José Miguel P. Ferreira de Oliveira

Proteomic analysis of the secretory response of Aspergillus niger to D-maltose and D-xylose

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on d-sorbitol, small amounts of d-maltose or d-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by d-maltose or d-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on d-maltose and β-xylosidase D on d-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra d-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of d-xylose or d-maltose. Furthermore, d-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers d-maltose and d-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by d-maltose or d-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for d-xylose induction, d-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.

Proteomics of industrial fungi: trends and insights for biotechnology

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.

Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction.

ABSTRACT Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which—many of them hypothetical proteins—were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.

Efficient cloning system for construction of gene silencing vectors in Aspergillus niger

An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on d-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR.

The CAG repeat within the androgen receptor gene and its relationship to cryptorchidism

PURPOSE We examined the significance of the CAG repeat polymorphism in the pathogenesis of cryptorchidism. MATERIALS AND METHODS Genomic deoxyribonucleic acid (DNA) was extracted from blood samples from 42 cryptorchid boys and from 31 non-cryptorchid control subjects. In the cryptorchid group, 7 had bilateral cryptorchidism and 6 had patent processus vaginalis in the contralateral side. To determine the number of CAG repeats, the DNA was amplified by polymerase chain reaction and sequenced. RESULTS The mean CAG repeat length in the AR gene was 22.5 (range 16 to 28) in patients and 21.5 (range 17 to 26) in controls (non-significant). Patients with bilateral cryptorchidism had a mean length of 24.3 (range 21 to 26) and patients with unilateral cryptorchidism and patent processus vaginalis in the contra lateral side had a mean of 25.2 (range 21 to 28), which was statistically different from controls (p = 0.015 and p = 0.005 respectively). CONCLUSION CAG repeat length of the AR gene does not seem to play a major role in patients with unilateral cryptorchidism. However, in patients with bilateral undescended testis, a less functional androgen receptor through a longer polyglutamine chain may have a role in its pathogenesis. In the same way, patients with unilateral cryptorchidism a contralateral patent processus vaginalis have longer CAG repeats that might be responsible for a slower testicular descent and incomplete closure of the processus vaginalis.

Analysis of Variance Components Reveals the Contribution of Sample Processing to Transcript Variation

ABSTRACT The proper design of DNA microarray experiments requires knowledge of biological and technical variation of the studied biological model. For the filamentous fungus Aspergillus niger, a fast, quantitative real-time PCR (qPCR)-based hierarchical experimental design was used to determine this variation. Analysis of variance components determined the contribution of each processing step to total variation: 68% is due to differences in day-to-day handling and processing, while the fermentor vessel, cDNA synthesis, and qPCR measurement each contributed equally to the remainder of variation. The global transcriptional response to d-xylose was analyzed using Affymetrix microarrays. Twenty-four statistically differentially expressed genes were identified. These encode enzymes required to degrade and metabolize d-xylose-containing polysaccharides, as well as complementary enzymes required to metabolize complex polymers likely present in the vicinity of d-xylose-containing substrates. These results confirm previous findings that the d-xylose signal is interpreted by the fungus as the availability of a multitude of complex polysaccharides. Measurement of a limited number of transcripts in a defined experimental setup followed by analysis of variance components is a fast and reliable method to determine biological and technical variation present in qPCR and microarray studies. This approach provides important parameters for the experimental design of batch-grown filamentous cultures and facilitates the evaluation and interpretation of microarray data.

Sulforaphane Induces Oxidative Stress and Death by p53-Independent Mechanism: Implication of Impaired Glutathione Recycling

