Kinome-Wide Synthetic Lethal Screen Identifies PANK4 as a Modulator of Temozolomide Resistance in Glioblastoma.

Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterized. Here, following a kinome-wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ-resistant GBM cell models, patient-derived GBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)-based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.

Definition of an Inflammatory Biomarker Signature in Plasma-Derived Extracellular Vesicles of Glioblastoma Patients.

Glioblastoma (GB) is an aggressive type of tumour for which therapeutic options and biomarkers are limited. GB diagnosis mostly relies on symptomatic presentation of the tumour and, in turn, brain imaging and invasive biopsy that can delay its diagnosis. Description of easily accessible and effective biomarkers present in biofluids would thus prove invaluable in GB diagnosis. Extracellular vesicles (EVs) derived from both GB and stromal cells are essential to intercellular crosstalk in the tumour bulk, and circulating EVs have been described as a potential reservoir of GB biomarkers. Therefore, EV-based liquid biopsies have been suggested as a promising tool for GB diagnosis and follow up. To identify GB specific proteins, sEVs were isolated from plasma samples of GB patients as well as healthy volunteers using differential ultracentrifugation, and their content was characterised through mass spectrometry. Our data indicate the presence of an inflammatory biomarker signature comprising members of the complement and regulators of inflammation and coagulation including VWF, FCGBP, C3, PROS1, and SERPINA1. Overall, this study is a step forward in the development of a non-invasive liquid biopsy approach for the identification of valuable biomarkers that could significantly improve GB diagnosis and, consequently, patients' prognosis and quality of life.

TMEM87a/Elkin1, a component of a novel mechanoelectrical transduction pathway, modulates melanoma adhesion and migration.

Mechanoelectrical transduction is a cellular signalling pathway where physical stimuli are converted into electro-chemical signals by mechanically activated ion channels. We describe here the presence of mechanically activated currents in melanoma cells that are dependent on TMEM87a, which we have renamed Elkin1. Heterologous expression of this protein in PIEZO1-deficient cells, that exhibit no baseline mechanosensitivity, is sufficient to reconstitute mechanically activated currents. Melanoma cells lacking functional Elkin1 exhibit defective mechanoelectrical transduction, decreased motility and increased dissociation from organotypic spheroids. By analysing cell adhesion properties, we demonstrate that Elkin1 deletion is associated with increased cell-substrate adhesion and decreased homotypic cell-cell adhesion strength. We therefore conclude that Elkin1 supports a PIEZO1-independent mechanoelectrical transduction pathway and modulates cellular adhesions and regulates melanoma cell migration and cell-cell interactions.

When cells receive signals about their surrounding environment, this initiates a chain of signals which generate a response. Some of these signalling pathways allow cells to sense physical and mechanical forces via a process called mechanotransduction. There are different types of mechanotransduction. In one pathway, mechanical forces open up specialized channels on the cell surface which allow charged particles to move across the membrane and create an electrical current. Mechanoelectrical transduction plays an important role in the spread of cancer: as cancer cells move away from a tumour they use these signalling pathways to find their way between cells and move into other parts of the body. Understanding these pathways could reveal ways to stop cancer from spreading, making it easier to treat. However, it remains unclear which molecules regulate mechanoelectrical transduction in cancer cells. Now, Patkunarajah, Stear et al. have studied whether mechanoelectrical transduction is involved in the migration of skin cancer cells. To study mechanoelectrical transduction, a fine mechanical input was applied to the skin cancer cells whilst measuring the flow of charged molecules moving across the membrane. This experiment revealed that a previously unknown protein named Elkin1 is required to convert mechanical forces into electrical currents. Deleting this newly found protein caused skin cancer cells to move more slowly and dissociate more easily from tumour-like clusters of cells. These findings suggest that Elkin1 is part of a newly identified mechanotransduction pathway that allows cells to sense mechanical forces from their surrounding environment. More work is needed to determine what role Elkin1 plays in mechanoelectrical transduction and whether other proteins are also involved. This could lead to new approaches that prevent cancer cells from dissociating from tumours and spreading to other body parts.

