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.

The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients.

In order to control the COVID-19 pandemic caused by SARS-CoV-2 infection, serious progress has been made to identify infected patients and to detect patients with a positive immune response against the virus. Currently, attempts to generate a vaccine against the coronavirus are ongoing. To understand SARS-CoV-2 immunoreactivity, we compared the IgG antibody response against SARS-CoV-2 in infected versus control patients by dot blot using recombinant viral particle proteins: N (Nucleocapsid), M (Membrane) and S (Spike). In addition, we used different protein fragments of the N and S protein to map immune epitopes. Most of the COVID-19 patients presented a specific immune response against the full length and fragments of the N protein and, to lesser extent, against a fragment containing amino acids 300-685 of the S protein. In contrast, immunoreactivity against other S protein fragments or the M protein was low. This response is specific for COVID-19 patients as very few of the control patients displayed immunoreactivity, likely reflecting an immune response against other coronaviruses. Altogether, our results may help develop method(s) for measuring COVID-19 antibody response, selectivity of methods detecting such SARS-CoV-2 antibodies and vaccine development.

PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks.

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.

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. In muscle cells, tropomyosin is subject to calcium regulation, but its regulation in nonmuscle 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 nonmuscle tropomyosins, which in fission yeast controls actin filament stability and division site placement.

Evidence for the nuclear import of histones H3.1 and H4 as monomers.

Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether-and-release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin-alpha proteins (predominantly IPO4), but not to histone-specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.

Rapid production of pure recombinant actin isoforms in Pichia pastoris.

Actins are major eukaryotic cytoskeletal proteins, and they are involved in 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 for biochemical studies of the non-muscle actin cytoskeleton. 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 ß4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin ß4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the ß- 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 dendritic actin 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.

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 ß-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.

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 (also known as MAPK1) 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 unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 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- and 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 that accompanies the transition between DCIS and a more invasive phenotype.

The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts.

Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential.

In vivo quantitative proteomics for the study of oncometabolism.

The active reprograming of cellular metabolism is a primary driver of oncogenesis and a hallmark of established neoplastic lesions. Much of this reprogramming depends on the expression levels and posttranslational modifications (PTMs) of metabolic enzymes. Stable isotope labeling of amino acids in culture (SILAC) is an amino acid-based labeling technique that can be used both in vitro and in vivo to comparatively assess the levels and PTMs of proteins. To this aim, SILAC-labeled cell lysates can be spiked into each sample as a standard, followed by the analysis of specimens by mass spectrometry (MS). Combined with appropriate protocols for the lysis and preparation of samples for MS, this technique allows for the accurate and in-depth quantification of the proteome of a wide variety of cell and tissue samples. In particular, SILAC can be employed to infer the metabolic state of neoplastic lesions and obtain a profound understanding of the proteomic alterations that accompany oncogenesis and tumor progression. Here, we describe a proteomic approach based on SILAC, high-resolution chromatography and high-accuracy MS for comparing levels and phosphorylation status of proteins between the samples of interest. This method can be applied not only to the proteomic study of oncometabolism in murine tissues, but also to the study of cellular samples and human specimens.

Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells.

Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function.

SILAC-based proteomics of human primary endothelial cell morphogenesis unveils tumor angiogenic markers.

Proteomics has been successfully used for cell culture on dishes, but more complex cellular systems have proven to be challenging and so far poorly approached with proteomics. Because of the complexity of the angiogenic program, we still do not have a complete understanding of the molecular mechanisms involved in this process, and there have been no in depth quantitative proteomic studies. Plating endothelial cells on matrigel recapitulates aspects of vessel growth, and here we investigate this mechanism by using a spike-in SILAC quantitative proteomic approach. By comparing proteomic changes in primary human endothelial cells morphogenesis on matrigel to general adhesion mechanisms in cells spreading on culture dish, we pinpoint pathways and proteins modulated by endothelial cells. The cell-extracellular matrix adhesion proteome depends on the adhesion substrate, and a detailed proteomic profile of the extracellular matrix secreted by endothelial cells identified CLEC14A as a matrix component, which binds to MMRN2. We verify deregulated levels of these proteins during tumor angiogenesis in models of multistage carcinogenesis. This is the most in depth quantitative proteomic study of endothelial cell morphogenesis, which shows the potential of applying high accuracy quantitative proteomics to in vitro models of vessel growth to shed new light on mechanisms that accompany pathological angiogenesis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000359.

Quantitative mass spectrometry-based proteomics in angiogenesis.

The process of new blood vessel formation from pre-existing ones is called angiogenesis. Beyond playing a critical role in the physiological development of the vascular system, angiogenesis is a well-recognised hallmark of cancer. Unbiased system-wide approaches are required to complement the current knowledge, and intimately understand the molecular mechanisms regulating this process in physiological and pathological conditions. In this review we describe the cellular and molecular dynamics regulating the physiological growth of vessels and their deregulation in cancer, survey in vitro and in vivo models currently exploited to investigate various aspects of angiogenesis and describe state-of-the-art and most widespread methods and technologies in MS shotgun proteomics. Finally, we focus on current applications of MS to better understand endothelial cell behaviour and propose how modern proteomics can impact on angiogenesis research.

Voltage-dependent anion channel as a resident protein of lipid rafts: post-transductional regulation by estrogens and involvement in neuronal preservation against Alzheimer's disease.

The voltage-dependent anion channel, VDAC, is present at the neuronal membrane, where it appears to participate, among others, in the extrinsic apoptotic pathway and in the modulation of amyloid-beta induced injury, suggesting the involvement of this channel in Alzheimer's disease (AD) neurotoxicity. VDAC is also highly concentrated in neuronal lipid raft microdomains of different mouse and human cognitive areas, where it has been shown associated with estrogen receptor alpha (ERα), as a part of a `signalosome' that may activate some intracellular signal transduction. At the plasma membrane level, estrogens and antiestrogens (tamoxifen) have been demonstrated to exert rapid antagonist effects on the activation of VDAC, through their distinct effects on the channel post-transductional modulation. Therefore, part of the alternative mechanisms of estrogen related to neuroprotection against amyloid-beta may involve VDAC phosphorylation, in order to maintain the channel in an unactivated (closing) state. Interestingly, VDAC-ERα association has been shown to be disrupted in neuronal lipid rafts of AD brains, in correlation with the aberrant lipid composition observed in these microstructures, suggesting that disturbance of protein interactions may be related to variation in the physico-chemical properties of these microdomains.