Simultaneous Control of Endogenous and User-Defined Genetic Pathways Using Unique ecDHFR Pharmacological Chaperones

Ramadurgum, Prerana; Woodard, Da Nae R.; Daniel, Steffi; Peng, Hui; Mallipeddi, Prema L.; Niederstrasser, Hanspeter; Mihelakis, Melina; Chau, Viet Q.; Douglas, Peter M.; Posner, Bruce A.; Hulleman, John D.

doi:10.1016/j.chembiol.2020.03.006

Cited by 13 publications

(38 citation statements)

References 53 publications

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“…While TMP is the canonical stabilizer for the ecDHFR-DD, we have found that non-antibiotic TMP derivatives ( Peng et al, 2019 ) or other compounds containing the 2,4-diaminopyrimidine (or triazine) ring with an aryl group ( Ramadurgum et al, 2020 ) can also promote ecDHFR-DD-POI abundance, and thus be used to conditionally control cellular signaling. Interestingly, the chemical space that can stabilize the ecDHFR-DD appears to be rather vast.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Based on our work thus far, such endogenous pathways include, but are not limited to cFMS kinase inhibition, PI3Kγ/δ inhibition, P2X 3 and P2X 2/3 , ENaC, and pharmacological chaperoning of other proteins. As a starting point, one can begin using already validated compounds described in ( Ramadurgum et al, 2020 ), or they could source from the list of virtually identified compounds (Table S1 in Ramadurgum et al, 2020 ) most of which were not validated. (see Figure 6 for example stabilizers) Proceed with performing dose-response curves and downstream evaluation as described above in “ Evaluating ecDHFR-DD-POI Basal Abundance and Dynamic Range in Newly Generated Stable Cell Lines “.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…While these are the most characterized DDs, in theory, one could generate a DD from a destabilized form of any protein that retains an ability to bind a stabilizing ligand, thereby promoting protein abundance. Nonetheless, due to its extensive use in the literature both in vitro ( Shoulders et al, 2013a , Shoulders et al, 2013b ) and in vivo ( Datta et al, 2018 , Peng et al, 2019 , Ramadurgum et al, 2020 ), we have focused on optimization and use of the ecDHFR-DD.…”

Section: Before You Beginmentioning

confidence: 99%

“…(see Figures 1 and 2 for example data). Looking ahead, for validation in vivo , researchers should design a plan for introducing their ecDHFR-DD-POI into vectors that are more suitable for long term, stable expression, such as the use of an adeno-associated virus (AAV) vectors containing a truncated chicken beta actin promoter (smCBA, ( Ramadurgum et al, 2020 )), or targeting vectors that could be used for the generation of knock-in mice at the ROSA26 or other loci. The user should also consider the size of their fusion protein, promoter, and reporter genes before choosing an expression vector.…”

Section: Before You Beginmentioning

confidence: 99%

“…Here, we describe a platform for achieving these goals. For complete details on the use and execution of this protocol, please refer to Ramadurgum et al (2020) .…”

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Protocol for Designing Small-Molecule-Regulated Destabilizing Domains for In Vitro Use

Ramadurgum

Hulleman

2020

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SUMMARY The use of destabilizing domains (DDs) to conditionally control the abundance of a protein of interest (POI) through a small-molecule stabilizer has gained increasing traction both in vitro and in vivo . Yet there are specific considerations for the development and accurate control of user-defined POIs via DDs, as well as the identification of novel (and potentially synergistic) small-molecule stabilizers. Here, we describe a platform for achieving these goals. For complete details on the use and execution of this protocol, please refer to Ramadurgum et al. (2020) .

