to repress Ras activity, indicating that the NF1 phenotype may be due to protein instability. In contrast, other variant proteins are incapable of repressing Ras activity, indicating that some do not functionally engage Ras and stimulate GTP-ase activity. We observed that stability and Ras activity can be mutually exclusive. These assays allow us to categorize variants by functional effects, may help to classify variants of unknown significance, and may have future implications for more directed therapeutics.

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Section: Assay Validation With Cdna Titrationsmentioning

confidence: 54%

Section: Assay Validation With Cdna Titrationsmentioning

confidence: 99%

Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine

Long

Liu

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…Recently, different solutions have been proposed to circumvent this problem. Wallis et al (2018) [163] developed and validated a mouse NF1 cDNA expression system, Cui and Morrison (2019) [164] published experimental conditions to clone the full length NF1 containing a mini-intron to eliminate its toxicity, and Sherekar et al (2020) [43] developed a codon-optimized human NF1 cDNA that is stable and non-toxic in E. coli and human cells. These constructs will be of great help in studying the entire neurofibromin protein instead of separate domains.…”

Section: Discussionmentioning

confidence: 99%

Neurofibromin Structure, Functions and Regulation

Bergoug

Doudeau

Godin

et al. 2020

Cells

102

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Neurofibromin is a large and multifunctional protein encoded by the tumor suppressor gene NF1, mutations of which cause the tumor predisposition syndrome neurofibromatosis type 1 (NF1). Over the last three decades, studies of neurofibromin structure, interacting partners, and functions have shown that it is involved in several cell signaling pathways, including the Ras/MAPK, Akt/mTOR, ROCK/LIMK/cofilin, and cAMP/PKA pathways, and regulates many fundamental cellular processes, such as proliferation and migration, cytoskeletal dynamics, neurite outgrowth, dendritic-spine density, and dopamine levels. The crystallographic structure has been resolved for two of its functional domains, GRD (GAP-related (GTPase-activating protein) domain) and SecPH, and its post-translational modifications studied, showing it to be localized to several cell compartments. These findings have been of particular interest in the identification of many therapeutic targets and in the proposal of various therapeutic strategies to treat the symptoms of NF1. In this review, we provide an overview of the literature on neurofibromin structure, function, interactions, and regulation and highlight the relationships between them.

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“…Currently, the crystal structure of NF1-LRD is not available due to its poor stability even with the used of codon-optimized constructs [71]. However, this issue may be overcome with the use of the cloning-friendly NF1 mini-genes by the Morrison’s group [72], which may also aid in identification of the potential interaction partner(s).…”

Section: Discussionmentioning

confidence: 99%

Pathogenic mutations in neurofibromin identifies a leucine-rich domain regulating glioma cell invasiveness

et al. 2019

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Glioblastoma (GBM) is the most aggressive tumor of the brain. NF1, a tumor suppressor gene and RAS-GTPase, is one of the highly mutated genes in GBM. Dysregulated NF1 expression promotes cell invasion, proliferation, and tumorigenesis. Loss of NF1 expression in glioblastoma is associated with increased aggressiveness of the tumor. Here, we show that NF1-loss in patient-derived glioma cells using shRNA increases self-renewal, heightens cell invasion, and promotes mesenchymal subtype and epithelial mesenchymal transition-specific gene expression that enhances tumorigenesis. The neurofibromin protein contains at least four major domains, with the GAP-related domain being the most well-studied. In this study, we report that the leucine-rich domain (LRD) of neurofibromin inhibits invasion of human glioblastoma cells without affecting their proliferation. Moreover, under conditions tested, the NF1-LRD fails to hydrolyze Ras-GTP to Ras-GDP, suggesting that its suppressive function is independent of Ras signaling. We further demonstrate that rare variants within the NF1-LRD domain found in a subset of the patients are pathogenic and reduce NF1-LRD’s invasion suppressive function. Taken together, our results show, for the first time, that NF1-LRD inhibits glioma invasion, and provides evidence of a previously unrecognized function of NF1-LRD in glioma biology.

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Construction of cloning-friendly minigenes for mammalian expression of full-length human NF1 isoforms

Cited by 10 publications

References 21 publications

Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine

Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine

Neurofibromin Structure, Functions and Regulation

Pathogenic mutations in neurofibromin identifies a leucine-rich domain regulating glioma cell invasiveness

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