Updates of the in-gel digestion method for protein analysis by mass spectrometry

The in‐gel digestion of proteins for analysis by liquid chromatograph mass spectrometry has been used since the early 1990s. Although several improvements have contributed to increasing the quality of the data obtained, many recent publications still use sub‐optimal approaches. We present updates of the in‐gel digestion protocol. We show that alternative reducing, alkylating agent reactions and tryptic digestion buffers increase peptide and protein identification and reduce incubation times. Our results indicate that a simultaneous and short, high temperature reduction and alkylation reaction using Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP) and chloroacetamide (CAA) with a subsequent gel wash improve protein identification and sequence coverage, diminish peptide side reactions. Additionally, use of 4‐(2‐Hydroxyethyl)piperazine‐1‐ethanesulfonic acid buffer (HEPES) allows a significant reduction in the digestion time improving trypsin performance and increasing the peptide recovery. The updates of the in‐gel digestion protocol described here are efficient and offer flexibility to be incorporated in any proteomic laboratory

A proteomic approach to identify endosomal cargoes controlling cancer invasiveness

We have previously shown that Rab17 - a small GTPase associated with epithelial polarity - is specifically suppressed by ERK2 signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this are not known. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp-8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma in situ (DCIS) and a more invasive phenotype. We developed an unbiased proteomic approach to elucidate the complement of receptors that redistributes between endosomes and the plasma membrane, and have pin-pointed neuropilin-2 (NRP2) as a key pro-invasive cargo of Rab17/Vamp8-regulated trafficking. Indeed, reduced Rab17 or Vamp8 levels lead to increased mobilisation of NRP2-containing late endosomes and upregulated cell surface expression of NRP2. Finally, we show that NRP2 is required for the basement membrane disruption which accompanies transition between DCIS and a more invasive phenotype

Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes

Cell cryopreservation is an essential tool for drug toxicity/function screening and transporting cell-based therapies, and is essential in most areas of biotechnology. There is a challenge, however, associated with the cryopreservation of cells in monolayer format (attached to tissue culture substrates) which gives far lower cell yields (<20% typically) compared to suspension freezing. Here we investigate the mechanisms by which the protective osmolyte L-proline enhances cell-monolayer cryopreservation. Pre-incubating A549 cells with proline, prior to cryopreservation in monolayers, increased post-thaw cell yields two-fold, and the recovered cells grow faster compared to cells cryopreserved using DMSO alone. Further increases in yield were achieved by adding polymeric ice recrystallization inhibitors, which gave limited benefit in the absence of proline. Mechanistic studies demonstrated a biochemical, rather than biophysical (i.e. not affecting ice growth) mode of action. It was observed that incubating cells with proline (before freezing) transiently reduced the growth rate of the cells, which was not seen with other osmolytes (betaine and alanine). Removal of proline led to rapid growth recovery, suggesting that proline pre-conditions the cells for cold stress, but with no impact on downstream cell function. Whole cell proteomics did not reveal a single pathway or protein target but rather cells appeared to be primed for a stress response in multiple directions, which together prepare the cells for freezing. These results support the use of proline alongside standard conditions to improve post-thaw recovery of cell monolayers, which is currently considered impractical. It also demonstrates that a chemical biology approach to discovering small molecule biochemical modulators of cryopreservation may be possible, to be used alongside traditional (solvent) based cryoprotectants

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

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 in vivo 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 FBS using two complementary methodologies developed herein; quartz crystal microbalance and ‘centrifugal capture’, coupled with nano-liquid 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, while strong binding with IAPP fibrils occurred for linear proteins or multi-domain 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

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

Rapid production of pure recombinant actin isoforms in Pichia pastoris

Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from S. cerevisiae, S. pombe, and the β- and γ- isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate actin dendritic networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton

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

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness‐induced CCN1 activates β‐catenin nuclear translocation and signaling and that this contributes to upregulate N‐cadherin levels on the surface of the endothelium, in vitro. This facilitates N‐cadherin‐dependent cancer cell–endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness‐induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis