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The nuclear receptor-related 1 protein, Nurr1, is a transcription factor critical for the development and maintenance of dopamine-producing neurons in the substantia nigra pars compacta, a cell population that progressively loses the ability to make dopamine and degenerates in Parkinson's disease. Recently, we demonstrated that Nurr1 binds directly to and is regulated by the endogenous dopamine metabolite 5,6-dihydroxyindole (DHI). Unfortunately, DHI is an unstable compound, and thus a poor tool for studying Nurr1 function. Here, we report that 5-chloroindole, an unreactive analog of DHI, binds directly to the Nurr1 ligand binding domain with micromolar affinity and stimulates the activity of Nurr1, including the transcription of genes governing the synthesis and packaging of dopamine.

Human MondoA requires glucose as well as other modulatory signals to function in transcription. One such signal is acidosis, which increases MondoA activity and also drives a protective gene signature in breast cancer. How low pH controls MondoA transcriptional activity is unknown. We found that low pH medium increases mitochondrial ATP (mtATP), which is subsequently exported from the mitochondrial matrix. Mitochondria-bound hexokinase transfers a phosphate from mtATP to cytoplasmic glucose to generate glucose-6-phosphate (G6P), which is an established MondoA activator. The outer mitochondrial membrane localization of MondoA suggests that it is positioned to coordinate the adaptive transcriptional response to a cells most abundant energy sources, cytoplasmic glucose and mtATP. In response to acidosis, MondoA shows preferential binding to just two targets, TXNIP and its paralog ARRDC4. Because these transcriptional targets are suppressors of glucose uptake, we propose that MondoA is critical for restoring metabolic homeostasis in response to high energy charge.

Triple-negative breast cancer (TNBC) is an aggressive subset of breast carcinomas that lack expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). Unlike other breast cancer subtypes, targeted therapy is presently unavailable for patients with TNBC. In spite of initial responses to chemotherapy, drug resistance tends to develop rapidly and the prognosis of metastatic TNBC is poor. Hence, there is an urgent need for novel-targeted treatment methods or development of safe and effective alternatives with recognized mechanism(s) of action. AMP-activated protein kinase (AMPK), an energy sensor, can regulate protein and lipid metabolism responding to alterations in energy supply. In the past 10 years, interest in AMPK has increased widely since it appeared as an attractive targeting molecule for cancer therapy. There has been a deep understanding of the possible role of abnormal AMPK signaling pathways in the regulation of growth and survival and the development of drug resistance in TNBC. The increasing popularity of using AMPK regulators for TNBC-targeted therapy is supported by a considerable development in ascertaining the molecular pathways implicated. This review highlights the available evidence for AMPK-targeted anti-TNBC activity of various agents or treatment strategies, with special attention placed on recent preclinical and clinical advances in the manipulation of AMPK in TNBC. The elaborative analysis of these AMPK-related signaling pathways will have a noteworthy impact on the development of AMPK regulators, resulting in efficacious treatments for this lethal disease.

Triple-negative breast cancer (TNBC) is an aggressive subset of breast carcinomas that lack expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). Unlike other breast cancer subtypes, targeted therapy is presently unavailable for patients with TNBC. In spite of initial responses to chemotherapy, drug resistance tends to develop rapidly and the prognosis of metastatic TNBC is poor. Hence, there is an urgent need for novel-targeted treatment methods or development of safe and effective alternatives with recognized mechanism(s) of action. AMP-activated protein kinase (AMPK), an energy sensor, can regulate protein and lipid metabolism responding to alterations in energy supply. In the past 10 years, interest in AMPK has increased widely since it appeared as an attractive targeting molecule for cancer therapy. There has been a deep understanding of the possible role of abnormal AMPK signaling pathways in the regulation of growth and survival and the development of drug resistance in TNBC. The increasing popularity of using AMPK regulators for TNBC-targeted therapy is supported by a considerable development in ascertaining the molecular pathways implicated. This review highlights the available evidence for AMPK-targeted anti-TNBC activity of various agents or treatment strategies, with special attention placed on recent preclinical and clinical advances in the manipulation of AMPK in TNBC. The elaborative analysis of these AMPK-related signaling pathways will have a noteworthy impact on the development of AMPK regulators, resulting in efficacious treatments for this lethal disease.

