Novel regulation from novel interactions: Identification of an RNA sponge that controls the levels, processing and efficacy of the RoxS riboregulator of central metabolism in Bacillus subtilis

Small RNAs (sRNAs) are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While potentially of importance, we know the function of few. This is in nosmall part because we lack global-scale methodology enabling target identification, this being especiallyacute in species without known RNA meeting point proteins (e.g. Hfq). We apply a combination ofpsoralen RNA cross-linking and Illumina-sequencing to identify RNA-RNA interacting pairs in vivo inBacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings arerare (compared with sRNA/mRNA), we identify a robust example involving the unusually conservedsRNA (RoxS/RsaE) and an unstudied sRNA that we term Regulator of small RNA A (RosA). Thisinteraction is found in independent samples across multiple conditions. Given the possibility of a novelassociated regulatory mechanism, and the rarity of well-characterised bacterial sRNA-sRNAinteractions, we mechanistically dissect RosA and its interactants. RosA we show to be a sponge RNA,the first to be described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxSand FsrA. Unexpectedly, it acts differently on each. As expected of a sponge RNA, FsrA is sequesteredby RosA. The RosA/RoxS interaction is more complex affecting not only the level of RoxS but also itsprocessing and efficacy. Importantly, RosA provides the condition-dependent intermediary betweenCcpA, the key regulator of carbon metabolism, and RoxS. This not only provides evidence for a novel,and functionally important, regulatory mechanism, but in addition, provides the missing link betweentranscriptional and post-transcriptional regulation of central metabolism

Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects.

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion

Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability

Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement

Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. Whereas, in muscle cells, tropomyosin is subject to calcium regulation, its regulation in non-muscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of non-muscle tropomyosins, which in fission yeast controls actin filament stability and division site placement

Beyond oil degradation: enzymatic potential of Alcanivorax to degrade natural and synthetic polyesters

Thematic Issue on Metal(loid) Microbiology.Pristine marine environments are highly oligotrophic ecosystems populated by well‐established specialized microbial communities. Nevertheless, during oil spills, low‐abundant hydrocarbonoclastic bacteria bloom and rapidly prevail over the marine microbiota. The genus Alcanivorax is one of the most abundant and well‐studied organisms for oil degradation. While highly successful under polluted conditions due to its specialized oil‐degrading metabolism, it is unknown how they persist in these environments during pristine conditions. Here, we show that part of the Alcanivorax genus, as well as oils, has an enormous potential for biodegrading aliphatic polyesters thanks to a unique and abundantly secreted alpha/beta hydrolase. The heterologous overexpression of this esterase proved a remarkable ability to hydrolyse both natural and synthetic polyesters. Our findings contribute to (i) better understand the ecology of Alcanivorax in its natural environment, where natural polyesters such as polyhydroxyalkanoates (PHA) are produced by a large fraction of the community and, hence, an accessible source of carbon and energy used by the organism in order to persist, (ii) highlight the potential of Alcanivorax to clear marine environments from polyester materials of anthropogenic origin as well as oils, and (iii) the discovery of a new versatile esterase with a high biotechnological potential.VZ was supported by CONICYT‐BECAS CHILE/Doctorado Becas Chile en el Extranjero, Folio 72160583. JAC‐O was supported by the NERC Independent Research Fellowship NE/K009044/1, NERC research project NE/S005501/1, and Ramón y Cajal contract RYC‐2017‐22452 (funded by the Ministry of Science, Innovation and Universities, the National Agency of Research, and the European Social Fund). RB was supported by the MINECO project CTM2015‐70180‐R (FEDER co‐funding)

Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability

Profiling the Serum Protein Corona of Fibrillar Human Islet Amyloid Polypeptide

Amyloids may be regarded as native nanomaterials that form in the presence of complex protein mixtures. By drawing an analogy with the physicochemical properties of nanoparticles in biological fluids, we hypothesized that amyloids should form a protein corona <i>in vivo</i> that would imbue the underlying amyloid with a modified biological identity. To explore this hypothesis, we characterized the protein corona of human islet amyloid polypeptide (IAPP) fibrils in fetal bovine serum using two complementary methodologies developed herein: quartz crystal microbalance and “centrifugal capture”, coupled with nanoliquid chromatography tandem mass spectroscopy. Clear evidence for a significant protein corona was obtained. No trends were identified for amyloid corona proteins based on their physicochemical properties, whereas strong binding with IAPP fibrils occurred for linear proteins or multidomain proteins with structural plasticity. Proteomic analysis identified amyloid-enriched proteins that are known to play significant roles in mediating cellular machinery and processing, potentially leading to pathological outcomes and therapeutic targets

Project PXD015051: "Identification of an RNA sponge that controls the levels, processing and efficacy of the RoxS riboregulator of central metabolism in B. subtilis"

Only a few small regulatory RNAs (sRNAs) have been characterized in B. subtilis, the paradigm of Gram-positive bacteria, and one of the major challenges is target identification. Here we use global in vivo RNA psoralen cross-linking to identify RNA-RNA partners in Bacillus subtilis. Two sRNAs, RoxS and FsrA, play key roles in balancing the metabolic state of the cell in response to carbon sources and iron limitation, respectively. In this study, we identify new mRNA targets for both RoxS and FsrA, and a small RNA (S345/RosA) that is able to interact with both sRNAs. We report that RosA controls the maturation and degradation of RoxS and acts as a sponge to limit the efficacy of RoxS on its targets. Expression of RosA is catabolically repressed by the transcription factor CcpA. We provide evidence that the RosA/RoxS interaction plays a key role in regulating metabolism in response to a switches in carbon source

PYCR1-dependent proline synthesis in cancer associated fibroblasts is required for the deposition of pro-tumorigenic extracellular matrix

Elevated production of collagen-rich extracellular matrix (ECM) is a hallmark of cancer associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. How to target ECM production to oppose cancer is yet unclear, since targeting CAFs can restrain but also promote cancer progression. Here we find that proline, which is a highly abundant amino acid in collagen proteins, is newly synthesised from glutamine to make tumour collagen in breast cancer xenografts, and that its production is elevated in mammary CAFs. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are enhanced by increased pyruvate dehydrogenase-derived acetyl-CoA levels, via gene expression regulation through the epigenetic regulator histone acetyl-transferase EP300. Our work unveils unprecedented roles of CAF metabolism to support pro-tumorigenic collagen production. PYCR1 is a cancer cell vulnerability and potential target for therapy, hence our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of pro-tumorigenic ECM