Molecular targets underlying SUMO-mediated neuroprotection in brain ischemia

SUMOylation (small ubiquitin-like modifier conjugation) is an important post-translational modification which is becoming increasingly implicated in the altered protein dynamics associated with brain ischemia. The function of SUMOylation in cells undergoing ischemic stress and the identity of small ubiquitin-like modifier (SUMO) targets remain in most cases unknown. However, the emerging consensus is that SUMOylation of certain proteins might be part of an endogenous neuroprotective response. This review brings together the current understanding of the underlying mechanisms and downstream effects of SUMOylation in brain ischemia, including processes such as autophagy, mitophagy and oxidative stress. We focus on recent advances and controversies regarding key central nervous system proteins, including those associated with the nucleus, cytoplasm and plasma membrane, such as glucose transporters (GLUT1, GLUT4), excitatory amino acid transporter 2 glutamate transporters, K+ channels (K2P1, Kv1.5, Kv2.1), GluK2 kainate receptors, mGluR8 glutamate receptors and CB1 cannabinoid receptors, which are reported to be SUMO-modified. A discussion of the roles of these molecular targets for SUMOylation could play following an ischemic event, particularly with respect to their potential neuroprotective impact in brain ischemia, is proposed.

Propeptide-mediated inhibition of myostatin increases muscle mass through inhibiting proteolytic pathways in aged mice

Mammalian aging is accompanied by a progressive loss of skeletal muscle, a process called sarcopenia. Myostatin, a secreted member of the transforming growth factor-β family of signaling molecules, has been shown to be a potent inhibitor of muscle growth. Here, we examined whether muscle growth could be promoted in aged animals by antagonizing the activity of myostatin through the neutralizing activity of the myostatin propeptide. We show that a single injection of an AAV8 virus expressing the myostatin propeptide induced an increase in whole body weights and all muscles examined within 7 weeks of treatment. Our cellular studies demonstrate that muscle enlargement was due to selective fiber type hypertrophy, which was accompanied by a shift toward a glycolytic phenotype. Our molecular investigations elucidate the mechanism underpinning muscle hypertrophy by showing a decrease in the expression of key genes that control ubiquitin-mediated protein breakdown. Most importantly, we show that the hypertrophic muscle that develops as a consequence of myostatin propeptide in aged mice has normal contractile properties. We suggest that attenuating myostatin signaling could be a very attractive strategy to halt and possibly reverse age-related muscle loss.

Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling

BACKGROUND: Brain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-alpha) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation. RESULTS: Here we describe for the first time the TNF-mediated signal transduction cascade in neural stem cells (NSCs) that results in increased proliferation. Moreover, we demonstrate IKK-alpha/beta-dependent proliferation and markedly up-regulated cyclin D1 expression after TNF treatment. The significant increase in proliferation in TNF-treated cells was indicated by increased neurosphere volume, increased bromodeoxyuridin (BrdU) incorporation and a higher total cell number. Furthermore, TNF strongly activated nuclear factor-kappa B (NF-kappaB) as measured by reporter gene assays and by an activity-specific antibody. Proliferation of control and TNF-treated NSCs was strongly inhibited by expression of the NF-kappaB super-repressor IkappaB-AA1. Pharmacological blockade of IkappaB ubiquitin ligase activity led to comparable decreases in NF-kappaB activity and proliferation. In addition, IKK-beta gene product knock-down via siRNA led to diminished NF-kappaB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFbeta-activated kinase 1 (TAK-1) is partially dispensable for TNF-mediated and endogenous proliferation. Understanding stem cell proliferation is crucial for future regenerative and anti-tumor medicine. CONCLUSION: TNF-mediated activation of IKK-beta resulted in activation of NF-kappaB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-kappaB pathway resulted in strongly increased proliferation of NSCs.

SUMOylation: novel neuroprotective approach for Alzheimer's disease?

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized in the brain by the formation of amyloid-beta (Aβ)-containing plaques and neurofibrillary tangles containing the microtubule-associated protein tau. Neuroinflammation is another feature of AD and astrocytes are receiving increasing attention as key contributors. Although some progress has been made, the molecular mechanisms underlying the pathophysiology of AD remain unclear. Interestingly, some of the main proteins involved in AD, including amyloid precursor protein (APP) and tau, have recently been shown to be SUMOylated. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to regulate APP and tau and may modulate other proteins implicated in AD. Here we present an overview of recent studies suggesting that protein SUMOylation might be involved in the underlying pathogenic mechanisms of AD and discuss how this could be exploited for therapeutic intervention.

