Tranexamic acid suppresses the release of mitochondrial DNA, protects the endothelial monolayer and enhances oxidative phosphorylation.

Damage-associated molecular patterns, including mitochondrial DNA (mtDNA) are released during hemorrhage resulting in the development of endotheliopathy. Tranexamic acid (TXA), an antifibrinolytic drug used in hemorrhaging patients, enhances their survival despite the lack of a comprehensive understanding of its cellular mechanisms of action. The present study is aimed to elucidate these mechanisms, with a focus on mitochondria. We found that TXA inhibits the release of endogenous mtDNA from granulocytes and endothelial cells. Furthermore, TXA attenuates the loss of the endothelial monolayer integrity induced by exogenous mtDNA. Using the Seahorse XF technology, it was demonstrated that TXA strongly stimulates mitochondrial respiration. Studies using Mitotracker dye, cells derived from mito-QC mice, and the ActivSignal IPAD assay, indicate that TXA stimulates biogenesis of mitochondria and inhibits mitophagy. These findings open the potential for improvement of the strategies of TXA applications in trauma patients and the development of more efficient TXA derivatives.

Structure of the human 20S U5 snRNP.

The 20S U5 small nuclear ribonucleoprotein particle (snRNP) is a 17-subunit RNA-protein complex and a precursor of the U4/U6.U5 tri-snRNP, the major building block of the precatalytic spliceosome. CD2BP2 is a hallmark protein of the 20S U5 snRNP, absent from the mature tri-snRNP. Here we report a high-resolution cryogenic electron microscopy structure of the 20S U5 snRNP, shedding light on the mutually exclusive interfaces utilized during tri-snRNP assembly and the role of the CD2BP2 in facilitating this process.

MMTV-NeuT/ATTAC mice: a new model for studying the stromal tumor microenvironment.

One of the central challenges in cancer prevention is the identification of factors in the tumor microenvironment (TME) that increase susceptibility to tumorigenesis. One such factor is stromal fibrosis, a histopathologic negative prognostic criterion for invasive breast cancer. Since the stromal composition of the breast is largely adipose and fibroblast tissue, it is important to understand how alterations in these tissues affect cancer progression. To address this question, a novel transgenic animal model was developed by crossing MMTV-NeuT mice containing a constitutively active ErbB2 gene into the FAT-ATTAC (fat apoptosis through targeted activation of caspase 8) background, which expresses an inducible caspase 8 fusion protein targeted to mammary adipose tissue. Upon caspase 8 activation, lipoatrophy of the mammary gland results in stromal fibrosis and acceleration of mammary tumor development with an increase in tumor multiplicity. Fibrosis was accompanied by an increase in collagen deposition, α-smooth muscle actin and CD31 expression in the tumor stroma as well as an increase in PD-L1-positive tumor cells, and infiltration by regulatory T cells, myeloid-derived suppressor cells and tumor-associated macrophages. Gene expression and signal transduction profiling indicated upregulation of pathways associated with cytokine signaling, inflammation and proliferation. This model should be useful for evaluating new therapies that target desmoplasia in the TME associated with invasive cancer.

Correction: MMTV-NeuT/ATTAC mice: a new model for studying the stromal tumor microenvironment.

[This corrects the article DOI: 10.18632/oncotarget.24233.].

Enolase takes part in a macromolecular complex associated to mitochondria in yeast.

In eucaryotes, glycolytic enzymes are classically regarded as being localised in the cytosol. Recently, we have shown that part of the cellular pool of the glycolytic enzyme, enolase, is tightly associated with the mitochondrial surface in the yeast Saccharomyces cerevisiae (N. Entelis, I. Brandina, P. Kamenski, I.A. Krasheninnikov, R.P. Martin and I. Tarassov, A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae, Genes Dev. 20 (2006) 1609-1620). Here, using enzymatic assays, we show that all glycolytic enzymes are associated with mitochondria in yeast, to extents similar to those previously reported for Arabidopsis cells. Using separation of mitochondrial complexes by blue-native/SDS-PAGE and coimmunoprecipitation of mitochondrial proteins with anti-enolase antibodies, we found that enolase takes part in a large macromolecular complex associated to mitochondria. The identified components included additional glycolytic enzymes, mitochondrial membrane carriers, and enzymes of the TCA cycle. We suggest a possible role of the enolase complex in the channeling of pyruvate, the terminal product of glycolysis, towards the TCA cycle within mitochondria. Moreover, we show that the mitochondrial enolase-containing complex also contains the cytosolic tRNA(CUU)Lys, which is mitochondrially-imported, and its presumed import carrier, the precursor of the mitochondrial lysyl-tRNA synthetase. This suggests an unsuspected novel function for this complex in tRNA mitochondrial import.

tRNA import into yeast mitochondria is regulated by the ubiquitin-proteasome system.

In Saccharomyces cerevisiae, one of two cytosolic lysine-tRNAs is partially imported into mitochondria. We demonstrate that three components of the ubiquitin/26S proteasome system (UPS), Rpn13p, Rpn8p and Doa1p interact with the imported tRNA and with the essential factor of its mitochondrial targeting, pre-Msk1p. Genetic and biochemical assays demonstrate that UPS plays a dual regulatory role, since the overall inhibition of cellular proteasome activity reduces tRNA import, while specific depletion of Rpn13p or Doa1p increases it. This result suggests a functional link between UPS and tRNA mitochondrial import in yeast and indicates on the existence of negative and positive import regulators.

The analysis of tRNA import into mammalian mitochondria.

Ribonucleic acid (RNA) import into mitochondria occurs in a variety of organisms. In mammalian cells, several small RNAs are imported in a natural manner; transfer RNAs (tRNAs) can be imported in an artificial way, following expression of corresponding genes from another organism (yeast) in the nucleus. We describe how to establish and to analyze such import mechanisms in cultured human cells. In detail, we describe (1) the construction of plasmids expressing importable yeast tRNA derivatives in human cells, (2) the procedure of transfection of either immortalized cybrid cell lines or primary patient's fibroblasts and downregulation of tRNA expression directed by small interfering RNA (siRNA) as a way to demonstrate the effect of import in vivo, (3) the methods of mitochondrial RNA isolation from the transfectants, and (4) approaches for quantification of RNA mitochondrial import.

A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae.

In many organisms, mitochondria import nuclear DNA-encoded small RNAs. In yeast Saccharomyces cerevisiae, one out of two cytoplasmic isoacceptor tRNAs(Lys) is partially addressed into the organelle. Mitochondrial targeting of this tRNA was shown to depend on interaction with the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. However, preMsk1p alone was unable to direct tRNA targeting, suggesting the existence of additional protein factor(s). Here, we identify the glycolytic enzyme, enolase, as such a factor. We demonstrate that recombinant enolase and preMSK1p are sufficient to direct tRNA import in vitro and that depletion of enolase inhibits tRNA import in vivo. Enzymatic and tRNA targeting functions of enolase appear to be independent. Three newly characterized properties of the enolase can be related to its novel function: (1) specific affinity to the imported tRNA, (2) the ability to facilitate formation of the complex between preMsk1p and the imported tRNA, and (3) partial targeting toward the mitochondrial outer membrane. We propose a model suggesting that the cell exploits mitochondrial targeting of the enolase in order to address the tRNA toward peri-mitochondrially synthesized preMsk1p. Our results indicate an alternative molecular chaperone function of glycolytic enzyme enolase in tRNA mitochondrial targeting.