Treatment of Yersinia similis with the cationic lipid DOTAP enhances adhesion to and invasion into intestinal epithelial cells - A proof-of-principle study.

The monocationic quaternary surfactant DOTAP has been used for the delivery of nucleic acids and peptides into mammalian cells. This study tested the applicability of DOTAP for the enhancement of adhesion and invasion frequencies of Yersinia (Y.) similis to enable the analysis of the effects of low-pathogenic bacteria on intestinal epithelial cells. Incubation of Y. similis with DOTAP ahead of infection of C2BBe1 intestinal epithelial cells increased invasion and adhesion frequency four- and five-fold, respectively, in plating assays. Proteomic approaches confirmed the increased bacterial load on infected cells: analysis of protein extracts by two-dimensional difference gel electrophoresis (2D-DIGE) revealed higher amounts of bacterial proteins present in the cells infected with DOTAP-treated bacteria. MALDI-TOF mass spectrometry of selected spots from gel-separated protein extracts confirmed the presence of both bacterial and human cell proteins in the samples. Label-free quantitative proteomics analysis identified 1170 human cell proteins and 699 bacterial proteins. Three times more bacterial proteins (279 vs. 93) were detected in C2BBe1 cells infected with DOTAP-treated bacteria compared to infections with untreated bacteria. Infections with DOTAP-treated Y. similis led to a significant upregulation of the stress-inducible ubiquitin-conjugating enzyme UBE2M in C2BBe1 cells. This points towards a stronger impact of the stress and infection responsive transcription factor AP-1 by enhanced bacterial load. DOTAP-treatment of uninfected C2BBe1 cells led to a significant downregulation of the transmembrane trafficking protein TMED10. The application of DOTAP could be helpful for investigating the impact of otherwise low adherent or invasive bacteria on cultivated mammalian cells without utilisation of genetic modifications.

Spatial UBE2N protein expression indicates genomic instability in colorectal cancers.

BACKGROUND: One major hallmark of colorectal cancers (CRC) is genomic instability with its contribution to tumor heterogeneity and therapy resistance. To facilitate the investigation of intra-sample phenotypes and the de novo identification of tumor sub-populations, imaging mass spectrometry (IMS) provides a powerful technique to elucidate the spatial distribution patterns of peptides and proteins in tissue sections. METHODS: In the present study, we analyzed an in-house compiled tissue microarray (n = 60) comprising CRCs and control tissues by IMS. After obtaining protein profiles through direct analysis of tissue sections, two validation sets were used for immunohistochemical evaluation. RESULTS: A total of 28 m/z values in the mass range 800-3500 Da distinguished euploid from aneuploid CRCs (p < 0.001, ROC AUC values < 0.385 or > 0.635). After liquid chromatograph-mass spectrometry identification, UBE2N could be successfully validated by immunohistochemistry in the initial sample cohort (p = 0.0274, ROC AUC = 0.7937) and in an independent sample set of 90 clinical specimens (p = 0.0070, ROC AUC = 0.6957). CONCLUSIONS: The results showed that FFPE protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value for improved molecular classification. Particularly, the protein expression of UBE2N was validated in an independent clinical cohort to distinguish euploid from aneuploid CRCs.

RNAi-mediated knockdown of the Rhau helicase preferentially depletes proteins with a Guanine-quadruplex motif in the 5'-UTR of their mRNA.