Neurodegenerative diseases are a class of age-associated proteopathies characterized by the accumulation of misfolded and/or aggregation-prone proteins. This imbalance has been attributed, in part, to an age-dependent decay in the capacity of protein turnover. Most proteins are degraded by the ubiquitin-proteasome system (UPS), which is composed of ubiquitin ligases and regulatory particles, such as the 19S, that deliver cargo to the proteolytically active 20S proteasome (20S) core. However, a subset of clients, especially intrinsically disordered proteins (IDPs), are also removed by the action of the ubiquitin-independent proteasome system (UIPS). What are the specific contributions of the UPS and UIPS in the context of neurodegeneration? Here, we explore how age-associated changes in the relative contribution of the UPS and UIPS, combined with the IDP-like structure of many neurodegenerative disease-associated proteins, might contribute. Strikingly, the 20S has been shown to predominate in older neurons and to preferentially act on relevant substrates, such as synuclein and tau. Moreover, pharmacological activation of the 20S has been shown to accelerate removal of aggregation-prone proteins in some models. Together, these recent studies are turning attention to the 20S and the UIPS as potential therapeutic targets in neurodegeneration.

Pathological cardiac hypertrophy, which is a compensatory mechanism established to maintain cardiac function in response to neurohormonal or mechanical stresses, becomes maladaptive with time and frequently leads to heart failure. AMP-activated protein kinase (AMPK) has been extensively described in the literature to act as a break in cardiac hypertrophy development. Its anti-hypertrophic action mostly correlates with the inhibition of several important players of cardiac hypertrophy including protein synthesis and pro-hypertrophic gene expression pathways involving the transcription factor nuclear factor of activated T cells (NFAT) and the mitogen-activated protein kinases ERK1/2. In this chapter, we describe methodologies designed to evaluate cardiomyocyte hypertrophy and its major molecular mechanisms in response to AMPK activation. Two different compounds, AICAr and the biguanide phenformin, were used to promote AMPK activation.

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a key regulator of protein synthesis in mammalian cells. It phosphorylates and inhibits eEF2, the translation factor necessary for peptide translocation during the elongation phase of protein synthesis. When cellular energy demand outweighs energy supply, AMP-activated protein kinase (AMPK) and eEF2K become activated, leading to eEF2 phosphorylation, which reduces the rate of protein synthesis, a process that consumes a large proportion of cellular energy under optimal conditions. The goal of the present study was to elucidate the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation to decrease protein synthesis. Using genetically modified mouse embryo fibroblasts (MEFs), effects of treatments with commonly used AMPK activators to increase eEF2 phosphorylation were compared with that of the novel compound 991. Bacterially expressed recombinant eEF2K was phosphorylated in vitro by recombinant activated AMPK for phosphorylation site-identification by mass spectrometry followed by site-directed mutagenesis of the identified sites to alanine residues to study effects on the kinetic properties of eEF2K. Wild-type eEF2K and a Ser491/Ser492 mutant were retrovirally re-introduced in eEF2K-deficient MEFs and effects of 991 treatment on eEF2 phosphorylation and protein synthesis rates were studied in these cells. AMPK activation leads to increased eEF2 phosphorylation in MEFs mainly by direct activation of eEF2K and partly by inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment of MEFs with AMPK activators can also lead to eEF2K activation independently of AMPK probably via a rise in intracellular Ca. AMPK activates eEF2K by multi-site phosphorylation and the newly identified Ser491/Ser492 is important for activation, leading to mTOR-independent inhibition of protein synthesis. Our study provides new insights into the control of eEF2K by AMPK, with implications for li

Angiotensin-converting enzyme (ACE) plays a dominant role in blood pressure regulating system. Synthetic ACE inhibitor is designed as an antihypertensive drug to restrict ACE activity. However, the usage of synthetic drug may cause several adverse effects. Hence, a natural food protein with ACE inhibitory activity may be promising as a safer alternative to the synthetic drug. Blue lupin (Lupinus angustifolius) is one of the legumes that rich in protein. It has the potential to be a good source of ACE inhibitory peptides. However, blue lupin is usually served as an animal feed and consumption by human is rare. Therefore, this study was carried out to investigate the nutritional, protein and amino acid composition of blue lupin flour, and to evaluate the ACE inhibitory activity of its protein hydrolysates. The results revealed that the lupin flour was abundant in protein (43.3 g/100 g) and dietary fibre (33.5 g/100 g). According to the Osborne classification, plant storage protein was characterised into four categories based on their solubility in different solvents. Results from sequential Osborne extraction procedure showed that lupin protein comprised of 46% salt-soluble globulin, 27% water-soluble albumin, 18% alkaline-soluble glutelin and 7% alcohol-soluble prolamin fractions. Furthermore, lupin protein was rich in lysine but limiting in methionine. In this study, Alcalase and Flavourzyme were used to hydrolyse the lupin protein isolate for different hydrolysis times (4, 10 and 16 h). Gel electrophoresis analysis demonstrated that protein hydrolysis catalysed by Alcalase was more effective compared with Flavourzyme as evidenced by the lower molecular weight peptides presented in the hydrolysates prepared using Alcalase. The ACE inhibitory activity of hydrolysates was determined using an in vitro method. Hydrolysates prepared using Alcalase exhibited higher ACE inhibitory activities compared with those prepared using Flavourzyme, showing IC50 values ranging from 0

