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Label-free LC-MS/MS analysis of proteins associated with expanded G4C2 hexanucleotide repeat-containing transcripts in human induced pluripotent stem cells

ABSTRACT: Proteins co-localizing with RNA targets of interest in human induced pluripotent stem cells (iPSCs) were identified using our recently optimized hybridization-proximity labeling approach (HyPro2) combined with label-free mass spectrometry. Briefly, fixed and permeabilized iPSCs from an ALS patient (DN19V4) or a healthy donor (C0053) were hybridized with a digoxigenin-labeled antisense oligonucleotide probe targeting transcripts containing the G4C2 hexanucleotide repeat expansion, which is present in the C9orf72 gene in DN19V4 but not in C0053. Alternatively, a control probe set was used to target ACTB pre-mRNA, expressed in both DN19V4 and C0053. The purified HyPro2 enzyme, which contains a digoxigenin-binding domain and a modified ascorbate peroxidase domain, was then recruited to the RNA targets, enabling in vitro biotinylation of target-proximal proteins. Following streptavidin pull-down, biotinylated proteins were fragmented using a Trypsin/Lys-C protease mix and identified by label-free LC-MS/MS analysis. All experiments were performed in triplicate

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Pluripotent Stem Cell

DISEASE(S): Amyotrophic Lateral Sclerosis

SUBMITTER: Steven Lynham

LAB HEAD: Eugene Makeyev

PROVIDER: PXD063192 | Pride | 2025-08-12

REPOSITORIES: Pride

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			Action	DRS
	PR687_THC1_10_CS3_4.msf	Msf
	PR687_THC1_10_CS3_4.mzid	Mzid
	PR687_THC1_10_CS3_4.mzid_PR687_THC1_10_CS3_4.MGF	Mzid
	PR687_THC1_10_CS3_4.raw	Raw
	PR687_THC1_11_CS3_5.msf	Msf

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Project description:Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular ageing. Sox9 (SRY-Box Transcription Factor 9) is a master regulator of chondrogenesis, also expressed in the vasculature, that has been implicated in vascular smooth muscle cell (VSMC) osteo-chondrogenic conversion. Here, we investigated the relationship between vascular ageing, calcification and Sox9-driven ECM regulation in VSMCs. Immunohistochemistry in human aortic samples showed that Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16. Analysis of Sox9 expression in vitro showed it was mechanosensitive with increased expression and nuclear translocation in senescent cells and on stiff matrices. Manipulation of Sox9 via overexpression and depletion, combined with atomic force microscopy (AFM) and proteomics, revealed that Sox9 regulates ECM stiffness and organisation by orchestrating changes in collagen expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs whereby senescent cells plated onto ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) was identified as a Sox9 target, and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles (EVs) and Sox9 promoted EV secretion, leading to increased LH3 deposition within the ECM. These findings identify cellular senescence and Sox9 as a key regulators of ECM stiffness during VSMC ageing and highlight a crucial role for ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle whereby cellular senescence and increased ECM stiffening promote Sox9 expression which drives further ECM modifications that act to accelerate vascular stiffening and cellular senescence.

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Label-free LC-MS/MS analysis of proteins associated with expanded G4C2 hexanucleotide repeat-containing transcripts in human induced pluripotent stem cells

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