ten Dijke, P. | bioRxiv Science

bioRxiv2019

Transforming growth factor-beta signaling via ALK1 and ALK5 regulates distinct functional pathways in vein graft intimal hyperplasia

RationaleTransforming growth factor-beta (TGF{beta}) is tightly regulated at multiple levels, with regulation at the receptor level now recognized as a key determinant of the cellular response to this pleiotropic cytokine. TGF{beta} promotes saphenous vein graft neointima formation after coronary artery bypass graft (CABG) surgery, inducing smooth muscle cell (SMC) hyperplasia and fibrosis by signaling via activin receptor-like kinase 5(ALK5). However, the role of the alternate TGF{beta} receptor ALK1 remains completely unknown. ObjectiveTo define the receptor pathways activated by TGF{beta} in SMCs and their mechanistic importance during CABG neointima formation. Methods and resultsRadioligand co-IP assays revealed direct interactions between TGF{beta}, ALK5 and ALK1 in primary saphenous vein graft SMC (HSVSMC) from patients undergoing CABG. Knockdown and pharmacological inhibition of ALK5 or ALK1 in HSVSMC significantly attenuated TGF{beta}-induced phosphorylation of receptor-regulated (R)-Smads 2/3 and 1/5, respectively. Microarray profiling followed by qRT-PCR validation showed that TGF{beta} induced distinct transcriptional networks downstream of ALK5 or ALK1, associated with HSVSMC contractility and migration, respectively and confirmed using migration assays as well as qRT-PCR and western blot assays of contractile SMC markers. scRNAseq analysis of TGF{beta}-treated HSVSMC identified distinct subgroups of cells showing ALK5 or ALK1 transcriptional responses, while RNA velocity analyses indicated divergence in differentiation towards ALK5 or ALK1-dominant lineages. ALK1, ALK5 and their downstream effectors pSmad1/5 and pSmad2/3 were localized to SMA+ neointimal SMCs in remodelled mouse vein grafts. Pharmacological inhibition or genetic ablation of Smad1/5 substantially reducing neointima formation following acute vascular injury. Notably, expression and activation of ALK1, ALK5 and their respective downstream R-Smads was already evident in hyperplastic saphenous veins prior to grafting. ConclusionsWhilst canonical TGF{beta} signaling via ALK5 promotes a contractile HSVSMC phenotype, transactivation of ALK1 by TGF{beta} induces neointima formation by driving cell migration. Restoring the balance between ALK1 and ALK5 in HSVSMC may represent a novel therapeutic strategy for vein graft failure.

cell biology↗

bioRxiv2019

A Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos

Many reagents have been emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement allowing investigations of the function of individual enzymes in a cellular context. We here report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and whole animals. The probe labels active Ubiquitin Carboxy-terminal Hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1-2% of total brain protein. UCHL1 variants have been linked with the neurodegenerative disorders Parkinsons and Alzheimers disease. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1 specific research tools exist. We show that the reagents reported here are specific for UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 irreversibly in an activity-dependent manner. Its use is demonstrated by labelling of UCHL1 both in vitro and in cells. We furthermore show that this probe can report UCHL1 activity during the development of zebrafish embryos.

biochemistry↗