Sulforaphane (SFN) is a naturally-occurring isothiocyanate best known for its role as an indirect antioxidant. Notwithstanding, in different cancer cell lines, SFN may promote the accumulation of reactive oxygen species (ROS) and cause cell death e.g. by apoptosis. Osteosarcoma often becomes chemoresistant, and new molecular targets to prevent drug resistance are needed. Here, we aimed to determine the effect of SFN on ROS levels and to identify key biomarkers leading to ROS unbalance and apoptosis in the p53-null MG-63 osteosarcoma cell line. MG-63 cells were exposed to SFN for up to 48 h. At 10 μM concentration or higher, SFN decreased cell viability, increased the%early apoptotic cells and increased caspase 3 activity. At these higher doses, SFN increased ROS levels, which correlated with apoptotic endpoints and cell viability decline. In exposed cells, gene expression analysis revealed only partial induction of phase-2 detoxification genes. More importantly, SFN inhibited ROS-scavenging enzymes and impaired glutathione recycling, as evidenced by inhibition of glutathione reductase (GR) activity and combined inhibition of glutathione peroxidase (GPx) gene expression and enzyme activity. In conclusion, SFN induced oxidative stress and apoptosis via a p53-independent mechanism. GPx expression and activity were found associated with ROS accumulation in MG-63 cells and are potential biomarkers for the efficacy of ROS-inducing agents e.g. as co-adjuvant drugs in osteosarcoma.

The use of comet assay in plant toxicology: recent advances

The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

Sulforaphane induces DNA damage and mitotic abnormalities in human osteosarcoma MG-63 cells: correlation with cell cycle arrest and apoptosis.

Osteosarcoma is a recalcitrant bone malignancy with poor responsiveness to treatments; therefore, new chemotherapeutic compounds are needed. Sulforaphane (SFN) has been considered a promising chemotherapeutic compound for several types of tumors by inducing apoptosis and cytostasis, but its effects (e.g., genotoxicity) in osteosarcoma cells remains exploratory. In this work, the MG-63 osteosarcoma cell line was exposed to SFN up to 20 μM for 24 and 48 h. SFN induced G2/M phase arrest and decreased nuclear division index, associated with disruption of cytoskeletal organization. Noteworthy, SFN induced a transcriptome response supportive of G2/M phase arrest, namely a decrease in Chk1- and Cdc25C-encoding transcripts, and an increase in Cdk1-encoding transcripts. After 48-h exposure, SFN at a dietary concentration (5 μM) contributed to genomic instability in the MG-63 cells as confirmed by increased number of DNA breaks, clastogenicity, and nuclear and mitotic abnormalities. The increased formation of nucleoplasmic bridges, micronuclei, and apoptotic cells positively correlated with loss of viability. These results suggest that genotoxic damage is an important step for SFN-induced cytotoxicity in MG-63 cells. In conclusion, SFN shows potential to induce genotoxic damage at low concentrations and such potential deserves further investigation in other tumor cell types.

Cadmium-induced genotoxicity in human osteoblast-like cells.

Cadmium (Cd) is a widespread heavy metal used in numerous industrial processes. Cd exerts toxicological effects mostly in kidney and liver. Bone is also an important target of Cd, however, the cellular mechanisms of Cd toxicological effects in the bone cells are still poorly understood. Therefore, the present work aimed to investigate the putative cytotoxic and genotoxic effects of Cd to human bone cells. For that, the osteoblast-like MG-63 cells were exposed to 20 and 50μM Cd for 24 and 48h. Results showed a dose-dependent increase in Cd accumulation in cells and a decrease in cell viability, especially after 48h. Cell cycle analysis showed a delay at S phase concomitant with a decrease in cells at G0/G1 phase. After 24h, Cd treatment downregulated the expression of CHEK1, CHEK2 and CDK2 genes and upregulated the expression of CCNE1 gene. After 48h, the expression of ATM and CCNB1 genes were downregulated. Also, a 3.3 fold increase on the expression of gene CCNE1 was detected. Both Cd doses induced DNA fragmentation at 48h, while an increase in micronuclei (MN) and nucleoplasmic bridges (NPBs) together with an increase in the percentage of apoptotic/necrotic cells was detected for both time periods. Overall, our results demonstrate the cytotoxicity and genotoxicity of Cd in human bone cells. Also, the cytokinesis-block micronucleus (CBMN) assay parameters (MN, NPBs and the percentage of cells under apoptosis or necrosis) together with the cell cycle appear as the most sensitive to Cd cyto- and genotoxicity, being early affected even with the lowest Cd dose. Therefore, these cyto-/genotoxic techniques may be selected for early detection of Cd-induced toxicity.