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. 2023;43(12):664-674.
doi: 10.1080/10985549.2023.2284147. Epub 2023 Dec 20.

Genetic and Pharmacological Modulation of Cellular Proteostasis Leads to Partial Functional Rescue of Homocystinuria-Causing Cystathionine-Beta Synthase Variants

Renata Collard  1 , Tomas Majtan  1   2
Affiliations

Genetic and Pharmacological Modulation of Cellular Proteostasis Leads to Partial Functional Rescue of Homocystinuria-Causing Cystathionine-Beta Synthase Variants

Renata Collard et al. Mol Cell Biol. 2023.

Abstract

Homocystinuria (HCU), an inherited metabolic disorder caused by lack of cystathionine beta-synthase (CBS) activity, is chiefly caused by misfolding of single amino acid residue missense pathogenic variants. Previous studies showed that chemical, pharmacological chaperones or proteasome inhibitors could rescue function of multiple pathogenic CBS variants; however, the underlying mechanisms remain poorly understood. Using Chinese hamster DON fibroblasts devoid of CBS and stably overexpressing human WT or mutant CBS, we showed that expression of pathogenic CBS variant mostly dysregulates gene expression of small heat shock proteins HSPB3 and HSPB8 and members of HSP40 family. Endoplasmic reticulum stress sensor BiP was found upregulated with CBS I278T variant associated with proteasomes suggesting proteotoxic stress and degradation of misfolded CBS. Co-expression of the main effector HSP70 or master regulator HSF1 rescued steady-state levels of CBS I278T and R125Q variants with partial functional rescue of the latter. Pharmacological proteostasis modulators partially rescued expression and activity of CBS R125Q likely due to reduced proteotoxic stress as indicated by decreased BiP levels and promotion of refolding as indicated by induction of HSP70. In conclusion, targeted manipulation of cellular proteostasis may represent a viable therapeutic approach for the permissive pathogenic CBS variants causing HCU.

Keywords: chaperone; cystathionine beta-synthase; homocystinuria; misfolding; pharmacological; proteostasis.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

FIG 1
FIG 1
Expression profile of molecular chaperones in HCU. (A) Differential expression of genes encoding HSPs and chaperones in A23 CBS I278T compared to A23 CBS WT control (n = 3 independent biological replicates for both test and control samples). Significantly up- and downregulated genes are named and shown as green and red points, respectively. (B) Representative Western blot analysis of soluble protein extracts from A23 CBS WT and I278T (50 µg/lane) evaluating expression of CBS and selected molecular chaperones, such as HSP70, BiP and HSP40.
FIG 2
FIG 2
Disturbed proteostasis in HCU. The A23 CBS WT (top) and I278T cells (bottom) were grown on cell culture slides, fixed, permeabilized and probed for the indicated combination of targets as described in Materials and Methods section. Different effectors of proteostasis network are shown in red channel (A – BiP, B – HSP70 and C – 20S Proteasome), CBS in a green channel and nuclei in a blue channel (shown only in a composite image). (D) Fluorescent intensities for CBS, BiP, HSP70 and 20S Proteasome were normalized to background fluorescence, quantified using ImageJ package and analyzed using GraphPad Prism software (statistical significance: **P < 0.01).
FIG 3
FIG 3
Partial rescue of CBS by co-expression of molecular chaperones. Western blot analysis (A) and CBS activity (B) in soluble fractions of A23 CBS WT, R125Q and I278T cells co-expressing either HSPA1A or HSF1 compared to mock-transfected controls. (A) Cell lysates (50 µg/lane) were resolved in 10% SDS-PAGE, transferred to PVDF membrane and probed with antibodies against CBS, actin, HSP70, HSF1 and V5 tag. (B) Cell lysates (150–500 µg) were analyzed by radiometric CBS activity assay in the absence and presence of 500 µM SAM. The results were compared to the respective mock-transfected controls and statistically analyzed. *P < 0.05, **P < 0.01.
FIG 4
FIG 4
Rescue of CBS by pharmacological modulation of proteostasis network. Western blot analysis (A, C) and CBS activity (B, D) in soluble fractions of A23 CBS R125Q treated overnight with 0.5 µg/mL tunicamycin, 200 nM trans-ISRIB, 10 µM TRC051384, 5 µM KIRA6, 5 µM Dbeq or 5 µM celastrol (A, B) or after 8 h treatment with 20 µM MG132 or 20 µM carfilzomib (C, D). (A, C) Cell lysates (50 µg/lane) were resolved in 10% SDS-PAGE, transferred to PVDF membrane and probed with antibodies against CBS and actin used as a loading control. (B, D) Cell lysates (500 µg) were analyzed by radiometric CBS activity assay in the absence and presence of 500 µM SAM. The results were compared to vehicle-transfected controls and statistically analyzed. **P < 0.01, ***P < 0.001.
FIG 5
FIG 5
Pharmacological modulation of CBS refolding. The A23 CBS R125Q were grown on cell culture slides in the presence of vehicle only (top), 10 µM TRC051384 (middle) or 5 µM celastrol (bottom), fixed, permeabilized and probed for the indicated combination of targets as described in Materials and Methods section. Different effectors of proteostasis network are shown in red channel (A – BiP, B – HSP70 and C – 20S Proteasome), CBS in a green channel and nuclei in a blue channel (shown only in a composite image). (D) Fluorescent intensities for CBS, BiP, HSP70 and 20S Proteasome were normalized to background fluorescence, quantified using ImageJ package and analyzed using GraphPad Prism software (statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001).
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Grants and funding

This work was supported by the American Heart Association (16SDG30040000), Travere Therapeutics (SRA-22444) and University of Fribourg Research pool (22-15). TM also appreciate support from Prof. Csaba Szabo and the Jerome Lejeune Foundation research grant (awarded to CS).
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