Affinity Purification of NF1 Protein–Protein Interactors Identifies Keratins and Neurofibromin Itself as Binding Partners

Carnes, Rachel M.; Kesterson, Robert A.; Korf, Bruce R.; Mobley, James A.; Wallis, Deeann

doi:10.3390/genes10090650

Cited by 14 publications

(11 citation statements)

References 62 publications

(91 reference statements)

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“…Hence, this region (exons 18-21) may be critical and should not be targeted for exon skipping. These data are compatible with the recent finding that neurofibromin forms a dimer/oligomer (24,25). While regions critical for dimerization have not yet been reported, it is possible that they overlap with these critical exons.…”

Section: Discussionsupporting

confidence: 92%

Exon Skipping as a Therapeutic for Neurofibromatosis Type I

Wallis¹,

Leier²,

Moore³

et al. 2021

Preprint

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We investigated the feasibility of utilizing an exon skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which NF1 exons might be skipped while maintaining neurofibromin function. Human neurofibromin is well-known as a GTPase activating protein (GAP), but outside of its GAP-related domain (GRD), it is unclear how critical other regions are for function. Initial in silico analysis predicted exons that can be skipped with minimal loss of neurofibromin function. Utilizing a novel Nf1 cDNA system, we performed a functional screen to determine the effects of exon skipping on in vitro neurofibromin expression and GRD function. Loss of single exons 12, 17, 25, 41, 47, or 52 maintained significant GRD function in at least two Ras activity assays. Exons 18/19, 20 and 28 are critical for GRD function; deletion of exons 20, 41, or 47 led to significantly lower levels of neurofibromin. As suggested by in silico analysis, skipping of exons 17 or 52 resulted in both the highest neurofibromin levels and the greatest suppression of Ras activity. Assessment of NF1 patient databases indicates that pathogenic variants resulting in deletion or skipping of exons 17, 25, and 52 have not been reported; and truncating pathogenic variants in each exon account for ~0.91, 0.94, and 0.25% of unrelated NF1 cases, respectively. Hence, we designed antisense phosphodiamitate morpholino oligos (PMOs) to skip exon 17 and evaluated them in human cell lines that we generated via CRISPR/Cas9 with a patient-specific truncating pathogenic variant, c.1885G>A. We down-selected oligos that efficiently caused skipping of exon 17 and restored NF1 expression and function. Further, homozygous deletion of exon 17 in a novel mouse model is compatible with viable and grossly healthy animals with normal lifespan and no tumor development, providing proof-of-concept that exon 17 is not essential for murine neurofibromin function. Mild phenotypes observed include abnormal nesting behavior and lymphoid hyperplasia with increased numbers of both B- and T-cells. Hence, exon skipping should be further investigated as a therapeutic approach for NF1 patients with treatment of individuals with pathogenic variants in exon 17.

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Section: Discussionsupporting

confidence: 92%

Exon Skipping as a Therapeutic for Neurofibromatosis Type I

Wallis¹,

Leier²,

Moore³

et al. 2021

Preprint

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“…A published report noted that human neurofibromin eluted from size-exclusion chromatography during purification as an apparent dimer (22), and other studies have proposed that multimerization could potentially explain dominant-negative effects of heterozygous truncation mutations of NF1 in patients (23). A recent paper published during the preparation of this manuscript demonstrated that tagged murine neurofibromin could co-IP human neurofibromin in HEK293 cell lines, suggesting that these highly-homologous proteins from the two species (Ͼ98% identity) could form dimers as well (24).…”

Section: Discussionmentioning

confidence: 76%

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

et al. 2019

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Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

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“…An additional complexity to NF1 interactions is its dimerization activity (Figure 1, left) (Carnes, Kesterson, Korf, Mobley, & Wallis, 2019;Sherekar et al, 2019). While the mechanistic significance has yet to be determined, dimerization provides a potential explanation for the phenotypes observed with many NF1 disease variants.…”

Section: Introductionmentioning

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

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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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Affinity Purification of NF1 Protein–Protein Interactors Identifies Keratins and Neurofibromin Itself as Binding Partners

Cited by 14 publications

References 62 publications

Exon Skipping as a Therapeutic for Neurofibromatosis Type I

Exon Skipping as a Therapeutic for Neurofibromatosis Type I

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

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

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