Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts

DNA damage induced by ultraviolet (UV) radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR) and the other specific for transcribed DNA (TCR), and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient) and XP-C (GGR-deficient) primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high), defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions.

Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt

Goncalves DA, Silveira WA, Lira EC, Gra a FA, Paula-Gomes S, Zanon NM, Kettelhut IC, Navegantes LC. Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt. Am J Physiol Endocrinol Metab 302: E123-E133, 2012. First published September 27, 2011; doi:10.1152/ajpendo.00188.2011.-Although it is well known that administration of the selective beta(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 mu M), a PKA activator. The in vitro addition of triciribine (10 mu M), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.

Exercise training prevents oxidative stress and ubiquitin-proteasome system overactivity and reverse skeletal muscle atrophy in heart failure

Background: Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF. Methods/Principal Findings: Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7th month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects' levels. Conclusions: Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.

Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice

Cunha TF, Moreira JB, Paixao NA, Campos JC, Monteiro AW, Bacurau AV, Bueno CR Jr., Ferreira JC, Brum PC. Aerobic exercise training upregulates skeletal muscle calpain and ubiquitin-proteasome systems in healthy mice. J Appl Physiol 112: 1839-1846, 2012. First published March 29, 2012; doi:10.1152/japplphysiol.00346.2011.-Aerobic exercise training (AET) is an important mechanical stimulus that modulates skeletal muscle protein turnover, leading to structural rearrangement. Since the ubiquitin-proteasome system (UPS) and calpain system are major proteolytic pathways involved in protein turnover, we aimed to investigate the effects of intensity-controlled AET on the skeletal muscle UPS and calpain system and their association to training-induced structural adaptations. Long-lasting effects of AET were studied in C57BL/6J mice after 2 or 8 wk of AET. Plantaris cross-sectional area (CSA) and capillarization were assessed by myosin ATPase staining. mRNA and protein expression levels of main components of the UPS and calpain system were evaluated in plantaris by real-time PCR and Western immunoblotting, respectively. No proteolytic system activation was observed after 2 wk of AET. Eight weeks of AET resulted in improved running capacity, plantaris capillarization, and CSA. Muscle RING finger-1 mRNA expression was increased in 8-wk-trained mice. Accordingly, elevated 26S proteasome activity was observed in the 8-wk-trained group, without accumulation of ubiquitinated or carbonylated proteins. In addition, calpain abundance was increased by 8 wk of AET, whereas no difference was observed in its endogenous inhibitor calpastatin. Taken together, our findings indicate that skeletal muscle enhancements, as evidenced by increased running capacity, plantaris capillarization, and CSA, occurred in spite of the upregulated UPS and calpain system, suggesting that overactivation of skeletal muscle proteolytic systems is not restricted to atrophying states. Our data provide evidence for the contribution of the UPS and calpain system to metabolic turnover of myofibrillar proteins and skeletal muscle adaptations to AET.

Ubiquitin proteasome system and the atypical kinase PfPK7 are involved in melatonin signaling in Plasmodium falciparum

We previously reported that melatonin modulates the Plasmodium falciparum erythrocytic cycle by increasing schizont stage population as well as diminishing ring stage population. In addition, the importance of calcium and cAMP in melatonin signaling pathway in P. falciparum was also demonstrated. Nevertheless, the molecular effectors of the indoleamine signaling pathway remain elusive. We now demonstrate by real-time PCR that melatonin treatment up-regulates genes related to ubiquitin/proteasome system (UPS) components and that luzindole, a melatonin receptor antagonist, inhibits UPS transcription modulation. We also show that protein kinase PfPK7, a P. falciparum orphan kinase, plays a crucial role in the melatonin transduction pathway, since following melatonin treatment of P. falciparum parasites where pfpk7 gene is disrupted (pfpk7- parasites) (i) the ratio of asexual stages remain unchanged, (ii) the increase in cytoplasmatic calcium in response to melatonin was strongly diminished and (iii) up-regulation of UPS genes did not occur. The wild-type melatonin-induced alterations in cell cycle features, calcium rise and UPS gene transcription were restored by re-introduction of a functional copy of the pfpk7 gene in the pfpk7- parasites.

The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development

Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.

Association of genetic variants in the promoter region of genes encoding p22phox (CYBA) and glutamate cysteine ligase catalytic subunit (GCLC) and renal disease in patients with type 1 diabetes mellitus