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Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

“…Here, we describe a platform for achieving these goals. For complete details on the use and execution of this protocol, please refer to Ramadurgum et al (2020) .…”

mentioning

confidence: 99%

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Protocol for Designing Small-Molecule-Regulated Destabilizing Domains for In Vitro Use

Ramadurgum

Hulleman

2020

STAR Protocols

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A loss‐of‐function cysteine mutant in fibulin‐3 (EFEMP1) forms aberrant extracellular disulfide‐linked homodimers and alters extracellular matrix composition

et al. 2022

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Fibulin-3 (F3 or EFEMP1) is a disulfide-rich, secreted glycoprotein necessary for maintaining extracellular matrix (ECM) and connective tissue integrity. Two studies have identified distinct autosomal recessive F3 mutations in individuals with Marfan Syndrome-like phenotypes. Herein we characterized how one of these mutations, c.163T>C; p.Cys55Arg (C55R), disrupts F3 secretion, quaternary structure, and function by forming unique extracellular disulfide-linked homodimers. Dual cysteine mutants suggest that the C55R-induced disulfide species forms because of new availability of Cys70 on adjacent F3 monomers. Surprisingly, mutation of single cysteines located near C55 (i.e., C29, C42, C48, C61, C70, C159, and C171) also produced similar extracellular disulfide-linked dimers, suggesting that this is not a phenomenon isolated to the C55R mutant. To assess C55R functionality, F3 knockout (KO) retinal pigmented epithelial (RPE) were generated, followed by reintroduction of wild-type (WT) or C55R F3. F3 KO cells produced lower levels of the ECM remodeling enzyme, matrix metalloproteinase 2 and reduced formation of collagen VI ECM filaments, both of which were partially rescued by WT F3 overexpression. However, C55R F3 was unable to compensate for these same ECM-related defects. Our results highlight the unique behavior of particular cysteine mutations in F3 and uncover potential routes to restore C55R F3 loss-of-function.

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Fibulin-3 knockout mice demonstrate corneal dysfunction but maintain normal retinal integrity

et al. 2020

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Fibulin-3 (F3) is an extracellular matrix glycoprotein found in basement membranes across the body. An autosomal dominant R345W mutation in F3 causes a macular dystrophy resembling dry age-related macular degeneration (AMD), whereas genetic removal of wild-type (WT) F3 protects mice from sub-retinal pigment epithelium (RPE) deposit formation. These observations suggest that F3 is a protein which can regulate pathogenic sub-RPE deposit formation in the eye. Yet the precise role of WT F3 within the eye is still largely unknown. We found that F3 is expressed throughout the mouse eye (cornea, trabecular meshwork (TM) ring, neural retina, RPE/choroid, and optic nerve). We next performed a thorough structural and functional characterization of each of these tissues in WT and homozygous (F3−/−) knockout mice. The corneal stroma in F3−/− mice progressively thins beginning at 2 months, and the development of corneal opacity and vascularization starts at 9 months, which worsens with age. However, in all other tissues (TM, neural retina, RPE, and optic nerve), gross structural anatomy and functionality were similar across WT and F3−/− mice when evaluated using SD-OCT, histological analyses, electron microscopy, scotopic electroretinogram, optokinetic response, and axonal anterograde transport. The lack of noticeable retinal abnormalities in F3−/− mice was confirmed in a human patient with biallelic loss-of-function mutations in F3. These data suggest that (i) F3 is important for maintaining the structural integrity of the cornea, (ii) absence of F3 does not affect the structure or function of any other ocular tissue in which it is expressed, and (iii) targeted silencing of F3 in the retina and/or RPE will likely be well-tolerated, serving as a safe therapeutic strategy for reducing sub-RPE deposit formation in disease.

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Simultaneous Control of Endogenous and User-Defined Genetic Pathways Using Unique ecDHFR Pharmacological Chaperones

Cited by 13 publications

References 53 publications

Protocol for Designing Small-Molecule-Regulated Destabilizing Domains for In Vitro Use

Protocol for Designing Small-Molecule-Regulated Destabilizing Domains for In Vitro Use

A loss‐of‐function cysteine mutant in fibulin‐3 (EFEMP1) forms aberrant extracellular disulfide‐linked homodimers and alters extracellular matrix composition

Fibulin-3 knockout mice demonstrate corneal dysfunction but maintain normal retinal integrity

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