Genesis and growth of extracellular vesicle-derived microcalcification in atherosclerotic plaques

Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification zones. We also show that calcification morphology and the plaque’s collagen content – two determinants of atherosclerotic plaque stability - are interlinked

Net, a negative Ras-switchable TCF, contains a second inhibition domain, the CID, that mediates repression through interactions with CtBP and de-acetylation.

Signalling cascades are integrated at the transcriptional level by the interplay between factors such as the ternary complex factors (TCFs) that interact with serum response factor (SRF) and the serum response element (SRE) of the fos promoter. Net is a negative TCF that is switched to a positive regulator by the Ras signal. To understand the mechanisms of repression by Net, we used a yeast two-hybrid screen to identify factors that interact with its inhibitory domain. We isolated mCtBP1, the murine homologue of huCtBP1, a factor implicated in negative regulation of transformation by E1A plus Ras. We show that mCtBP1 interacts strongly with Net both in vitro and in vivo. The CtBP interaction domain of Net, the CID, mediates repression independently of the previously identified negative element, the NID. The CID inhibits by recruiting the co-repressor mCtBP1. The CID and mCtBP1 need to use de-acetylase activity for repression, whereas the NID apparently represses by other mechanisms. Finally, we provide evidence that CtBP and de-acetylation repress the c-fos SRE in low serum when it is inactive, but not in high serum when it is active. These results provide insights into the cross-talk between pathways that inhibit and stimulate transformation at the level of Net, a regulator of gene expression

Hdm2 recruits a hypoxia-sensitive corepressor to negatively regulate p53-dependent transcription

The transcription factor p53 lies at the center of a protein network that controls cell cycle progression and commitment to apoptosis. p53 is inactive in proliferating cells, largely because of negative regulation by the Hdm2/Mdm2 oncoprotein, with which it physically associates. Release from this negative regulation is sufficient to activate p53 and can be triggered in cells by multiple stimuli through diverse pathways. This diversity is achieved in part because Hdm2 uses multiple mechanisms to inactivate p53; it targets p53 for ubiquitination and degradation by the proteosome, shuttles it out of the nucleus and into the cytoplasm, prevents its interaction with transcriptional coactivators, and contains an intrinsic transcriptional repressor activity. Here we show that Hdm2 can also repress p53 activity through the recruitment of a known transcriptional corepressor, hCtBP2. This interaction, and consequent repression of p53-dependent transcription, is relieved under hypoxia or hypoxia-mimicking conditions that are known to increase levels of intracellular NADH. CtBP proteins can undergo an NADH-induced conformational change, which we show here results in a loss of their Hdm2 binding ability. This pathway represents a novel mechanism whereby p53 activity can be induced by cellular stress.<br/

C-terminal binding proteins: Emerging roles in cell survival and tumorigenesis

Within a cell, the levels and activity of multiple pro- and anti-apoptotic molecules act in concert to regulate commitment to apoptosis. Whilst the balance between survival and death can be tipped by the effects of single molecules, cellular apoptosis control pathways very often incorporate key transcription factors that co-ordinately regulate the expression of multiple apoptosis control genes. C-terminal binding proteins (CtBPs), which were originally identified through their binding to the Adenovirus E1A oncoprotein, have been described as such transcriptional regulators of the apoptosis program. Specifically, CtBPs function as transcriptional co-repressors, and have been demonstrated to promote cell survival by suppressing the expression of several pro-apoptotic genes. In this review we summarize the evidence supporting a key role for CtBP proteins in cell survival. We also describe the known mechanisms of transcriptional control by CtBPs, and review the multiplicity of intracellular signaling and transcriptional control pathways with which they are known to be involved. Finally we consider these findings in the context of additional known roles of CtBP molecules, and the potential implications that this combined knowledge may have for our comprehension of diseases of cell survival, notably cance

Physical and Functional Interactions between the Corepressor CtBP and the Epstein-Barr Virus Nuclear Antigen EBNA3C

CtBP has been shown to be a highly conserved corepressor of transcription. E1A and all the various transcription factors to which CtBP binds contain a conserved PLDLS CtBP-interacting domain, and EBNA3C includes a PLDLS motif (amino acids [aa] 728 to 732). Here we show that EBNA3C binds to CtBP both in vitro and in vivo and that the interaction requires an intact PLDLS. The C terminus of EBNA3C (aa 580 to 992) has modest trans-repressor activity when it is fused to the DNA-binding domain of Gal4, and deletion or mutation of the PLDLS sequence ablates this and unmasks a transactivation function within the fragment. However, loss of the CtBP interaction motif had little effect on the ability of full-length EBNA3C to repress transcription. A striking correlation between CtBP binding and the capacity of EBNA3C to cooperate with (Ha-)Ras in the immortalization and transformation of primary rat embryo fibroblasts was also revealed