The Cochaperone BAG2 Sweeps Paired Helical Filament-Insoluble Tau from the Microtubule

Tau inclusions are a prominent feature of many neurodegenerative diseases including Alzheimer`s disease. Their accumulation in neurons as ubiquitinated filaments suggests a failure in the degradation limb of the Tau pathway. The components of a Tau protein triage system consisting of CHIP/Hsp70 and other chaperones have begun to emerge. However, the site of triage and the master regulatory elements are unknown. Here, we report an elegant mechanism of Tau degradation involving the cochaperone BAG2. The BAG2/Hsp70 complex is tethered to the microtubule and this complex can capture and deliver Tau to the proteasome for ubiquitin-independent degradation. This complex preferentially degrades Sarkosyl insoluble Tau and phosphorylated Tau. BAG2 levels in cells are under the physiological control of the microRNA miR-128a, which can tune paired helical filament Tau levels in neurons. Thus, we propose that ubiquitinated Tau inclusions arise due to shunting of Tau degradation toward a less efficient ubiquitin-dependent pathway.

Hepatic denervation impairs the assembly and secretion of VLDL-TAG

VLDL secretion is a regulated process that depends on the availability of lipids, apoB and MTP. Our aim was to investigate the effect of liver denervation upon the secretion of VLDL and the expression of proteins involved in this process. Denervation was achieved by applying a 85% phenol solution onto the portal tract, while control animals were treated with 9% NaCl. VLDL secretion was evaluated by the Tyloxapol method. The hepatic concentration of TAG and cholesterol, and the plasma concentration of TAG, cholesterol, VLDL-TAG, VLDL-cholesterol and HDL-cholesterol were measured, as well as mRNA expression of proteins involved in the process of VLDL assembly. Hepatic acinar distribution of MTP and apoB was evaluated by immunohistochemistry. Denervation increased plasma concentration of cholesterol (125.3 +/- 10.1 vs. 67.1 +/- 4.9 mg dL(-1)) and VLDL-cholesterol (61.6 +/- 5.6 vs. 29.4 +/- 3.3 mg dL(-1)), but HDL-cholesterol was unchanged (45.5 +/- 6.1 vs. 36.9 +/- 3.9 mg dL(-1)). Secretion of VLDL-TAG (47.5 +/- 23.8 vs. 148.5 +/- 27.4 mg dL h(-1)) and mRNA expression of CPT I and apoB were reduced (p < 0.01) in the denervated animals. MTP and apoB acinar distribution was not altered in the denervated animals, but the intensity of the reaction was reduced in relation to controls. Copyright (C) 2008 John Wiley & Sons, Ltd.

In vivo effects of TGF beta 1 on the the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGF beta) wanes in the milk of lactating rat, an increase in TGF beta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGF beta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGF beta 1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGF beta 1 while T beta RII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p < 0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p < 0.05). Importantly, TGF beta 1 inhibited cell proliferation (p < 0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p < 0.05). Also, TGF beta 1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p < 0.001), and by morphological features at 12 h (p < 0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGF beta 1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TG beta 1 in gastric growth during postnatal development, (C) 2007 Elsevier B.V. All rights reserved.

Neurofilament heavy subunit in cerebrospinal fluid: A biomarker of amyotrophic lateral sclerosis?

The objectives of this study were to investigate the presence of the three neurofilament subunits, ubiquitin, proteasome and 3-nitrotyrosine, in CSF samples of ALS patients. CSF samples were obtained by lumbar puncture from 10 ALS patients and six controls. All samples were analysed by Western blotting. Results revealed that neurofilament heavy subunit was identified in 70% of ALS cases and we conclude that this subunit may be a promising biomarker for clinical diagnosis of ALS.

Intracellular peptides as natural regulators of cell signaling

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 mu M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R-CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

Exercise Training Reduces Insulin Resistance and Upregulates the mTOR/p70S6k Pathway in Cardiac Muscle of Diet-Induced Obesity Rats

Obesity and insulin resistance are rapidly expanding public health problems. These disturbances are related to many diseases, including heart pathology. Acting through the Akt/mTOR pathway, insulin has numerous and important physiological functions, such as the induction of growth and survival of many cell types and cardiac hypertrophy. However, obesity and insulin resistance can alter mTOR/p70S6k. Exercise training is known to induce this pathway, but never in the heart of diet-induced obesity subjects. To evaluate the effect of exercise training on mTOR/p70S6k in the heart of obese Wistar rats, we analyzed the effects of 12 weeks of swimming on obese rats, induced by a high-fat diet. Exercise training reduced epididymal fat, fasting serum insulin and plasma glucose disappearance. Western blot analyses showed that exercise training increased the ability of insulin to phosphorylate intracellular molecules such as Akt (2.3-fold) and Foxo1 (1.7-fold). Moreover, reduced activities and expressions of proteins, induced by the high-fat diet in rats, such as phospho-JNK (1.9-fold), NF-kB (1.6-fold) and PTP-1B (1.5-fold), were observed. Finally, exercise training increased the activities of the transduction pathways of insulin-dependent protein synthesis, as shown by increases in Raptor phosphorylation (1.7-fold), p70S6k phosphorylation (1.9-fold), and 4E-BP1 phosphorylation (1.4-fold) and a reduction in atrogin-1 expression (2.1-fold). Results demonstrate a pivotal regulatory role of exercise training on the Akt/ mTOR pathway, in turn, promoting protein synthesis and antagonizing protein degradation. J. Cell. Physiol. 226: 666-674, 2011. (C) 2010 Wiley-Liss, Inc.