Guanine-quadruplex (G-quadruplex) structures in mRNAs have been shown to modulate gene expression. However, the overall biological relevance of this process is under debate, as cellular helicases unwind G-quadruplex structures. The helicase Rhau (encoded by the DHX36 gene) was reported to be the major source of RNA G-quadruplex resolving activity in lysates of human cells. In the current study, we depleted Rhau by RNAi-mediated silencing and analyzed the effect on proteins whose mRNAs harbor a G-quadruplex motif in their 5'-UTRs. A targeted investigation of the proto-oncogenes Bcl-2 and NRAS, which are well-known examples for the translational repression of G-quadruplex structures, did not reveal effects caused by Rhau silencing. We therefore carried out a global analysis of changes in protein levels by label-free quantification using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Following Rhau knockdown, of all the identified proteins, only 1.9% were significantly downregulated to at least 70%. According to a bioinformatic analysis with the QGRS mapper, 33% of the downregulated proteins were predicted to harbor a G-quadruplex motif in the 5'-UTR of their respective mRNAs, compared to only 11% in the complete dataset. This indicates that in an unexpectedly small set of genes, in which G-quadruplex motifs are unusually common in the 5'-UTR of their mRNAs, Rhau helicase is responsible for the regulation of their expression.

Protein Corona Formation on Colloidal Polymeric Nanoparticles and Polymeric Nanogels: Impact on Cellular Uptake, Toxicity, Immunogenicity, and Drug Release Properties.

The adsorption of biomolecules to the surface of nanoparticles (NPs) following administration into biological environments is widely recognized. In particular, the "protein corona" is well understood in terms of formation kinetics and impact upon the biological interactions of NPs. Its presence is an essential consideration in the design of therapeutic NPs. In the present study, the protein coronas of six polymeric nanoparticles of prospective therapeutic use were investigated. These included three colloidal NPs-soft core-multishell (CMS) NPs, plus solid cationic Eudragit RS (EGRS), and anionic ethyl cellulose (EC) nanoparticles-and three nanogels (NGs)-thermoresponsive dendritic-polyglycerol (dPG) nanogels (NGs) and two amino-functionalized dPG-NGs. Following incubation with human plasma, protein coronas were characterized and their biological interactions compared with pristine NPs. All NPs demonstrated protein adsorption and increased hydrodynamic diameters, although the solid EGRS and EC NPs bound notably more protein than the other tested particles. Shifts toward moderately negative surface charges were also observed for all corona bearing NPs, despite varied zeta potentials in their pristine states. While the uptake and cellular adhesion of the colloidal NPs in primary human keratinocytes and human umbilical vein endothelial cells were significantly decreased when bearing the protein corona, no obvious impact was seen in the NGs. By contrast, corona bearing NGs induced marked increases in cytokine release from primary human macrophages not seen with corona bearing colloidal NPs. Despite this, no apparent enhancement to in vitro toxicity was noted. Finally, drug release from EGRS and EC NPs was assessed, where a decrease was seen in the EGRS NPs alone. Together these results provide a direct comparison of the physical and biological impact the protein corona has on NPs of widely varied character and in particular highlights a distinction between the corona's effects on NGs and colloidal NPs.

Proteomic analysis reveals differential protein expression in variants of papillary thyroid carcinoma.

INTRODUCTION: Fine Needle Aspiration Biopsy (FNAB) allows the cytological differentiation of benign and malignant thyroid nodules. However, the method itself is not adequate in determining some cases. For example, the diagnosis of Follicular Variant Papillary Thyroid Carcinoma (FV-PTC) can be challenging. In the current study we investigate the protein profiles of FV-PTC and classical variant PTC (CV-PTC) with no lymph node metastasis and compare it with benign thyroid tissue. METHOD: We used CV-PTC (n = 6), FV-PTC (n = 6) and benign thyroid tissues (n = 6) to prepare tissue lysates. Proteins from each group were trypsin and lys-C digested. The samples were analyzed on a Q Exactive Orbitrap mass spectrometer. RESULTS: We identified 2560 proteins across all 18 specimens. Protein profiles revealed that there was no clear distinction between benign and FV-PTC samples. However, further examination of our data showed that proteins in energy metabolism have altered in FV-PTC. Proteomic pathway analysis showed marked alteration of the actin cytoskeleton proteins, especially several members of Arp2/3 complex were significantly increased in CV-PTC. We made the novel observation that IQGAP1 protein was significantly increased in CV-PTC, whereas IQGAP2 protein was highly expressed in FV-PTC lesions, suggesting differential roles of IQGAP proteins in thyroid pathology. CONCLUSION: In the present study, mass spectrometry based label free quantification approach was applied to investigate the protein profiles of FV-PTC, CV-PTC and benign thyroid tissues. This study pointed out that actin cytoskeleton proteins, IQGAP proteins and changes in energy metabolism play predominant roles in thyroid pathology.

