GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology

DiCesare, Sophia M.; Ortega, Antonio J.; Collier, Gracen E.; Daniel, Steffi; Thompson, Krista N.; McCoy, Melissa K.; Posner, Bruce A.; Hulleman, John D.

doi:10.1101/2023.12.14.571757

Cited by 2 publications

(2 citation statements)

References 90 publications

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“… 2 Compared to conventional reporters, such as green fluorescent protein (GFP), firefly luciferase, or even small full-length luciferases such as Gaussia luciferase, 3 NanoLuc has shown exceptional sensitivity and versatility, making it ideal for use in multiple biological studies. 4 , 5 , 6 , 7 …”

Section: Before You Beginmentioning

confidence: 99%

“…This method can be used in a variety of different cell lines and on a large number of target proteins. The ease and high sensitivity of the NanoBiT assay makes this method favorable for endogenous protein investigations and studies involving bioluminescence imaging, 10 protein-protein interaction studies, 8 monitoring protein degradation in real-time, 11 and high throughput screening (HTS), 5 such as compound and genetic screens. We have decided to use our HiBiT tagging of the cholesterol ester transfer protein (CETP), a protein associated with age-related macular degeneration (AMD) 12 , 13 , 14 , 15 and atherosclerosis, 16 in HepG2 cells as an example workflow for this method and the subsequent analysis/validation of CRISPR editing.…”

Section: Before You Beginmentioning

confidence: 99%

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Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing

Lankford,

Hulleman

2024

STAR Protocols

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Section: Before You Beginmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing

Lankford,

Hulleman

2024

STAR Protocols

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A spectrum of clinically-identified cysteine mutations in fibulin-3 (EFEMP1) highlight its disulfide bonding complexity and potential to induce stress response activation

Collier,

Hulleman

2024

Preprint

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Fibulin-3 (FBLN3), also known as EFEMP1, is a secreted extracellular matrix (ECM) glycoprotein that contains forty cysteine residues. These cysteines, which are distributed across one atypical and five canonical calcium-binding epidermal growth factor (EGF) domains, are important for regulating FBLN3 structure, secretion, and presumably function. As evidence of this importance, a rare homozygous p.C55R mutation in FBLN3 negates its function, alters disulfide bonding, and causes marfanoid syndrome. Additional studies suggest that heterozygous premature stop codon mutations in FBLN3 may also cause similar, albeit less severe, connective tissue disorders. Interestingly, a series of twenty-four cysteine mutations in FBLN3 have been identified in the human population and published in the Clinical Variation (ClinVar) and gnomAD databases. We tested how seven of these cysteine mutants (five loss-of-cysteine variants: C42Y, C190R, C218R, C252F, and C365S, two gain-of-cysteine variants: R358C, Y369C) and two newly developed mutations (G57C and Y397C) altered FBLN3 secretion, disulfide bonding, MMP2 zymography, and stress response activation Surprisingly, we found a wide variety of biochemical behaviors: i) loss-of-cysteine variants correlated with an increased likelihood of disulfide dimer formation, ii) N-terminal mutations were less likely to disrupt secretion, and were less prone to aggregation, iii) in contrast to wild-type FBLN3, multiple, but not all variants failed to induce MMP2 levels in cell culture, and iv) C-terminal mutations (either loss or gain of cysteines) were more prone to significant secretion defects, intracellular accumulation/misfolding, and stress response activation. These results provide molecular and biochemical insight into FBLN3 folding, secretion, and function for many cysteine mutations found in the human population, some of which may increase the likelihood of subclinical connective tissue or other FBLN3-associated haploinsufficiency diseases.

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GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology

Cited by 2 publications

References 90 publications

Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing

Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing

A spectrum of clinically-identified cysteine mutations in fibulin-3 (EFEMP1) highlight its disulfide bonding complexity and potential to induce stress response activation

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