A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

BRCA1 Mutation Leads to Deregulated Ubc9 Levels which Triggers Proliferation and Migration of Patient-Derived High Grade Serous Ovarian Cancer and Triple Negative Breast Cancer Cells

Women who carry a germline mutation in BRCA1 gene typically develop triple negative breast cancers (TNBC) and high grade serous ovarian cancers (HGSOC). Previously, we reported that wild type BRCA1 proteins, unlike the disease-associated mutant BRCA1 proteins to bind the sole sumo E2-conjugating enzyme Ubc9. In this study, we have used clinically relevant cell lines with known BRCA1 mutations and report the in-vivo association of BRCA1 and Ubc9 in normal mammary epithelial cells but not in BRCA1 mutant HGSOC and TNBC cells by immunofluorescence analysis. BRCA1-mutant HGSOC / TNBC cells and ovarian tumor tissues showed increased expression of Ubc9 compared to BRCA1 reconstituted HGSOC, normal mammary epithelial cells and matched normal ovarian tissues. Knockdown of Ubc9 expression resulted in decreased proliferation and migration of BRCA1 mutant TNBC and HGSOC cells. This is the first study demonstrating the functional link between BRCA1 mutation, high Ubc9 expression and increased migration of HGSOC and TNBC cells. High Ubc9 expression due to BRCA1 mutation may trigger an early growth and transformation advantage to normal breast and ovarian epithelial cells resulting in aggressive cancers. Future work will focus on studying whether Ubc9 expression could show a positive correlation with BRCA1 linked HGSOC and basal like TNBC phenotype

Truncated and Helix-Constrained Peptides with High Affinity and Specificity for the cFos Coiled-Coil of AP-1

Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i-->i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, alpha-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable alpha-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ~9 kcal/mol, but this was compensated by increased conformational entropy (TDS ≤ 7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by alpha-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases

Combinations of β-lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101) and MRSA (n = 115). Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI), plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin) displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin) in 87-89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA infections. We demonstrated that plectasin strongly rejuvenates the therapeutic potencies of existing antibiotics in vitro and in vivo. This is a novel strategy that can have major clinical implications in our fight against bacterial infections

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

High Protein Binding and Cidal Activity against Penicillin-Resistant S. pneumoniae: A Cefditoren In Vitro Pharmacodynamic Simulation

BACKGROUND: Although protein binding is a reversible phenomenon, it is assumed that antibacterial activity is exclusively exerted by the free (unbound) fraction of antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Activity of cefditoren, a highly protein bound 3(rd) generation cephalosporin, over 24h after an oral 400 mg cefditoren-pivoxil bid regimen was studied against six S. pneumoniae strains (penicillin/cefditoren MICs; microg/ml): S1 (0.12/0.25), S2 (0.25/0.25), S3 and S4 (0.5/0.5), S5 (1/0.5) and S6 (4/0.5). A computerized pharmacodynamic simulation with media consisting in 75% human serum and 25% broth (mean albumin concentrations = 4.85+/-0.12 g/dL) was performed. Protein binding was measured. The cumulative percentage of a 24h-period that drug concentrations exceeded the MIC for total (T > MIC) and unbound concentrations (fT > MIC), expressed as percentage of the dosing interval, were determined. Protein binding was 87.1%. Bactericidal activity (> or = 99.9% initial inocula reduction) was obtained against strains S1 and S2 at 24h (T > MIC = 77.6%, fT > MIC = 23.7%). With T > MIC of 61.6% (fT > MIC = 1.7%), reductions against S3 and S4 ranged from 90% to 97% at 12h and 24h; against S5, reduction was 45.1% at 12h and up to 85.0% at 24h; and against S6, reduction was 91.8% at 12h, but due to regrowth of 52.9% at 24h. Cefditoren physiological concentrations exerted antibacterial activity against strains exhibiting MICs of 0.25 and 0.5 microg/ml under protein binding conditions similar to those in humans. CONCLUSIONS/SIGNIFICANCE: The results of this study suggest that, from the pharmacodynamic perspective, the presence of physiological albumin concentrations may not preclude antipneumococcal activity of highly bound cephalosporins as cefditoren

Reduction of metastasis using a non-volatile buffer

The tumor microenvironment is acidic as a consequence of upregulated glycolysis and poor perfusion and this acidity, in turn, promotes invasion and metastasis. We have recently demonstrated that chronic consumption of sodium bicarbonate increased tumor pH and reduced spontaneous and experimental metastases. This occurred without affecting systemic pH, which was compensated. Additionally, these prior data did not rule out the possibility that bicarbonate was working though effects on carbonic anhydrase, and not as a buffer per se. Here, we present evidence that chronic ingestion of a non-volatile buffer, 2-imidazole-1-yl-3-ethoxycarbonylpropionic acid (IEPA) with a pKa of 6.9 also reduced metastasis in an experimental PC3M prostate cancer mouse model. Animals (n = 30) were injected with luciferase expressing PC3M prostate cancer cells either subcutaneously (s.c., n = 10) or intravenously (i.v., n = 20). Four days prior to inoculations, half of the animals for each experiment were provided drinking water containing 200 mM IEPA buffer. Animals were imaged weekly to follow metastasis, and these data showed that animals treated with IEPA had significantly fewer experimental lung metastasis compared to control groups (P < 0.04). Consistent with prior work, the pH of treated tumors was elevated compared to controls. IEPA is observable by in vivo magnetic resonance spectroscopy and this was used to measure the presence of IEPA in the bladder, confirming that it was orally available. The results of this study indicate that metastasis can be reduced by non-volatile buffers as well as bicarbonate and thus the effect appears to be due to pH buffering per se

Current views on the role of Notch signaling and the pathogenesis of human leukemia

The Notch signaling pathway is highly conserved from Drosophila to humans and plays an important role in the regulation of cellular proliferation, differentiation and apoptosis

Protective effects of a compound herbal extract (Tong Xin Luo) on free fatty acid induced endothelial injury: Implications of antioxidant system

<p>Abstract</p> <p>Background</p> <p>Tong-Xin-Luo (TXL) – a mixture of herbal extracts, has been used in Chinese medicine with established therapeutic efficacy in patients with coronary artery disease.</p> <p>Methods</p> <p>We investigated the protective role of TXL extracts on endothelial cells injured by a known risk factor – palmitic acid (PA), which is elevated in metabolic syndrome and associated with cardiovascular complications. Human aortic endothelial cells (HAECs) were preconditioned with TXL extracts before exposed to PA for 24 hours.</p> <p>Results</p> <p>We found that PA (0.5 mM) exposure induced 73% apoptosis in endothelial cells. However, when HAECs were preconditioned with ethanol extracted TXL (100 μg/ml), PA induced only 7% of the endothelial cells into apoptosis. Using antibody-based protein microarray, we found that TXL attenuated PA-induced activation of p38-MAPK stress pathway. To investigate the mechanisms involved in TXL's protective effects, we found that TXL reduced PA-induced intracellular oxidative stress. Through AMPK pathway, TXL restored the intracellular antioxidant system, which was depressed by the PA treatment, with an increased expression of thioredoxin and a decreased expression of the thioredoxin interacting protein.</p> <p>Conclusion</p> <p>In summary, our study demonstrates that TXL protects endothelial cells from PA-induced injury. This protection is likely mediated by boosting intracellular antioxidant capacity through AMPK pathway, which may account for the therapeutic efficacy in TXL-mediated cardiovascular protection.</p