A comparative study of carbonic anhydrase activity in lymphocytes from colorectal cancer tissues and adjacent healthy counterparts.

Several carbonic anhydrase (CA, EC 4.2.1.1) isoforms play an essential role in processes connected to tumorigenesis, as they efficiently accelerate the hydration of carbon dioxide to bicarbonate and proton. In this context, examples are CA IX and CA XII, which were proved to be upregulated in many solid malignancies. On the other hand, cancer and the immune system are inextricably linked, and targeting the immune checkpoints recently was shown to efficiently improve the treatment of malignancies. In this study, we have investigated the expression of CA isoforms in tumour-infiltrating lymphocytes (TILs) that, according to the immunosurveillance theory, were suggested to have a crucial role in the development of colorectal cancer (CRC). T lymphocytes isolated from healthy surrounding mucosa showed a higher CA activity compared to those present in tumour and peripheral blood in the same patients. CA I and II were confirmed as enzyme isoforms involved in the process, as determined by proteomic analysis of corresponding TIL samples. These preliminary findings suggest a dysregulation of the local immune response in the CRC tissues and a loss of effective anticancer mechanisms mediated by CAs therein.

Tuberomics: a molecular profiling for the adaption of edible fungi (Tuber magnatum Pico) to different natural environments.

BACKGROUND: Truffles are symbiotic fungi that develop underground in association with plant roots, forming ectomycorrhizae. They are primarily known for the organoleptic qualities of their hypogeous fruiting bodies. Primarily, Tuber magnatum Pico is a greatly appreciated truffle species mainly distributed in Italy and Balkans. Its price and features are mostly depending on its geographical origin. However, the genetic variation within T. magnatum has been only partially investigated as well as its adaptation to several environments. RESULTS: Here, we applied an integrated omic strategy to T. magnatum fruiting bodies collected during several seasons from three different areas located in the North, Center and South of Italy, with the aim to distinguish them according to molecular and biochemical traits and to verify the impact of several environments on these properties. With the proteomic approach based on two-dimensional electrophoresis (2-DE) followed by mass spectrometry, we were able to identify proteins specifically linked to the sample origin. We further associated the proteomic results to an RNA-seq profiling, which confirmed the possibility to differentiate samples according to their source and provided a basis for the detailed analysis of genes involved in sulfur metabolism. Finally, geographical specificities were associated with the set of volatile compounds produced by the fruiting bodies, as quantitatively and qualitatively determined through proton transfer reaction-mass spectrometry (PTR-MS) and gas-chromatography-mass spectrometry (GC-MS). In particular, a partial least squares-discriminant analysis (PLS-DA) model built from the latter data was able to return high confidence predictions of sample source. CONCLUSIONS: Results provide a characterization of white fruiting bodies by a wide range of different molecules, suggesting the role for specific compounds in the responses and adaptation to distinct environments.

An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population.

The H1069Q substitution is the most frequent mutation of the Cu transporter ATP7B that causes Wilson disease in the Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes, but, in the presence of excessive Cu, it relocates to the endo-lysosomal compartment to excrete Cu via bile canaliculi. In contrast, ATP7B-H1069Q is strongly retained in the ER, does not reach the Golgi complex and fails to move to the endo-lysosomal compartment in the presence of excessive Cu, thus causing toxic Cu accumulation. We have previously shown that, in transfected cells, the small heat-shock protein αB-crystallin is able to correct the mislocalization of ATP7B-H1069Q and its trafficking in the presence of Cu overload. Here, we first show that the α-crystallin domain of αB-crystallin mimics the effect of the full-length protein, whereas the N- and C-terminal domains have no such effect. Next, and most importantly, we demonstrate that a twenty-residue peptide derived from the α-crystallin domain of αB-crystallin fully rescues Golgi localization and the trafficking response of ATP7B-H1069Q in the presence of Cu overload. In addition, we show that this peptide interacts with the mutant transporter in the live cell. These results open the way to attempt developing a pharmacologically active peptide to specifically contrast the Wilson disease form caused by the ATP7B-H1069Q mutant.

Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach.