Background Oxidative stress is recognized as a major pathogenic factor of cellular damage caused by hyperglycemia. NOX/NADPH oxidases generate reactive oxygen species and NOX1, NOX2 and NOX4 isoforms are expressed in kidney and require association with subunit p22phox (encoded by the CYBA gene). Increased expression of p22phox was described in animal models of diabetic nephropathy. In the opposite direction, glutathione is one of the main endogenous antioxidants whose plasmatic concentrations were reported to be reduced in diabetes patients. The aim of the present investigation was to test whether functional single nucleotide polymorphisms (SNPs) in genes involved in the generation of NADPH-dependent O2•- (-675 T → A in CYBA, unregistered) and in glutathione metabolism (-129 C → T in GCLC [rs17883901] and -65 T → C in GPX3 [rs8177412]) confer susceptibility to renal disease in type 1 diabetes patients. Methods 401 patients were sorted into two groups according to the presence (n = 104) or absence (n = 196) of overt diabetic nephropathy or according to glomerular filtration rate (GFR) estimated by Modification of Diet in Renal Disease (MDRD) equation: ≥ 60 mL (n = 265) or < 60 mL/min/1.73 m2 (n = 136) and were genotyped. Results No differences were found in the frequency of genotypes between diabetic and non-diabetic subjects. The frequency of GFR < 60 mL/min was significantly lower in the group of patients carrying CYBA genotypes T/A+A/A (18.7%) than in the group carrying the T/T genotype (35.3%) (P = 0.0143) and the frequency of GFR < 60 mL/min was significantly higher in the group of patients carrying GCLC genotypes C/T+T/T (47.1%) than in the group carrying the C/C genotype (31.1%) (p = 0.0082). Logistic regression analysis identified the presence of at least one A allele of the CYBA SNP as an independent protection factor against decreased GFR (OR = 0.38, CI95% 0.14-0.88, p = 0.0354) and the presence of at least one T allele of the GCLC rs17883901 SNP as an independent risk factor for decreased GFR (OR = 2.40, CI95% 1.27-4.56, p = 0.0068). Conclusions The functional SNPs CYBA -675 T → A and GCLC rs17883901, probably associated with cellular redox imbalances, modulate the risk for renal disease in the studied population of type 1 diabetes patients and require validation in additional cohorts.

1031-1034delTAAC (Leu125Stop): a novel familial UBE3A mutation causing Angelman syndrome in two siblings showing distinct phenotypes

Background More than 50 mutations in the UBE3A gene (E6-AP ubiquitin protein ligase gene) have been found in Angelman syndrome patients with no deletion, no uniparental disomy, and no imprinting defect. Case Presentation We here describe a novel UBE3A frameshift mutation in two siblings who have inherited it from their asymptomatic mother. Despite carrying the same UBE3A mutation, the proband shows a more severe phenotype whereas his sister shows a milder phenotype presenting the typical AS features. Conclusions We hypothesized that the mutation Leu125Stop causes both severe and milder phenotypes. Potential mechanisms include: i) maybe the proband has an additional problem (genetic or environmental) besides the UBE3A mutation; ii) since the two siblings have different fathers, the UBE3A mutation is interacting with a different genetic variant in the proband that, by itself, does not cause problems but in combination with the UBE3A mutation causes the severe phenotype; iii) this UBE3A mutation alone can cause either typical AS or the severe clinical picture seen in the proband.

FGFR4 Profile as a Prognostic Marker in Squamous Cell Carcinoma of the Mouth and Oropharynx

Background: Fibroblast growth factor receptor 4 (FGFR4) is a member of a receptor tyrosine kinase family of enzymes involved in cell cycle control and proliferation. A common single nucleotide polymorphism (SNP) Gly388Arg variant has been associated with increased tumor cell motility and progression of breast cancer, head and neck cancer and soft tissue sarcomas. The present study evaluated the prognostic significance of FGFR4 in oral and oropharynx carcinomas, finding an association of FGFR4 expression and Gly388Arg genotype with tumor onset and prognosis. Patients and Methods: DNA from peripheral blood of 122 patients with oral and oropharyngeal squamous cell carcinomas was used to determine FGFR4 genotype by PCR-RFLP. Protein expression was assessed by immunohistochemistry (IHC) on paraffin-embedded tissue microarrays. Results: Presence of allele Arg388 was associated with lymphatic embolization and with disease related premature death. In addition, FGFR4 low expression was related with lymph node positivity and premature relapse of disease, as well as disease related death. Conclusion: Our results propose FGFR4 profile, measured by the Gly388Arg genotype and expression, as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx.

Redox Control of 20S Proteasome Gating

The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin-and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities. Aims: The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation. Results: Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal alpha 5 subunit of yeast cells grown to the stationary phase in glucose-containing medium. Innovation: We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity. Conclusion: S-glutathiolation is a post-translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption. Antioxid. Redox Signal. 16, 1183-1194.

Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.

Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response. IMPORTANCE Athlete's foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete's foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host's immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.

Protein quality control disruption by PKC beta II in heart failure: rescue by the selective PKC beta II inhibitor, beta IIV5-3

Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform beta II (PKC beta II) in disrupting PQC. We show that active PKC beta II directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKC beta II, using a selective PKC beta II peptide inhibitor (beta IIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKC beta II increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, beta IIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKC beta II activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKC beta II as a novel inhibitor of proteasomal function. PQC disruption by increased PKC beta II activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKC beta II inhibition may benefit patients with heart failure. (218 words)