Angiotensin-converting enzyme (ACE) plays a dominant role in blood pressure regulating system. Synthetic ACE inhibitor is designed as an antihypertensive drug to restrict ACE activity. However, the usage of synthetic drug may cause several adverse effects. Hence, a natural food protein with ACE inhibitory activity may be promising as a safer alternative to the synthetic drug. Blue lupin (Lupinus angustifolius) is one of the legumes that rich in protein. It has the potential to be a good source of ACE inhibitory peptides. However, blue lupin is usually served as an animal feed and consumption by human is rare. Therefore, this study was carried out to investigate the nutritional, protein and amino acid composition of blue lupin flour, and to evaluate the ACE inhibitory activity of its protein hydrolysates. The results revealed that the lupin flour was abundant in protein (43.3 g/100 g) and dietary fibre (33.5 g/100 g). According to the Osborne classification, plant storage protein was characterised into four categories based on their solubility in different solvents. Results from sequential Osborne extraction procedure showed that lupin protein comprised of 46% salt-soluble globulin, 27% water-soluble albumin, 18% alkaline-soluble glutelin and 7% alcohol-soluble prolamin fractions. Furthermore, lupin protein was rich in lysine but limiting in methionine. In this study, Alcalase and Flavourzyme were used to hydrolyse the lupin protein isolate for different hydrolysis times (4, 10 and 16 h). Gel electrophoresis analysis demonstrated that protein hydrolysis catalysed by Alcalase was more effective compared with Flavourzyme as evidenced by the lower molecular weight peptides presented in the hydrolysates prepared using Alcalase. The ACE inhibitory activity of hydrolysates was determined using an in vitro method. Hydrolysates prepared using Alcalase exhibited higher ACE inhibitory activities compared with those prepared using Flavourzyme, showing IC50 values ranging from 0

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-2-3 activator, the MC2562 compound we synthesized recently. Both resveratrol and MC2562 were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing keratinocyte proliferation. In vitro experiments revealed that the activation of SIRTs stimulated keratinocyte proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC2 was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC2 detachment from the promoter region of insulin growth factor I (IGF-I), fibroblast growth factor 10 (FGF-10), and Epithelial Growth Factor (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

OBJECTIVE: Adiponectin (APN) system malfunction is causatively related to increased cardiovascular morbidity/mortality in diabetic patients. The aim of the current study was to investigate molecular mechanisms responsible for APN transmembrane signaling and cardioprotection. METHODS AND RESULTS: Compared with wild-type mice, caveolin-3 knockout (Cav-3KO) mice exhibited modestly increased myocardial ischemia/reperfusion injury (increased infarct size, apoptosis, and poorer cardiac function recovery; P CONCLUSIONS: Taken together, these results demonstrated for the first time that Cav-3 plays an essential role in APN transmembrane signaling and APN anti-ischemic/cardioprotective actions.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.

Monoaminergic neurotransmission is a key element in the physiopathology of depressive disorders, but information is still sparse on animal models of this disease. Here, we used the olfactory bulbectomy (OBX) model of depression to characterize cAMP-second messenger signaling pathways, i.e., adenylyl cyclase activity (basal, sodium fluoride (NaF)- and forskolin-stimulated conditions) as well as Gi and Gs protein levels in different regions of the limbic system. Two weeks after surgery and compared to sham controls, OBX rats displayed reduced NaF-stimulated adenylyl cyclase activity and increased Gi/Gs ratios in the hypothalamus, pre-frontal and cingulate cortices but not in the amygdala, hippocampus and caudate nucleus. No differences were found in basal or forskolin-stimulated conditions. The observed reduction of adenylyl cyclase activity induced by NaF and the increase in the Gi/Gs ratio could explain the changes in neurotransmission in OBX rats as well as in humans with depression.

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects. (c) 2008 IUBMB IUBMB Life, 2008.