Caloric restriction and intermittent fasting alter hepatic lipid droplet proteome and diacylglycerol species and prevent diabetes in NZO mice.

Caloric restriction and intermittent fasting are known to improve glucose homeostasis and insulin resistance in several species including humans. The aim of this study was to unravel potential mechanisms by which these interventions improve insulin sensitivity and protect from type 2 diabetes. Diabetes-susceptible New Zealand Obese mice were either 10% calorie restricted (CR) or fasted every other day (IF), and compared to ad libitum (AL) fed control mice. AL mice showed a diabetes prevalence of 43%, whereas mice under CR and IF were completely protected against hyperglycemia. Proteomic analysis of hepatic lipid droplets revealed significantly higher levels of PSMD9 (co-activator Bridge-1), MIF (macrophage migration inhibitor factor), TCEB2 (transcription elongation factor B (SIII), polypeptide 2), ACY1 (aminoacylase 1) and FABP5 (fatty acid binding protein 5), and a marked reduction of GSTA3 (glutathione S-transferase alpha 3) in samples of CR and IF mice. In addition, accumulation of diacylglycerols (DAGs) was significantly reduced in livers of IF mice (P=0.045) while CR mice showed a similar tendency (P=0.062). In particular, 9 DAG species were significantly reduced in response to IF, of which DAG-40:4 and DAG-40:7 also showed significant effects after CR. This was associated with a decreased PKCε activation and might explain the improved insulin sensitivity. In conclusion, our data indicate that protection against diabetes upon caloric restriction and intermittent fasting associates with a modulation of lipid droplet protein composition and reduction of intracellular DAG species.

Alzheimer amyloid peptide aß42 regulates gene expression of transcription and growth factors.

The pathogenesis of Alzheimer's disease (AD) is characterized by the aggregation of amyloid-ß (Aß) peptides leading to deposition of senile plaques and a progressive decline of cognitive functions, which currently remains the main criterion for its diagnosis. Robust biomarkers for AD do not yet exist, although changes in the cerebrospinal fluid levels of tau and Aß represent promising candidates in addition to brain imaging and genetic risk profiling. Although concentrations of soluble Aß42 correlate with symptoms of AD, less is known about the biological activities of Aß peptides which are generated from the amyloid-ß protein precursor. An unbiased DNA microarray study showed that Aß42, at sub-lethal concentrations, specifically increases expression of several genes in neuroblastoma cells, notably the insulin-like growth factor binding proteins 3 and 5 (IGFBP3/5), the transcription regulator inhibitor of DNA binding, and the transcription factor Lim only domain protein 4. Using qRT-PCR, we confirmed that mRNA levels of the identified candidate genes were exclusively increased by the potentially neurotoxic Aß42 wild-type peptide, as both the less toxic Aß40 and a non-toxic substitution peptide Aß42 G33A did not affect mRNA levels. In vivo immunohistochemistry revealed a corresponding increase in both hippocampal and cortical IGFBP5 expression in an AD mouse model. Proteomic analyses of human AD cerebrospinal fluid displayed increased in vivo concentrations of IGFBPs. IGFBPs and transcription factors, as identified here, are modulated by soluble Aß42 and may represent useful early biomarkers.

IPG strip-based peptide fractionation for shotgun proteomics.

Efficient fractionation of peptides is an essential prerequisite for comprehensive analysis of complex protein mixtures by shotgun mass spectrometry. The separation of peptides by isoelectric focusing is particularly attractive due to its orthogonality to reverse-phase HPLC. Here, we present a protocol for in-gel peptide isoelectric focusing using immobilized pH gradient strips. The method shows high resolving power for up to 1 mg of sample and is highly reproducible.