MuRF1 is a muscle fiber-type II associated factor and together with MuRF2 regulates type-II fiber trophicity and maintenance

MuRF1 is a member of the RBCC (RING, B-box, coiled-coil) superfamily that has been proposed to act as an atrogin during muscle wasting. Here, we show that MuRF1 is preferentially induced in type-II muscle fibers after denervation. Fourteen days after denervation, MuRF1 protein was further elevated but remained preferentially expressed in type-II muscle fibers. Consistent with a fiber-type dependent function of MuRF1, the tibialis anterior muscle (rich in type-II muscle fibers) was considerably more protected in MuRF1-KO mice from muscle wasting when compared to soleus muscle with mixed fiber-types. We also determined fiber-type distributions in MuRF1/MuRF2 double-deficient KO (dKO) mice, because MuRF2 is a close homolog of MuRF1. MuRF1/MuRF2 dKO mice showed a profound loss of type-II fibers in soleus muscle. As a potential mechanism we identified the interaction of MuRF1/MuRF2 with myozenin-1, a calcineurin/NFAT regulator and a factor required for maintenance of type-II muscle fibers. MuRF1/MuRF2 dKO mice had lost myozenin-1 expression in tibialis anterior muscle, implicating MuRF1/MuRF2 as regulators of the calcineurin/NFAT pathway. In summary, our data suggest that expression of MuRF1 is required for remodeling of type-II fibers under pathophysiological stress states, whereas MuRF1 and MuRF2 together are required for maintenance of type-II fibers, possibly via the regulation of myozenin-1. (C) 2010 Elsevier Inc. All rights reserved.

Identification of genes associated with local aggressiveness and metastatic behavior in soft tissue tumors

Soft tissue tumors represent a group of neoplasia with different histologic and biological presentations varying from benign, locally confined to very aggressive and metastatic tumors. The molecular mechanisms responsible for such differences are still unknown. The understanding of these molecular alterations mechanism will be critical to discriminate patients who need systemic treatment from those that can be treated only locally and could also guide the development of new drugs` against this tumors. Using 102 tumor samples representing a large spectrum of these tumors, we performed expression profiling and defined differentially expression genes that are likely to be involved in tumors that are locally aggressive and in tumors with metastatic potential. We described a set of 12 genes (SNRPD3, MEGF9, SPTAN-1, AFAP1L2, ENDOD1, SERPIN5, ZWINTAS, TOP2A, UBE2C, ABCF1, MCM2, and ARL6IP5) showing opposite expression when these two conditions were compared. These genes are mainly related to cell-cell and cell-extracellular matrix interactions and cell proliferation and might represent helpful tools for a more precise classification and diagnosis as well as potential drug targets.

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.

The Molecular Chaperone Hsp70 Family Members Function by a Bidirectional Heterotrophic Allosteric Mechanism

The Hsp70 family is one of the most important and conserved molecular chaperone families. It is well documented that Hsp70 family members assist many cellular processes involving protein quality control, as follows: protein folding, transport through membranes, protein degradation, escape from aggregation, intracellular signaling, among several others. The Hsp70 proteins act as a cellular pivot capable of receiving and distributing substrates among the other molecular chaperone families. Despite the high identity of the Hsp70 proteins, there are several homologue Hsp70 members that do not have the same role in the cell, which allow them to develop and participate in such large number of activities. The Hsp70 proteins are composed of two main domains: one that binds ATP and hydrolyses it to ADP and another which directly interacts with substrates. These domains present bidirectional heterotrophic allosteric regulation allowing a fine regulated cycle of substrate binding and release. The general mechanism of the Hsp70s cycle is under the control of ATP hydrolysis that modulates the low (ATP-bound state) and high (ADP-bound state) affinity states of Hsp70 for substrates. An important feature of the Hsp70s cycle is that they have several co-chaperones that modulate their cycle and that can also interact and select substrates. Here, we review some known details of the bidirectional heterotrophic allosteric mechanism and other important features for Hsp70s regulating cycle and function.

Beacon interacts with cdc2/cdc28-like kinases

Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.