INTRODUCTION: MicroRNAs (miRs) regulate gene expression to support important physiological functions. Significant evidences suggest that miRs play a crucial role in many pathological events and in the cell response to various stresses. METHODS: With the aim to identify new miRs induced by perturbation of intracellular calcium homeostasis, we analysed miR expression profiles of thapsigargin (TG)-treated cells by microarray. In order to identify miR-663a-regulated genes, we evaluated proteomic changes in miR-663a-overexpressing cells by two-dimensional differential in-gel electrophoresis coupled to mass spectrometric identification of the differentially represented proteins. Microarray and proteomic analyses were supported by biochemical validation. RESULTS: Results of microarray revealed 24 differentially expressed miRs; among them, miR-663a turned out to be by ER stress and under the control of the PERK pathway of the unfolded protein response. Proteomic analysis revealed that PLOD3, which is the gene encoding for collagen-modifying lysyl hydroxylase 3 (LH3), is regulated by miR-663a. Luciferase reporter assays demonstrated that miR-663a indeed reduces LH3 expression by targeting to 3'-UTR of PLOD3 mRNA. Interestingly, miR-663a inhibition of LH3 expression generates reduced extracellular accumulation of type IV collagen, thus suggesting the involvement of miR-663a in modulating collagen 4 secretion in physiological conditions and in response to ER stress. CONCLUSION: The finding of the ER stress-induced PERK-miR-663a pathway may have important implications in the understanding of the molecular mechanisms underlying the function of this miR in normal and/or pathological conditions.

Inhibition of PID1/NYGGF4/PCLI1 gene expression highlights its role in the early events of the cell cycle in NIH3T3 fibroblasts.

The PID1/NYGGF4/PCLI1 gene encodes for a protein with a phosphotyrosine-binding domain, which interacts with the lipoprotein receptor-related protein 1. Previous work by us and others suggested a function of the gene in cell proliferation of NIH3T3 fibroblasts and 3T3-L1 pre-adipocytes. The molecular characterization of PCLI1 protein, ectopically expressed in NIH3T3 fibroblasts, revealed two phosphorylation sites at Ser154 and Ser165. In order to clarify the functions of this gene, we analyzed the effects of its downregulation on cellular proliferation and cell cycle progression in NIH3T3 cell cultures. Downregulation of PID1/NYGGF4/PCLI1 mRNA levels by short hairpin RNAs (shRNAs) elicited decreased proliferation rate in mammalian cell lines; cell cycle analysis of serum-starved, synchronized NIH3T3 fibroblasts showed an increased accumulation of shRNA-interfered cells in the G1 phase. Decreased levels of FOS and MYC mRNAs were accordingly associated with these events. The molecular scenario emerging from our data suggests that PID1/NYGGF4/PCLI1 controls cellular proliferation and cell cycle progression in NIH3T3 cells.

The cytosolic chaperone α-crystallin B rescues folding and compartmentalization of misfolded multispan transmembrane proteins.

The α-crystallin B chain (CRYAB or HspB5) is a cytosolic chaperone belonging to the small heat shock protein family, which is known to help in the folding of cytosolic proteins. Here we show that CRYAB binds the mutant form of at least two multispan transmembrane proteins (TMPs), exerting an anti-aggregation activity. It rescues the folding of mutant Frizzled4, which is responsible for a rare autosomal dominant form of familial exudative vitreoretinopathy (Fz4-FEVR), and the mutant ATP7B Cu transporter (ATP7B-H1069Q) associated with a common form of Wilson's disease. In the case of Fz4-FEVR, CRYAB prevents the formation of inter-chain disulfide bridges between the lumenal ectodomains of the aggregated mutant chains, which enables correct folding and promotes appropriate compartmentalization on the plasma membrane. ATP7B-H1069Q, with help from CRYAB, folds into the proper conformation, moves to the Golgi complex, and responds to copper overload in the same manner as wild-type ATP7B. These findings strongly suggest that CRYAB plays a pivotal role, previously undetected, in the folding of multispan TMPs and, from the cytosol, is able to orchestrate folding events that take place in the lumen of the ER. Our results contribute to the explanation of the complex scenario behind multispan TMP folding; additionally, they serve to expose interesting avenues for novel therapeutic approaches.

Non-enzymatic glycation and glycoxidation protein products in foods and diseases: an interconnected, complex scenario fully open to innovative proteomic studies.

The Maillard reaction includes a complex network of processes affecting food and biopharmaceutical products; it also occurs in living organisms and has been strictly related to cell aging, to the pathogenesis of several (chronic) diseases, such as diabetes, uremia, cataract, liver cirrhosis and various neurodegenerative pathologies, as well as to peritoneal dialysis treatment. Dozens of compounds are involved in this process, among which a number of protein-adducted derivatives that have been simplistically defined as early, intermediate and advanced glycation end-products. In the last decade, various bottom-up proteomic approaches have been successfully used for the identification of glycation/glycoxidation protein targets as well as for the characterization of the corresponding adducts, including assignment of the modified amino acids. This article provides an updated overview of the mass spectrometry-based procedures developed to this purpose, emphasizing their partial limits with respect to current proteomic approaches for the analysis of other post-translational modifications. These limitations are mainly related to the concomitant sheer diversity, chemical complexity, and variable abundance of the various derivatives to be characterized. Some challenges to scientists are finally proposed for future proteomic investigations to solve main drawbacks in this research field.

Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts.

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (P≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-ß-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.

Glomerular autoimmune multicomponents of human lupus nephritis in vivo: α-enolase and annexin AI.

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum.

Phenotypical and biochemical characterization of tomato plants treated with triacontanol.

Biostimulants are heterogeneous products designed to support plant development and to improve the yield and quality of crops. Here, we focused on the effects of triacontanol, a promising biostimulant found in cuticle waxes, on tomato growth and productivity. We examined various phenological traits related to vegetative growth, flowering and fruit yield, the metabolic profile of fruits, and the response of triacontanol-treated plants to salt stress. Additionally, a proteomic analysis was conducted to clarify the molecular mechanisms underlying triacontanol action. Triacontanol application induced advanced and increased blooming without affecting plant growth. Biochemical analyses of fruits showed minimal changes in nutritional properties. The treatment also increased the germination rate of seeds by altering hormone homeostasis and reduced salt stress-induced damage. Proteomics analysis of leaves revealed that triacontanol increased the abundance of proteins related to development and abiotic stress, while down-regulating proteins involved in biotic stress resistance. The proteome of the fruits was not significantly affected by triacontanol, confirming that biostimulation did not alter the nutritional properties of fruits. Overall, our findings provide evidence of the effects of triacontanol on growth, development, and stress tolerance, shedding light on its mechanism of action and providing new insights into its potential in agricultural practices.

Downregulation of praja2 restrains endocytosis and boosts tyrosine kinase receptors in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC will provide clues to treat this aggressive malignant tumor. Here, we report that the ubiquitin ligase praja2 forms a complex with-and ubiquitylates the AP2 adapter complex, contributing to receptor endocytosis and clearance. In human RCC tissues and cells, downregulation of praja2 by oncogenic miRNAs (oncomiRs) and the proteasome markedly impairs endocytosis and clearance of the epidermal growth factor receptor (EGFR), and amplifies downstream mitogenic and proliferative signaling. Restoring praja2 levels in RCC cells downregulates EGFR, rewires cancer cell metabolism and ultimately inhibits tumor cell growth and metastasis. Accordingly, genetic ablation of praja2 in mice upregulates RTKs (i.e. EGFR and VEGFR) and induces epithelial and vascular alterations in the kidney tissue.In summary, our findings identify a regulatory loop between oncomiRs and the ubiquitin proteasome system that finely controls RTKs endocytosis and clearance, positively impacting mitogenic signaling and kidney cancer growth.

Increased anaerobic metabolism is a distinctive signature in a colorectal cancer cellular model of resistance to antiepidermal growth factor receptor antibody.

Cetuximab is a chimeric antibody approved for the treatment of metastatic colorectal cancer that selectively targets epidermal growth factor receptor (EGFR) signaling. Treatment efficacy with this drug is often impaired by acquired resistance and poor information has been accumulated on the mechanisms underlying such a phenomenon. By taking advantage of a syngenic cellular system of sensitivity and acquired resistance to anti-EGFR therapy in the colorectal carcinoma GEO cell line, we profiled protein expression differences between Cetuximab-sensitive and -resistant cells. Combined 2D DIGE and MS analyses revealed a main proteomic signature resulting from selective deregulation of various metabolic enzymes, including glucose-6-phosphate dehydrogenase, transketolase, lactate dehydrogenase B, and pyruvate dehydrogenase E1, which was also confirmed by Western blotting experiments. Lactate dehydrogenase B downregulation has been already related to an increased anaerobic utilization of glucose by tumor cells; accordingly, we verified that Cetuximab-resistant cells have a significantly higher production of lactate. Resistant cells also showed decreased nicotinamide adenine dinucleotide phosphate (NADPH) levels. Observed protein deregulations were not related to functional alterations of the hypoxia-inducible factor 1-associated pathways. Our data demonstrate that increased anaerobic metabolism is a prominent feature observed in the GEO syngenic model of acquired resistance to anti-EGFR therapy in colorectal cancer.

Transcriptomic and proteomic analysis of a compatible tomato-aphid interaction reveals a predominant salicylic acid-dependent plant response.

BACKGROUND: Aphids are among the most destructive pests in temperate climates, causing significant damage on several crops including tomato. We carried out a transcriptomic and proteomic study to get insights into the molecular mechanisms and dynamics of the tomato response to the Macrosyphum euphorbiae aphid. RESULTS: The time course analysis of aphid infestation indicated a complex, dynamic pattern of gene expression. Several biological functions were affected and genes related to the stress and defence response were the most represented. The Gene Ontology categories of the differentially expressed genes (899) and identified proteins (57) indicated that the tomato response is characterized by an increased oxidative stress accompanied by the production of proteins involved in the detoxification of oxygen radicals. Aphids elicit a defense reaction based on the cross-communication of different hormone-related signaling pathways such as those related to the salicylic acid (SA), jasmonic acid (JA), ethylene and brassinosteroids. Among them, the SA-signaling pathway and stress-responsive SA-dependent genes play a dominant role. Furthermore, tomato response is characterized by a reduced accumulation of photosynthetic proteins and a modification of the expression of various cell wall related genes. CONCLUSIONS: Our work allowed a more comprehensive understanding of the signaling events and the defense dynamics of the tomato response to aphids in a compatible interaction and, based on experimental data, a model of the tomato-aphid molecular interaction was proposed. Considering the rapid advancement of tomato genomics, this information will be important for the development of new protection strategies.

AUF1 Recognizes 8-Oxo-Guanosine Embedded in DNA and Stimulates APE1 Endoribonuclease Activity.

Aims: The existence of modified ribonucleotide monophosphates embedded in genomic DNA, as a consequence of oxidative stress conditions, including 8-oxo-guanosine and ribose monophosphate abasic site (rAP), has been recently highlighted by several works and associated with oxidative stress conditions. Although human apurinic-apyrimidinic endodeoxyribonuclease 1 (APE1), a key enzyme of the base-excision repair pathway, repairs rAP sites and canonical deoxyribose monophosphate abasic sites with similar efficiency, its incision-repairing activity on 8-oxo-guanosine is very weak. The aims of this work were to: (i) identify proteins able to specifically bind 8-oxo-guanosine embedded in DNA and promote APE1 endoribonuclease activity on this lesion, and (ii) characterize the molecular and biological relevance of this interaction using human cancer cell lines. Results: By using an unbiased proteomic approach, we discovered that the AU-rich element RNA-binding protein 1 (AUF1) actively recognizes 8-oxo-guanosine and stimulates the APE1 enzymatic activity on this DNA lesion. By using orthogonal approaches, we found that: (i) the interaction between AUF1 and APE1 is modulated by H2O2-treatment; (ii) depletion of APE1 and AUF1 causes the accumulation of single- and double- strand breaks; and (iii) both proteins are involved in modulating the formation of DNA:RNA hybrids. Innovation: These results establish unexpected functions of AUF1 in modulating genome stability and improve our knowledge of APE1 biology with respect to 8-oxo-guanosine embedded in DNA. Conclusion: By showing a novel function of AUF1, our findings shed new light on the process of genome stability in mammalian cells toward oxidative stress-related damages. Antioxid. Redox Signal. 39, 411-431.

Fructose Diet-Associated Molecular Alterations in Hypothalamus of Adolescent Rats: A Proteomic Approach.

BACKGROUND: The enhanced consumption of fructose as added sugar represents a major health concern. Due to the complexity and multiplicity of hypothalamic functions, we aim to point out early molecular alterations triggered by a sugar-rich diet throughout adolescence, and to verify their persistence until the young adulthood phase. METHODS: Thirty days old rats received a high-fructose or control diet for 3 weeks. At the end of the experimental period, treated animals were switched to the control diet for further 3 weeks, and then analyzed in comparison with those that were fed the control diet for the entire experimental period. RESULTS: Quantitative proteomics identified 19 differentially represented proteins, between control and fructose-fed groups, belonging to intermediate filament cytoskeleton, neurofilament, pore complex and mitochondrial respiratory chain complexes. Western blotting analysis confirmed proteomic data, evidencing a decreased abundance of mitochondrial respiratory complexes and voltage-dependent anion channel 1, the coregulator of mitochondrial biogenesis PGC-1α, and the protein subunit of neurofilaments α-internexin in fructose-fed rats. Diet-associated hypothalamic inflammation was also detected. Finally, the amount of brain-derived neurotrophic factor and its high-affinity receptor TrkB, as well as of synaptophysin, synaptotagmin, and post-synaptic protein PSD-95 was reduced in sugar-fed rats. Notably, deregulated levels of all proteins were fully rescued after switching to the control diet. CONCLUSIONS: A short-term fructose-rich diet in adolescent rats induces hypothalamic inflammation and highly affects mitochondrial and cytoskeletal compartments, as well as the level of specific markers of brain function; above-reported effects are reverted after switching animals to the control diet.

FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

FKBP51 plays a relevant role in sustaining cancer cells, particularly melanoma. This cochaperone participates in several signaling pathways. FKBP51 forms a complex with Akt and PHLPP, which is reported to dephosphorylate Akt. Given the recent discovery of a spliced FKBP51 isoform, in this paper, we interrogate the canonical and spliced isoforms in regulation of Akt activation. We show that the TPR domain of FKBP51 mediates Akt ubiquitination at K63, which is an essential step for Akt activation. The spliced FKBP51, lacking such domain, cannot link K63-Ub residues to Akt. Unexpectedly, PHLPP silencing does not foster phosphorylation of Akt, and its overexpression even induces phosphorylation of Akt. PHLPP stabilizes levels of E3-ubiquitin ligase TRAF6 and supports K63-ubiquitination of Akt. The interactome profile of FKBP51 from melanoma cells highlights a relevant role for PHLPP in improving oncogenic hallmarks, particularly, cell proliferation.

Combinatorial peptide ligand libraries for the analysis of low-expression proteins: Validation for normal urine and definition of a first protein MAP.

In this review, we report the evolution on experimental conditions for the analysis of normal urine based on combinatorial peptide ligand library (CPLL) treatment and successive 2-DE and 2-DE/MS analysis. The main topics are (i) definition of the urine sample requirements, (ii) optimization of the urine/ligand ratio, (iii) essay conditions, (iv) en bloc elution. Overall, normal urine protein composition as studied by 2-DE includes over 2600 spots. Relevant data on inter and intraessay reproducibility obtained by the analysis of different normal urines repeated several times are also here presented. We found a 73% reproducibility upon analysis of the same sample and 68% correspondence of protein composition among different normal urine samples. Based on the above results, we are completing the characterization with LC-MS of 249 spots. The composition of normal urine proteins after CPLLs is finally shown with the indication of those spots which are currently under identification. This map will be completed in a near future; in the meantime this would represent the basic reference sample for newly developed studies on human diseases.

Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions.

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein-protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

Targeted inhibition of ubiquitin signaling reverses metabolic reprogramming and suppresses glioblastoma growth.

Glioblastoma multiforme (GBM) is the most frequent and aggressive form of primary brain tumor in the adult population; its high recurrence rate and resistance to current therapeutics urgently demand a better therapy. Regulation of protein stability by the ubiquitin proteasome system (UPS) represents an important control mechanism of cell growth. UPS deregulation is mechanistically linked to the development and progression of a variety of human cancers, including GBM. Thus, the UPS represents a potentially valuable target for GBM treatment. Using an integrated approach that includes proteomics, transcriptomics and metabolic profiling, we identify praja2, a RING E3 ubiquitin ligase, as the key component of a signaling network that regulates GBM cell growth and metabolism. Praja2 is preferentially expressed in primary GBM lesions expressing the wild-type isocitrate dehydrogenase 1 gene (IDH1). Mechanistically, we found that praja2 ubiquitylates and degrades the kinase suppressor of Ras 2 (KSR2). As a consequence, praja2 restrains the activity of downstream AMP-dependent protein kinase in GBM cells and attenuates the oxidative metabolism. Delivery in the brain of siRNA targeting praja2 by transferrin-targeted self-assembling nanoparticles (SANPs) prevented KSR2 degradation and inhibited GBM growth, reducing the size of the tumor and prolonging the survival rate of treated mice. These data identify praja2 as an essential regulator of cancer cell metabolism, and as a potential therapeutic target to suppress GBM growth.

Mapping phosphoproteins in Neisseria meningitidis serogroup A.

To investigate the phosphorylation capability of serogroup A Neisseria meningitidis (MenA) and to implement our knowledge in meningococcal biology and in bacterial post-translational modifications, cell extracts were separated by 2-DE and 51 novel phosphoproteins were revealed by the use of the highly specific Ser/Thr/Tyr-phosphorylated proteins staining by Pro-Q Diamond and identified by MALDI-ToF/MS. Our results indicate that phosphorylation in MenA is comparable to that of other bacterial species. A first functional characterization of the identified modified proteins was also given, in order to understand their role in meningococcal physiopathology.